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

Abstract

The present invention discloses a physically cross-linked chitosan-based hemostatic sponge and a preparation method thereof. The sponge uses chitosan as the main raw material. An alkaline solution is first added dropwise to a chitosan acid solution and then centrifuged to obtain chitosan gel. The chitosan gel is mixed and stirred with a carboxymethyl chitosan aqueous solution, and freeze-dried to obtain the hemostatic sponge. No cross-linking agent is used herein. Chitosan gel is used as the main component. The chitosan gel and carboxymethyl chitosan are cross-linked through hydrogen bonds, thereby ensuring the mechanical properties of the sponge. The liquid absorption rate of the chitosan / carboxymethyl chitosan hemostatic sponge can reach more than 40 times, and the hemostatic effect is equivalent to that of gauze. In addition, the pore structure is uniform, the biocompatibility is good, and there is no biological toxicity.

Description

Technical Field

[0001] The invention relates to a physically cross-linked chitosan-based hemostatic sponge and a preparation method thereof, belonging to the technical field of biomaterials. Background Art

[0002] Sponges are solid, porous preparations with high absorbency, light weight, and low density. They can stop bleeding and absorb wound exudate, and have good gas permeability, demonstrating their superior hemostatic properties. Chitosan, a natural polymer produced by deacetylation of chitin, has potential applications in biology and medicine due to its broad bioactivity, good biocompatibility, complete biodegradability, and low toxicity. However, due to its poor solubility in water and high solubility in acid, the preparation of chitosan sponges is complex and requires alkaline washing or secondary freeze-drying, limiting their application. To improve the preparation of chitosan-based sponges, many researchers have focused on carboxylated chitosan, modifying chitosan to produce water-soluble carboxylated chitosan and reacting it with crosslinkers to prepare hemostatic sponges. However, these hemostatic sponges still suffer from poor hemostatic efficacy and low liquid absorption. Therefore, developing a hemostatic sponge with improved hemostatic properties and a streamlined process is of great significance. Summary of the Invention

[0003] The purpose of the present invention is to provide a physically cross-linked chitosan-based hemostatic sponge to address the problems of existing chitosan sponges such as long process flow, poor water absorption, and poor hemostatic effect. The hemostatic sponge does not require secondary freezing during its preparation process, has good hemostatic performance, and has a short process flow.

[0004] One of the purposes of the present invention is to provide a method for preparing a physically cross-linked chitosan-based hemostatic sponge, comprising the following steps:

[0005] (1) Add chitosan to the acid solution and stir until it is completely dissolved to obtain a chitosan acid solution;

[0006] (2) slowly adding an alkaline solution to the chitosan acid solution obtained in step (1) until no more white flocculent precipitates are precipitated, and centrifuging and washing until the supernatant is neutral to obtain chitosan gel;

[0007] (3) The chitosan gel in step (2) is mixed with the carboxymethyl chitosan aqueous solution, stirred evenly, and the obtained mixture is allowed to stand and freeze-dried to prepare a chitosan-based hemostatic sponge.

[0008] 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 to simplify the process, the present invention provides a method for preparing a physically cross-linked chitosan-based hemostatic sponge, namely, first preparing a chitosan neutral gel, and then cross-linking the chitosan gel with a carboxymethyl chitosan gel to obtain the sponge. In the process of preparing the chitosan neutral gel, an alkaline solution is used to neutralize the acid in the chitosan acid solution so that no acid residue is left in the gel. The chitosan gel and the carboxymethyl chitosan are cross-linked to further increase the stability of the gel, and the number of freeze-drying times can be reduced when preparing the hemostatic sponge. The prepared hemostatic sponge has excellent water absorption and in vitro coagulation effect.

[0009] In one embodiment of the present invention, in step (3), before pouring the mixed solution into the watch glass, an aqueous solution of an additive is added to the mixed solution, wherein the additive comprises at least one of silk fibroin, gelatin, and glycerol. The addition of the additive helps to further enhance the flexibility and water absorption of the chitosan sponge.

[0010] In one embodiment of the present invention, in step (3), the mixed solution is allowed to stand at 1-5°C for 20-30 hours before freeze-drying. The purpose of standing is to allow the chitosan gel to fully react with the carboxymethyl chitosan to form a gel.

[0011] In one embodiment of the present invention, the auxiliary agent is glycerol, and the mass concentration of the glycerol aqueous solution is 5-30%.

[0012] In one embodiment of the present invention, the viscosity of the chitosan is 50-800 CPS.

[0013] In one embodiment of the present invention, the carboxymethyl chitosan is O-carboxymethyl chitosan and / or N-carboxymethyl chitosan.

[0014] In one embodiment of the present invention, the acid includes acetic acid and / or citric acid, and the volume percentage of the acid solution is 1-2%. The base includes sodium hydroxide, and the mass percentage of the base solution is 2-5%.

[0015] In one embodiment of the present invention, the mass percentage of the chitosan acid solution is 0.2-3%.

[0016] In one embodiment of the present invention, the mass percentage of the carboxymethyl chitosan aqueous solution is 0.1-5%, preferably 0.2-2%.

[0017] A second object of the present invention is to provide a hemostatic sponge obtained according to the preparation method of claim 1.

[0018] In one embodiment of the present invention, the components of the hemostatic sponge include 1 to 5 parts of chitosan gel and 1 to 20 parts of carboxymethyl chitosan, by weight.

[0019] In one embodiment of the present invention, the components of the hemostatic sponge include, by weight, 1 to 5 parts of chitosan gel, 1 to 20 parts of carboxymethyl chitosan, and 0.5 to 10 parts of glycerol.

[0020] Beneficial effects of the present invention

[0021] (1) Chitosan is dissolved in an acid solution, neutralized with an alkali to form a gel, and the neutral gel is cross-linked with carboxymethyl chitosan. The chitosan-based hemostatic sponge is prepared after freeze-drying. The hemostatic sponge prepared by the preparation method of the present invention does not contain substances such as acetic acid that are irritating to the human body.

[0022] (2) When making the hemostatic sponge, chitosan gel and carboxymethyl chitosan undergo a cross-linking reaction, which further increases the stability of chitosan. The hemostatic sponge made by the method of the present invention has a higher liquid absorption rate;

[0023] (3) The formula provided by the present invention is simple, the materials used are non-biotoxic, the preparation process is simple to operate, no secondary freeze-drying is required, and the reaction is easy to control. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The present invention is a flow chart of the preparation process of the chitosan-based hemostatic sponge;

[0025] Figure 2 The SEM images of the hemostatic sponges prepared in Examples 1 to 4 and Comparative Examples 1 to 2 are shown;

[0026] Figure 3 The in vitro coagulation time test results of the hemostatic sponges prepared in Examples 1 to 4 and Comparative Examples 1 to 2;

[0027] Figure 4 The water absorption test results of the hemostatic sponges prepared in Examples 1 to 4 and Comparative Examples 1 to 2 are shown;

[0028] Figure 5 The figures are the hemolysis rate test results of the hemostatic sponges prepared in Examples 1 to 4 and Comparative Examples 1 to 2. DETAILED DESCRIPTION

[0029] The following describes preferred embodiments of the present invention. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0030] Test method:

[0031] Whole blood clotting time test: Add 10mg of hemostatic sponge to a centrifuge tube and preheat to 37°C. Then, add 1mL of anticoagulated whole blood and incubate for another 3 minutes. Immediately thereafter, add 100µL of 0.2mol / L CaCl2 solution to the tube. Gently tilt the tube every 3-5 seconds until the blood stops flowing when tilted 90°. This indicates coagulation and the clotting time is recorded. A blank control group containing no anticoagulated whole blood was used.

[0032] Water absorption test: Weigh a certain mass of hemostatic sponge, record the mass as M. Immerse it in a beaker of distilled water at room temperature. After absorbing enough water, gently pinch a corner of the sponge with tweezers to remove it from the water. Hold the sponge lightly on the water surface with the tweezers to drain for 1 minute, then weigh it again, record it as M1, and calculate the water absorption rate A according to formula (1):

[0033] (1)

[0034] Hemolysis rate test: Soak the hemostatic sponge sample in 0.9% saline for 24 hours to obtain the sample extract. Mix 0.5 mL of sample extract with 0.5 mL of red blood cell suspension and incubate at 37°C for 3 hours. Centrifuge the mixture at 1600 rpm for 10 minutes, take the supernatant and measure the absorbance value at 540 nm using a microplate reader. Mix 0.5 mL of ultrapure water with 0.5 mL of 2% red blood cell suspension and incubate at 37°C for 3 hours as a positive control, and mix 0.5 mL of 0.9% saline with 0.5 mL of 2% red blood cell suspension and incubate at 37°C for 3 hours as a negative control. Calculate the hemolysis rate according to formula (2):

[0035] (2)

[0036] Example 1

[0037] A method for preparing a physically cross-linked chitosan-based hemostatic sponge comprises the following steps:

[0038] (1) Take 196 mL of ultrapure water, add 4 mL of acetic acid, stir for 3 minutes, add 2 g of chitosan, and stir until completely dissolved to obtain chitosan acetic acid solution;

[0039] (2) Slowly add a 4% sodium hydroxide solution to the chitosan acetic acid solution prepared in step (1) until a white precipitate is completely precipitated, and wash the white precipitate to neutrality by centrifugation to obtain a chitosan neutral gel.

[0040] (3) Take 5 parts of the chitosan neutral gel obtained in step (2), mix it with 4 parts of a 1% carboxymethyl chitosan aqueous solution, stir it thoroughly for 0.5 h to obtain a mixed solution, then add 1 part of a 10% glycerol aqueous solution dropwise to the mixed solution, stir it for 0.5 h, and transfer the mixed solution to a 4°C refrigerator and let it stand for 24 h. Then, freeze-dry the system at -60°C for 24 h to obtain hemostatic sponge MG1.

[0041] Example 2

[0042] A method for preparing a physically cross-linked chitosan-based hemostatic sponge comprises the following steps:

[0043] (1) Take 196 mL of ultrapure water, add 4 mL of acetic acid, stir for 3 minutes, add 2 g of chitosan, and stir until completely dissolved to obtain chitosan acetic acid solution;

[0044] (2) slowly adding a 4% sodium hydroxide solution to the chitosan acetic acid solution obtained in step (1) until a white precipitate is completely precipitated, and washing the white precipitate to neutrality by centrifugation to obtain a chitosan neutral gel;

[0045] (3) Take 5 parts of the chitosan neutral gel obtained in step (2), mix them with 4 parts of a 0.5% mass concentration of carboxymethyl chitosan aqueous solution, and stir them thoroughly for 0.5 h to obtain a mixed solution. Then, add 1 part of a 20% mass concentration of glycerol aqueous solution dropwise to the mixed solution, stir for 0.5 h, and transfer the mixed solution to a 4°C refrigerator and let it stand for 24 h. Then, freeze-dry the system at -60°C for 24 h to obtain hemostatic sponge MG2.

[0046] Example 3

[0047] A method for preparing a physically cross-linked chitosan-based hemostatic sponge comprises the following steps:

[0048] (1) Take 196 mL of ultrapure water, add 4 mL of acetic acid, stir for 3 minutes, add 2 g of chitosan, and stir until completely dissolved to obtain chitosan acetic acid solution;

[0049] (2) slowly adding a 4% sodium hydroxide solution to the chitosan acetic acid solution obtained in step (1) until a white precipitate is completely precipitated, and washing the white precipitate to neutrality by centrifugation to obtain a chitosan neutral gel;

[0050] (3) Take 5 parts of the chitosan neutral gel obtained in step (2), mix it with 4 parts of carboxymethyl chitosan with a mass concentration of 1%, and stir it thoroughly for 0.5 h to obtain a mixed solution. Then, add 1 part of a 20% glycerol aqueous solution dropwise to the mixed solution, stir it for 0.5 h, and transfer the mixed solution to a 4°C refrigerator and let it stand for 24 h. Then, freeze-dry the system at -60°C for 24 h to obtain hemostatic sponge MG3.

[0051] Example 4

[0052] A method for preparing a physically cross-linked chitosan-based hemostatic sponge comprises the following steps:

[0053] (1) Take 196 mL of ultrapure water, add 4 mL of acetic acid, stir for 3 minutes, add 2 g of chitosan, and stir until completely dissolved to obtain chitosan acetic acid solution;

[0054] (2) slowly adding a 4% sodium hydroxide solution to the chitosan acetic acid solution obtained in step (1) until a white precipitate is completely precipitated, and washing the white precipitate to neutrality by centrifugation to obtain a chitosan neutral gel;

[0055] (3) Take 5 parts of the chitosan neutral gel obtained in step (2), mix it with 4 parts of carboxymethyl chitosan with a mass concentration of 0.5%, and stir it thoroughly for 0.5 h to obtain a mixed solution. Then, add 1 part of a 10% glycerol aqueous solution dropwise to the mixed solution, stir it for 0.5 h, and transfer the mixed solution to a 4°C refrigerator and let it stand for 24 h. Then, freeze-dry the system at -60°C for 24 h to obtain hemostatic sponge MG4.

[0056] Comparative Example 1

[0057] A method for preparing a chitosan-based hemostatic sponge comprises the following steps:

[0058] (1) Take 180 mL of ultrapure water, add 3.6 mL of acetic acid, stir for 3 minutes, add 4 g of chitosan, and stir until completely dissolved to obtain chitosan acetic acid solution;

[0059] (2) adding 15 parts of a 5.3% carboxymethyl chitosan solution dropwise to the chitosan acetic acid solution prepared in step (1), and stirring the mixture for 1 hour to obtain a mixed solution;

[0060] (3) Add 5 parts of a 40% glycerol aqueous solution dropwise to the mixed solution prepared in step (2), stir for 1 hour, and transfer the mixed solution to a 4°C refrigerator and let it stand for 24 hours. Then, freeze-dry the system at -60°C for 24 hours to obtain a precursor sponge;

[0061] (4) Immersing the prepolymer sponge prepared in step (3) in a sodium hydroxide solution having a mass concentration of 4% for 0.5 h, and then rinsing with ultrapure water until neutral to obtain a gel sponge;

[0062] (5) The gel sponge prepared in step (4) was freeze-dried at -60°C for 24 h to obtain hemostatic sponge MG5.

[0063] Comparative Example 2

[0064] A method for preparing a chitosan-based hemostatic sponge comprises the following steps:

[0065] (1) Take 180 mL of ultrapure water, add 3.6 mL of acetic acid, stir for 3 minutes, add 4 g of chitosan, and stir until completely dissolved to obtain chitosan acetic acid solution;

[0066] (2) adding 15 parts of a 5.3% carboxymethyl chitosan solution dropwise to the chitosan acetic acid solution prepared in step (1), and stirring the mixture for 1 hour to obtain a mixed solution;

[0067] (3) Five parts of a 40% glycerol aqueous solution were added dropwise to the mixed solution prepared in step (2), stirred for 1 hour, and the mixed solution was transferred to a 4°C refrigerator and allowed to stand for 24 hours. The system was then freeze-dried at -60°C for 24 hours to obtain hemostatic sponge MG6.

[0068] Take the hemostatic sponges prepared in Examples 1 to 4 and Comparative Examples 1 to 2, observe their appearance, and observe their microstructures by scanning electron microscopy. Figure 2 .from Figure 2 The results show that sponges MG1-MG4 and MG6 have a smooth appearance, while MG5 has a collapsed appearance. Sponges MG1-MG6 all exhibit a porous structure. Sponges MG1-MG4 have small pores and good connectivity, allowing them to quickly absorb lost blood and stop bleeding. Sponges MG5 and MG6, on the other hand, have large pores but poor connectivity, making them less susceptible to liquid infiltration.

[0069] from Figure 3 and Figure 4 The results show that sponges MG1 to MG4 have shorter in vitro whole blood coagulation times and higher water absorption rates. Although sponge MG5 also has a higher water absorption rate, its preparation process is long and requires secondary freezing. At the same time, its in vitro whole blood coagulation time is longer. Figure 5 It can be seen that the hemolysis rates of all samples were lower than 5%, indicating that they all had good blood compatibility.

[0070] Although the present invention has been disclosed as above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the patent document.

Claims

1. A method for preparing a physically cross-linked chitosan-based hemostatic sponge, characterized in that: The following steps are involved: (1) adding chitosan to the acid solution and stirring until completely dissolved to obtain a chitosan acid solution, wherein the mass concentration of the chitosan acid solution is 0.2-3%; (2) slowly adding an alkaline solution to the chitosan acid solution obtained in step (1) until no more white flocculent precipitates are precipitated, and centrifuging and washing until the supernatant is neutral to obtain chitosan gel; (3) The chitosan gel in step (2) is mixed with the carboxymethyl chitosan aqueous solution, stirred evenly, and the resulting mixture is allowed to stand at 4°C for 24 hours, and freeze-dried at -60°C for 24 hours to prepare a chitosan-based hemostatic sponge, which comprises 5 parts of chitosan gel, 4 parts of carboxymethyl chitosan, and 1 part of glycerol in parts by weight; Before the mixed solution is allowed to stand, a glycerol aqueous solution is added to the mixed solution, wherein the mass concentration of the glycerol aqueous solution is 10% and the mass concentration of the carboxymethyl chitosan aqueous solution is 1%.

2. The method for preparing the hemostatic sponge according to claim 1, wherein The viscosity of the chitosan is 50-800 CPS.

3. The method for preparing the hemostatic sponge according to claim 1, wherein The carboxymethyl chitosan includes O-carboxymethyl chitosan and / or N-carboxymethyl chitosan.

4. The method for preparing the hemostatic sponge according to claim 1, wherein The acid includes acetic acid and / or citric acid, and the volume percentage of the acid solution is 1-2%. The alkali includes sodium hydroxide, and the mass percentage of the alkali solution is 2-5%.

5. The hemostatic sponge obtained by the preparation method according to claim 1.

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