CN116585522A - A physically cross-linked chitosan-based hemostatic sponge and preparation method thereof - Google Patents

Abstract

The invention discloses a physically cross-linked chitosan-based hemostatic sponge and a preparation method thereof. The sponge uses chitosan as a main raw material, and first drips an alkali solution into the chitosan acid solution and then centrifuges to obtain the chitosan gel. Gel, chitosan gel and carboxymethyl chitosan aqueous solution are mixed and stirred, freeze-dried to prepare the hemostatic sponge. In this paper, no cross-linking agent is used, and chitosan gel is used as the main component. Chitosan gel and carboxymethyl chitosan are cross-linked by hydrogen bonds to ensure the mechanical properties of the sponge. Chitosan/carboxymethyl chitosan hemostatic sponge has a liquid absorption rate of more than 40 times, and the hemostatic effect is equivalent to that of gauze, and has a uniform pore structure, good biocompatibility, and no biotoxicity.

Description

A physically cross-linked chitosan-based hemostatic sponge and preparation method thereof

technical field

The invention relates to a physically cross-linked chitosan-based hemostatic sponge and a preparation method thereof, belonging to the technical field of biological materials.

Background technique

Sponge is a solid porous preparation with strong absorption capacity, light weight and low density, which can stop bleeding and absorb wound exudate, and has good gas permeability, showing better advantages in hemostasis. Chitosan is a natural high molecular polymer, which is prepared by deacetylation of chitin. Because of its wide range of biological activities, good biocompatibility, complete biodegradability and low toxicity, it has a wide range of biological and medical fields. potential application prospects. However, because chitosan is not easily soluble in water and acid, the preparation process of chitosan sponge is relatively long, requiring alkaline washing or secondary freeze-drying, and its application range is limited. In order to improve the preparation process of chitosan-based sponge, many researchers have devoted themselves to the study of carboxylated chitosan, that is, modifying chitosan to prepare water-soluble carboxylated chitosan, and using carboxylated chitosan Prepare hemostatic sponge by reacting with cross-linking agent. It has been found through research that this type of hemostatic sponge still has the problems of poor hemostatic effect and low liquid absorption multiple. Therefore, it is of great significance to study a hemostatic sponge with good hemostatic performance and short technological process.

Contents of the invention

The object of the present invention is to provide a physically cross-linked chitosan-based hemostatic sponge for the existing chitosan sponges, which have problems such as long technological process, poor water absorption, and poor hemostatic effect. Secondary freezing, good hemostatic performance, and short process flow.

One of object of the present invention is to provide a kind of preparation method of the chitosan-based hemostatic sponge of physical crosslinking, comprises the following steps:

(1) add chitosan in acid solution, stir until dissolving completely, obtain chitosan acid solution;

(2) in the chitosan acid solution that step (1) makes, slowly drip alkaline solution to white flocculent precipitation and no longer separate out, centrifugal washing is neutral to supernatant, obtains chitosan gel;

(3) Mix the chitosan gel in the step (2) with the carboxymethyl chitosan aqueous solution, stir evenly, leave the obtained mixed solution to stand and freeze-dry to prepare the chitosan-based hemostatic sponge.

In order to solve the problems of poor hemostatic effect and low water absorption of the chitosan hemostatic sponge prepared by the prior art, and simplify the process, the invention provides a method for preparing a physically cross-linked chitosan-based hemostatic sponge, that is, the shell is prepared first. Polysan neutral gel is prepared by cross-linking chitosan gel and carboxymethyl chitosan gel. In the process of preparing the chitosan neutral gel, an alkali solution is used to neutralize the acid in the chitosan acid solution, so that there is no acid residue in the gel. The cross-linking reaction between chitosan gel and carboxymethyl chitosan helps to further increase the stability of the gel, and can reduce the number of freeze-drying when preparing the hemostatic sponge. The prepared hemostatic sponge has excellent water absorption and in vitro Coagulation effect.

In one embodiment of the present invention, in the step (3), before the mixed solution is poured into a watch glass, an auxiliary agent aqueous solution is added to the mixed solution, and the auxiliary agent includes silk fibroin, gelatin, glycerin at least one. The addition of additives helps to further enhance the flexibility and water absorption of chitosan sponge.

In one embodiment of the present invention, in the step (3), before freeze-drying, the mixed solution is allowed to stand at 1-5° C. for 20-30 hours. The purpose of standing still is to make chitosan gel fully react with carboxymethyl chitosan to form gel.

In one embodiment of the present invention, the auxiliary agent is glycerin, and the mass concentration of the aqueous glycerin solution is 5-30%.

In one embodiment of the present invention, the viscosity of the chitosan is 50-800 CPS.

In one embodiment of the present invention, the carboxymethyl chitosan is O-carboxymethyl chitosan and/or N-carboxymethyl chitosan.

In one embodiment of the present invention, the acid comprises acetic acid and/or citric acid, the volume percentage of the acid solution is 1-2%, the alkali comprises sodium hydroxide, and the mass percentage of the alkali solution is 2 to 5%.

In one embodiment of the present invention, the mass percentage of the chitosan acid solution is 0.2-3%.

In one embodiment of the present invention, the mass percentage of the carboxymethyl chitosan aqueous solution is 0.1-5%, preferably 0.2-2%.

The second object of the present invention is to provide a hemostatic sponge obtained according to the preparation method described in claim 1.

In one embodiment of the present invention, in parts by weight, the components of the hemostatic sponge include 1-5 parts of chitosan gel and 1-20 parts of carboxymethyl chitosan.

In one embodiment of the present invention, in parts by weight, the components of the hemostatic sponge include 1-5 parts of chitosan gel, 1-20 parts of carboxymethyl chitosan, and 0.5-10 parts of glycerin.

Beneficial effects of the present invention

(1) Dissolve chitosan in acid solution, neutralize it with alkali to make gel, then carry out cross-linking reaction between neutral gel and carboxymethyl chitosan, and freeze-dry to obtain chitosan Sugar-based hemostatic sponge. The hemostatic sponge prepared by the preparation method of the present invention does not contain acetic acid and other substances that have a stimulating effect on the human body.

(2) when making hemostatic sponge, chitosan gel and carboxymethyl chitosan have crosslinked reaction, have further increased the stability of chitosan, adopt the hemostatic sponge that the inventive method makes has higher liquid absorption magnification;

(3) The formula provided by the present invention is simple, the materials used are all non-biologically toxic, the preparation process is simple to operate, no secondary freeze-drying is required, and the reaction is easy to control.

Description of drawings

Fig. 1 is the preparation process flowchart of chitosan-based hemostatic sponge of the present invention;

Fig. 2 is the SEM picture of the hemostatic sponge prepared by Examples 1-4 and Comparative Examples 1-2;

Fig. 3 is the in vitro coagulation time test result of the hemostatic sponge prepared in Examples 1-4 and Comparative Examples 1-2;

Fig. 4 is the water absorption test result of the hemostatic sponge prepared in Examples 1-4 and Comparative Examples 1-2;

Fig. 5 is the hemolysis rate test results of the hemostatic sponges prepared in Examples 1-4 and Comparative Examples 1-2.

Detailed ways

Preferred embodiments of the present invention are described below, and it should be understood that the embodiments are for better explaining the present invention, and are not intended to limit the present invention.

Test Methods:

Whole blood clotting time test: Add 10 mg hemostatic sponge into a centrifuge tube and preheat to 37°C. Then add 1 mL of anticoagulated whole blood and continue to incubate for 3 min, then immediately add 100 μL of 0.2 mol/L CaCl ₂ solution to the test tube. Gently tilt the test tube every 3 to 5 seconds until the test tube is tilted 90° and the blood no longer flows, which means coagulation, and record the coagulation time. Anticoagulated whole blood without samples was used as the blank control group.

Water absorption test: Weigh a certain mass of hemostatic sponge, and record the mass as M. Immerse it in a beaker filled with distilled water at room temperature. After absorbing enough water, gently hold a corner with tweezers to take it out of the water. Hold the tweezers gently to drain water on the water surface for 1 minute, then weigh it again, record it as M ₁ , according to the formula (1) Calculate the water absorption rate A:

Hemolysis rate test: Soak the hemostatic sponge sample in 0.9% physiological saline for 24 hours to obtain the sample extract. Mix 0.5mL sample extract with 0.5mL red blood cell suspension, and incubate at 37°C for 3h. The mixture was centrifuged at 1600 rpm for 10 min, and the supernatant was taken to measure the absorbance at 540 nm with a microplate reader. Mix 0.5mL ultrapure water with 0.5mL 2% erythrocyte suspension and incubate at 37°C for 3 hours as a positive control; mix 0.5mL 0.9% normal saline with 0.5mL 2% erythrocyte suspension and incubate at 37°C 3h as a negative control. Calculate the hemolysis rate according to formula (2):

Example 1

A preparation method of physically cross-linked chitosan-based hemostatic sponge, comprising the following steps:

(1) Get 196mL ultrapure water, add 4mL acetic acid, stir for 3 minutes, add 2g chitosan, stir until completely dissolving, obtain chitosan acetic acid solution;

(2) in the chitosan acetic acid solution that step (1) makes, slowly drip the sodium hydroxide solution that mass concentration is 4% to white precipitation and separate out completely, after white precipitation is centrifuged and washed to neutrality, chitosan is obtained Neutral gel.

(3) get 5 parts of chitosan neutral gels obtained in step (2), with 4 parts of mass concentration of 1% carboxymethyl chitosan aqueous solution, fully stir for 0.5h to obtain the mixed solution, Then, 1 part of glycerol aqueous solution with a mass concentration of 10% was added dropwise to the mixed solution, stirred for 0.5 h, and the mixed solution was transferred to a refrigerator at 4° C. for 24 h, and then the system was freeze-dried at -60° C. for 24 h to obtain Hemostatic sponge MG1.

Example 2

A preparation method of physically cross-linked chitosan-based hemostatic sponge, comprising the following steps:

(1) Get 196mL ultrapure water, add 4mL acetic acid, stir for 3 minutes, add 2g chitosan, stir until completely dissolving, obtain chitosan acetic acid solution;

(2) in the chitosan acetic acid solution that step (1) makes, slowly drip the sodium hydroxide solution that mass concentration is 4% to white precipitation and separate out completely, after white precipitation is centrifuged and washed to neutrality, chitosan is obtained Neutral gel;

(3) Get 5 parts of the chitosan neutral gel obtained in step (2), mix it with 4 parts of mass concentration of 0.5% carboxymethyl chitosan aqueous solution, fully stir for 0.5h, and obtain the mixed solution , and then dropwise added 1 part of glycerin aqueous solution with a mass concentration of 20% to the mixed solution, stirred for 0.5 h, and transferred the mixed solution to a refrigerator at 4° C. for 24 h, and then freeze-dried the system at -60° C. for 24 h, Get the hemostatic sponge MG2.

Example 3

A preparation method of physically cross-linked chitosan-based hemostatic sponge, comprising the following steps:

(1) Get 196mL ultrapure water, add 4mL acetic acid, stir for 3 minutes, add 2g chitosan, stir until completely dissolving, obtain chitosan acetic acid solution;

(2) in the chitosan acetic acid solution that step (1) makes, slowly drip the sodium hydroxide solution that mass concentration is 4% to white precipitation and separate out completely, after white precipitation is centrifuged and washed to neutrality, chitosan is obtained Neutral gel;

(3) get 5 parts of chitosan neutral gels obtained in step (2), mix it with 4 parts of mass concentration of 1% carboxymethyl chitosan and fully stir for 0.5h to obtain the mixed solution, then Add 1 part of glycerol aqueous solution with a mass concentration of 20% to the mixed solution dropwise, stir for 0.5 h, transfer the mixed solution to a refrigerator at 4°C and let it stand for 24 h, then freeze-dry the system at -60°C for 24 h to obtain hemostasis Sponge MG3.

Example 4

A preparation method of physically cross-linked chitosan-based hemostatic sponge, comprising the following steps:

(1) Get 196mL ultrapure water, add 4mL acetic acid, stir for 3 minutes, add 2g chitosan, stir until completely dissolving, obtain chitosan acetic acid solution;

(2) in the chitosan acetic acid solution that step (1) makes, slowly drip the sodium hydroxide solution that mass concentration is 4% to white precipitation and separate out completely, after white precipitation is centrifuged and washed to neutrality, chitosan is obtained Neutral gel;

(3) get 5 parts of chitosan neutral gels obtained in step (2), mix it with 0.5% carboxymethyl chitosan with 4 parts of mass concentration and fully stir for 0.5h to obtain the mixed solution, then Add 1 part of 10% glycerol aqueous solution to the mixture dropwise, stir for 0.5 h, transfer the mixed solution to a refrigerator at 4°C for 24 h, and then freeze-dry the system at -60°C for 24 h to obtain hemostasis Sponge MG4.

Comparative example 1

A preparation method of chitosan-based hemostatic sponge, comprising the following steps:

(1) Get 180mL ultrapure water, add 3.6mL acetic acid, stir for 3 minutes, add 4g chitosan, stir until completely dissolving, obtain chitosan acetic acid solution;

(2) Adding 15 parts of carboxymethyl chitosan solutions with a mass concentration of 5.3% dropwise to the chitosan acetic acid solution prepared in step (1) mixed fully for 1 h to obtain a mixed solution;

(3) Add 5 parts of aqueous glycerol solutions with a mass concentration of 40% dropwise to the mixed solution prepared in step (2), stir for 1 h, and transfer the mixed solution to a refrigerator at 4°C for 24 h, and then the system Freeze-dried at -60°C for 24 hours to obtain the precursor sponge

(4) immersing the prepolymer sponge prepared in step (3) in a sodium hydroxide solution with a mass concentration of 4% for 0.5 h and then rinsing with ultrapure water to neutrality to obtain a gel sponge;

(5) The gel sponge prepared in step (4) was freeze-dried at -60° C. for 24 hours to obtain the hemostatic sponge MG5.

Comparative example 2

A preparation method of chitosan-based hemostatic sponge, comprising the following steps:

(1) Get 180mL ultrapure water, add 3.6mL acetic acid, stir for 3 minutes, add 4g chitosan, stir until completely dissolving, obtain chitosan acetic acid solution;

(2) Adding 15 parts of carboxymethyl chitosan solutions with a mass concentration of 5.3% dropwise to the chitosan acetic acid solution prepared in step (1) mixed fully for 1 h to obtain a mixed solution;

(3) Add 5 parts of aqueous glycerol solutions with a mass concentration of 40% dropwise to the mixed solution prepared in step (2), stir for 1 h, and transfer the mixed solution to a refrigerator at 4°C for 24 h, and then the system Freeze-dry at -60°C for 24 hours to obtain the hemostatic sponge MG6.

Take the hemostatic sponges prepared in Examples 1-4 and Comparative Examples 1-2, observe their appearance, and observe their microstructure through a scanning electron microscope. The SEM image is shown in FIG. 2 . From the results shown in Figure 2, it can be seen that the appearance of sponges MG1-MG4 and sponge MG6 is flat, while the appearance of MG5 is collapsed. Sponge MG1-6 all present a porous structure. Among them, sponges MG1-4 have small pore size and good connectivity, so they can quickly absorb the lost blood to stop bleeding; while sponges MG5 and MG6 have large pore size but poor connectivity, and are not easy to immerse in liquid.

From the results shown in Figure 3 and Figure 4, it can be seen that the sponges MG1-MG4 have shorter in vitro whole blood coagulation time and higher water absorption rate. Although the sponge MG5 also has a higher water absorption rate, its preparation process is long. Secondary freezing is required, and at the same time, the coagulation time of whole blood in vitro is longer. It can be seen from Fig. 5 that the hemolysis rate of all samples is lower than 5%, and all samples have good blood compatibility.

Although the present invention has been disclosed above with preferred examples, it is not intended to limit the present invention. Any person familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the present invention The scope of protection of an invention should be defined in the power of attorney.

Claims (10)

1. a preparation method of physically cross-linked chitosan-based hemostatic sponge, characterized in that, may further comprise the steps: (1) add chitosan in acid solution, stir until dissolving completely, obtain chitosan acid solution; (2) in the chitosan acid solution that step (1) makes, slowly drip alkaline solution to white flocculent precipitation and no longer separate out, centrifugal washing is neutral to supernatant, obtains chitosan gel; (3) Mix the chitosan gel in the step (2) with the carboxymethyl chitosan aqueous solution, stir evenly, let the resulting mixture stand and freeze-dry at -60°C for 24 hours to obtain chitosan-based Hemostatic sponge. 2. the preparation method of hemostatic sponge according to claim 1, is characterized in that, in described step (3), before mixed solution is poured into watch glass, in mixed solution, add auxiliary agent aqueous solution, described auxiliary agent Including at least one of silk fibroin, gelatin and glycerin. 3. The preparation method of the hemostatic sponge according to claim 2, characterized in that, the auxiliary agent is glycerin, and the mass concentration of the glycerin aqueous solution is 5-30%. 4. The preparation method of the hemostatic sponge according to any one of claims 1-3, characterized in that the viscosity of the chitosan is 50-800CPS. 5. according to the preparation method of the hemostatic sponge described in any one in claim 1～3, it is characterized in that, described carboxymethyl chitosan comprises O-carboxymethyl chitosan and/or N-carboxymethyl chitosan Chitosan. 6. The preparation method of the hemostatic sponge according to any one of claims 1 to 3, wherein the acid comprises acetic acid and/or citric acid, and the volume percentage of the acid solution is 1 to 2%, so The alkali includes sodium hydroxide, and the mass percentage of the alkali solution is 2-5%. 7. The preparation method of the hemostatic sponge according to any one of claims 1-3, characterized in that, the mass concentration of the chitosan acid solution is 0.2-3%. 8. The preparation method of the hemostatic sponge according to any one of claims 1-3, characterized in that the mass concentration of the carboxymethyl chitosan aqueous solution is 0.1-5%. 9. the hemostatic sponge obtained by the preparation method according to claim 1. 10. The hemostatic sponge according to claim 9, characterized in that, calculated in parts by weight, its components include 1-5 parts of chitosan gel and 1-20 parts of carboxymethyl chitosan.