CN115386135A

CN115386135A - Carboxymethylated zymosan sponge and preparation method and application thereof

Info

Publication number: CN115386135A
Application number: CN202210992351.XA
Authority: CN
Inventors: 周宗宝; 肖将尉; 高博韬; 关淑文
Original assignee: Institute of Biological and Medical Engineering of Guangdong Academy of Sciences
Current assignee: Institute of Biological and Medical Engineering of Guangdong Academy of Sciences
Priority date: 2022-08-18
Filing date: 2022-08-18
Publication date: 2022-11-25
Anticipated expiration: 2042-08-18
Also published as: CN115386135B

Abstract

The invention discloses carboxymethylation zymosan sponge and a preparation method and application thereof, wherein the preparation method comprises the following steps: and (2) blending carboxymethylated zymosan and a high molecular material, then performing electrostatic spinning, and then sequentially performing freeze drying, crushing, homogenizing, and freeze drying to obtain the carboxymethylated zymosan sponge. The carboxymethylated zymosan sponge prepared by the preparation method can accelerate wound hemostasis, can regulate phagocytosis of macrophages, and is beneficial to anti-infection treatment. In addition, the carboxymethylated zymosan used in the sponge can promote the migration of cells and accelerate the healing of wounds. Compared with the chitosan hemostatic in the prior art, the carboxymethylated zymosan sponge has excellent mechanical property and water absorption property, and has better anti-infection capability and capability of accelerating fibroblast migration.

Description

Carboxymethylated zymosan sponge and preparation method and application thereof

Technical Field

The invention belongs to the field of medicines, and particularly relates to carboxymethylated zymosan sponge and a preparation method and application thereof.

Background

Bleeding during surgery can lead to a variety of adverse effects, including increased transfusion requirements, hypovolemic shock and cryocoagulopathy. Therefore, rapid hemostasis is of great importance during trauma surgery or long-term surgery. Hemostasis is a physiological process, also the first stage of wound healing, that stops bleeding from damaged blood vessels by forming blood clots. However, most of the major bleeding caused by trauma or surgery is often difficult to control, and thus, an external hemostatic agent is required to prevent a great loss of blood, thereby endangering life. Currently, research on hemostatic agents has generally focused on natural polysaccharides, bioprotein macromolecules, and inorganic particles, and various hemostatic agents have been used clinically, such as Surgicel, celox, hemCon bandage, mRDH, and the like. However, in the face of the severe challenges of the great amount of blood loss and hypertension caused by the current trunk, artery and intracavity bleeding, the existing hemostatic lacks sufficient tissue adhesion property and is slow to be absorbed, so that it is difficult to meet the requirements. Furthermore, the ability to resist infection during hemostasis is often overlooked, and continued infection of wounds can result in immeasurable losses, such as amputation, abscess formation, and prolonged wound healing time. Therefore, there is an urgent need to develop new and useful anti-infective hemostatic agents that are able to rapidly and effectively control major bleeding.

Natural polysaccharide is a renewable biomedical material that has been widely used in clinical studies. Among them, chitosan is favored for its superior biocompatibility, low toxicity and low sensitization, biodegradability, antibacterial activity, tissue adhesiveness and hemostatic activity in hemostasis. Chitosan is a naturally positively charged polysaccharide consisting of randomly distributed D-glucosamine and N-acetyl-D-glucosamine, and the amino groups present on the chitosan polysaccharide backbone are capable of electrostatic interactions with cell membranes and proteins in the blood stream, leading to strong blood clotting. However, it is noted that chitosan, which is inherently cationic in nature, is susceptible to induce hemolytic effects, causing hemoglobin extravasation, resulting in a sustained inflammatory response and thrombosis. In addition, alginate and carrageenan are two natural polysaccharides commonly used in hemostasis, but they do not possess anti-infective properties themselves and are therefore difficult to use in major hemorrhages.

The zymosan is a natural polysaccharide polymer and exists in fungal cell walls, and researches show that the zymosan can stimulate the immune system of a human body, enhance the resistance of the human body, effectively promote the repair of wounds and has an excellent biodegradation function. However, there is currently no zymosan product used for hemostasis; the possible reason is that pure zymosan is poorly soluble in water. Based on the consideration, carboxymethylated zymosan has been developed, however, the original mechanical properties of zymosan hydrogel are weakened or disappeared by carboxymethylated modification, and the requirements of hemostasis and high strength in application cannot be met. Therefore, how to make carboxymethylated zymosan system have good controllable bioactivity under the premise of keeping good elasticity and mechanical strength is a key technical problem which needs to be solved before clinical application.

Aerogels are currently the lightest solid materials known and have been widely used in industrial production. Generally, aerogels are a class of nanomaterials with low density and high porosity, and this characteristic of aerogels makes them capable of absorbing large amounts of moisture. Currently, the combination of electrospinning technology with aerogel is considered as a new high strength sponge preparation strategy.

The electrostatic spinning nanofiber has high porosity, an extracellular matrix-like structure, flexible components and a malleable form, shows excellent performance in tissue repair, and has wide prospects as a nanoscale construction unit of a multifunctional wound dressing. However, electrospinning water-soluble carboxymethylated polymers followed by the preparation of hemostatic aerogels remains a technical challenge. At present, no research on electrospinning hemostatic aerogels has been reported.

Disclosure of Invention

In order to overcome the problems in the prior art, the invention aims to provide a preparation method of carboxymethylated zymosan sponge, which is simple and has easily obtained raw materials.

The second purpose of the invention is to provide carboxymethylated zymosan sponge, the sponge of the invention has light weight and excellent mechanical property, has sufficient capacity of absorbing liquid exuded from wounds, and overcomes the defect of insufficient capacity of absorbing blood of the existing hemostatic hydrogel and gauze; meanwhile, the hemostatic gauze can generate extremely strong adhesive capacity with wound tissues, and prevent the sponge from falling off in the hemostatic process; in addition, the sponge can activate the immune system in vivo and resist infection.

The invention also aims to provide the application of the carboxymethylated zymosan sponge in a hemostatic product, an anti-infective medicament or a tissue repair product.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

the first aspect of the present invention provides a method for preparing carboxymethylated zymosan sponge, which comprises the following steps:

blending carboxymethylated zymosan and a high polymer material, performing electrostatic spinning, and then sequentially performing freeze drying, crushing, homogenizing, and freeze drying to obtain the carboxymethylated zymosan sponge.

Preferably, the step of electrostatic spinning after blending the carboxymethylated zymosan and the high molecular material specifically comprises the following steps: the carboxymethylated zymosan solution and the high molecular material solution are blended and then are subject to electrostatic spinning.

Preferably, the concentration of the carboxymethylated zymosan solution is 2-5 wt%; further preferably, the concentration of the carboxymethylated zymosan solution is 2 to 4wt%; still further preferably, the concentration of the carboxymethylated zymosan solution is 3 to 4wt%.

Preferably, the concentration of the polymer material solution is 10-15 wt%; further preferably, the concentration of the polymer material solution is 11 to 14wt%; still more preferably, the concentration of the polymer material solution is 12 to 14wt%.

Preferably, the pulverizing step is grinding into powder by using a freezing pulverizer.

Preferably, the homogenizing step is to mix the powder and tert-butyl alcohol uniformly and homogenize the mixture to obtain homogeneous slurry with the concentration of 50 to 100 g/L.

Preferably, the polymer material comprises at least one of polyvinyl alcohol, polyethylene oxide and polycaprolactone.

Preferably, the electrospinning conditions are: the voltage is 18.0-26.0 eV; the injection speed is 0.1-0.5 mL/h; the rotating speed of the receiver is as follows: 100-300 rpm.

Preferably, the freeze-drying is freeze-drying with liquid nitrogen.

Preferably, the mass ratio of the carboxymethylated zymosan to the high molecular material is 1 (1-4); further preferably, the mass ratio of the carboxymethylated zymosan to the high molecular material is 1 (1-3); still further preferably, the mass ratio of the carboxymethylated zymosan to the high molecular material is 1 (2-3).

Preferably, the preparation method of the carboxymethylated zymosan comprises the following steps: mixing zymosan and chloroacetic acid to react under alkaline condition to obtain carboxymethylated zymosan.

Preferably, the preparation method of the carboxymethylated zymosan specifically comprises the following steps: dispersing zymosan in isopropanol, stirring uniformly, adjusting pH to be alkaline by using a sodium hydroxide solution, adding chloroacetic acid for reaction, filtering and collecting filter residue, then cleaning, and freeze-drying to obtain the carboxymethylated zymosan; further preferably, the preparation method of the carboxymethylated zymosan specifically comprises the following steps: dispersing zymosan into isopropanol, uniformly stirring to prepare a zymosan solution with the concentration of 2-5%, adjusting the pH to 9-14 by using a sodium hydroxide solution with the concentration of 30-50 wt%, adding chloroacetic acid for reaction, filtering and collecting filter residues, then cleaning, and freeze-drying to prepare the carboxymethylated zymosan;

preferably, the pH is adjusted to be alkaline by using a sodium hydroxide solution, specifically, the pH is adjusted to be 10-13 by using a sodium hydroxide solution.

Preferably, the concentration of the sodium hydroxide solution is 30-40 wt%; further preferably, the concentration of the sodium hydroxide solution is 30 to 38wt%; still more preferably, the concentration of the sodium hydroxide solution is 30 to 35wt%.

Preferably, the mass ratio of the chloroacetic acid to the zymosan is (1-4): 1; more preferably, the mass ratio of the chloroacetic acid to the zymosan is (1-3.5): 1; still more preferably, the mass ratio of the chloroacetic acid to the zymosan is (1-3): 1.

preferably, the temperature of the mixing reaction is 45-65 ℃; further preferably, the temperature of the mixing reaction is 50 to 60 ℃.

Preferably, the time of the mixing reaction is 2 to 8 hours; further preferably, the mixing reaction time is 3-7 h; still more preferably, the time of the mixing reaction is 3 to 6 hours.

In a second aspect, the invention provides a carboxymethylated zymosan sponge prepared by the preparation method provided by the first aspect of the invention.

A third aspect of the invention provides the use of a carboxymethylated zymosan sponge according to the second aspect of the invention in a haemostatic product, an anti-infective agent or a tissue repair product.

The beneficial effects of the invention are: the preparation method combines the electrostatic spinning technology with the aerogel preparation technology, so that the sponge with excellent mechanical property and water absorption capacity is prepared.

The carboxymethylated zymosan sponge prepared by the preparation method can accelerate wound hemostasis, can regulate phagocytosis of macrophages, and is beneficial to anti-infection treatment. In addition, carboxymethylated zymosan used in the sponge can promote the migration of cells and accelerate the healing of wounds. Compared with the chitosan hemostatic in the prior art, the carboxymethylated zymosan sponge has excellent mechanical property and water absorption property, and has better anti-infection capacity and capacity of accelerating fibroblast migration.

Drawings

FIG. 1 is a schematic representation of the carboxymethylated zymosan sponges from example 2 and comparative example 1 according to the invention.

FIG. 2 is a microscopic topography of carboxymethylated zymosan sponges in example 2 of the present invention and comparative example 1.

FIG. 3 is a graph of the mechanical properties of carboxymethylated zymosan sponges in example 2 according to the invention and comparative example 1.

FIG. 4 is a graph showing the sponge and Celox obtained in example 2 of the present invention and comparative example 1 ^TM The hemostasis performance test chart.

FIG. 5 is a culture profile of the carboxymethylated zymosan sponge biocompatibility test in example 2.

FIG. 6 is a graph showing the hemolysis rate test of carboxymethylated zymosan sponge in example 2 of the present invention.

FIG. 7 is an SEM image of red blood cells after treatment with PBS and 800. Mu.g/mL carboxymethylated zymosan aerogel sponge.

FIG. 8 is a graph showing the test results of the bacterial infection resistance of the zymosan and carboxymethylated zymosan according to the present invention.

FIG. 9 is a schematic representation of fibroblast migration of zymosan and carboxymethylated zymosan of the present invention.

FIG. 10 is a graph showing the UV absorption of zymosan of the present invention and carboxymethylated zymosan of examples 1 to 4.

FIG. 11 is a graph of the infrared absorption of monochloroacetic acid, zymosan and carboxymethylated zymosan from examples 1-4.

Detailed Description

Specific embodiments of the present invention are described in further detail below with reference to the figures and examples, but the practice and protection of the present invention is not limited thereto. It is noted that the following processes, if not described in particular detail, are all realizable or understandable by those skilled in the art with reference to the prior art. The reagents or apparatus used are not indicated to the manufacturer, and are considered to be conventional products available by commercial purchase.

Example 1

The carboxymethylated zymosan sponge for rapid hemostasis in the embodiment is prepared by the following preparation method, and specifically comprises the following steps:

(1) 100g of yeast powder is suspended in 500mL of pure water, heated to boil, filtered while hot, and the filtrate is collected. And standing the high-temperature filtrate, completely separating out zymosan after the temperature is reduced to the room temperature, filtering again, and collecting filter residues to obtain zymosan (YG).

(2) 1g of zymosan is dispersed in 20mL of isopropanol and stirred at 3000 rpm, followed by the slow dropwise addition of 30wt% sodium hydroxide solution (3 g of sodium hydroxide in 10mL of deionized water), adjusting the pH to 12.0 and stirring for a further 30min. After most of the zymosan was dissolved, 1g of monochloroacetic acid (CAM) was added to the reaction solution while the reaction temperature was adjusted to 50 ℃ and the reaction was terminated for 5 hours. Then placing the reaction solution into a dialysis membrane (molecular weight cut-off 8000 DA), dialyzing in 5wt% sodium bicarbonate water solution and deionized water for 48 hours respectively, and freeze-drying to obtain carboxymethylated zymosan, which is recorded as CMYG-1.

(3) 0.5g carboxymethylated zymosan (CMYG-1) and 1g polyethylene oxide (PEO) were added to 10mL deionized water and stirred overnight to give a clear PEO/CMYG-1 electro-spun solution, which was then electro-spun using a high pressure electro-spinning machine under the conditions: the positive voltage range was 26.0eV, the injection rate was 0.5mL/h, and the receiver rotation speed was 150rpm. And obtaining the electrostatic spinning film based on carboxymethylated zymosan after the electric spinning solution is used.

(4) Freezing the carboxymethylated zymosan-based electrospun membrane obtained in step (3) by using liquid nitrogen, and grinding the frozen membrane into 200-mesh electrospun powder. And then 1g of electrospinning powder is put into 200mL of anhydrous tert-butyl alcohol, the mixture is homogenized at the speed of 30000rpm, the homogenate is poured into a cylindrical mold, the cylindrical mold is frozen at the temperature of minus 80 ℃, and the cylindrical mold is freeze-dried in a freeze-drying machine for 48 hours, so that the carboxymethylated zymosan aerogel sponge in the embodiment is obtained.

Example 2

(1) 1g of zymosan is dispersed in 20mL of isopropanol and stirred at 3000 rpm, followed by the slow dropwise addition of 30wt% sodium hydroxide solution (3 g of sodium hydroxide in 10mL of deionized water), adjusting the pH to 12.0 and stirring for a further 30min. After the major part of the zymosan was dissolved, 0.75g of monochloroacetic acid was added to the reaction solution, and the reaction temperature was adjusted to 50 ℃ at the same time, and the reaction was terminated for 5 hours. Then placing the reaction solution in a dialysis membrane (molecular weight cut-off of 8000 DA), dialyzing in 5wt% sodium bicarbonate water solution and deionized water for 48 hours respectively, and freeze-drying to obtain carboxymethylated zymosan, which is recorded as CMYG-2.

(2) 0.5g carboxymethylated zymosan (CMYG-2) and 1g polyethylene oxide (PEO) were added to 10mL deionized water and stirred overnight to give a clear PEO/CMYG-2 electro-spun solution, which was then electrospun using a high-pressure electrospinning machine under the following conditions: the positive voltage range was 26.0eV, the injection rate was 0.5mL/h, and the receiver rotation rate was 150rpm. And obtaining the electrostatic spinning film based on carboxymethylated zymosan after the electric spinning solution is used.

(3) And (3) freezing the electrostatic spinning film obtained in the step (2) by using liquid nitrogen, and grinding the electrostatic spinning film into 200-mesh electrostatic spinning powder. And then 1g of electrospinning powder is put into 200mL of anhydrous tert-butyl alcohol, the mixture is homogenized at the speed of 30000rpm, the homogenate is poured into a cylindrical mold, the cylindrical mold is frozen at the temperature of minus 80 ℃, and the cylindrical mold is freeze-dried in a freeze-drying machine for 48 hours, so that the carboxymethylated zymosan aerogel sponge in the embodiment is obtained.

Example 3

(1) 1g of zymosan was dispersed in 20mL of isopropanol and stirred at 3000 rpm, followed by slow dropwise addition of 30wt% sodium hydroxide solution (3 g of sodium hydroxide in 10mL of deionized water), pH adjusted to 12.0 and stirring continued for 30min. After most of the zymosan is dissolved, 0.5g of monochloroacetic acid is added into the reaction solution, the reaction temperature is adjusted to 50 ℃, and the reaction is finished for 5 hours. Then placing the reaction solution into a dialysis membrane (molecular weight cut-off 8000 DA), dialyzing in 5wt% sodium bicarbonate water solution and deionized water for 48 hours respectively, and freeze-drying to obtain carboxymethylated zymosan, which is recorded as CMYG-3.

(2) 0.5g carboxymethylated zymosan (CMYG-3) and 1g polyethylene oxide (PEO) were added to 10mL deionized water and stirred overnight to give a clear PEO/CMYG-3 electro-spun solution, which was then electrospun using a high-pressure electrospinning machine under the following conditions: the positive voltage range was 26.0eV, the injection rate was 0.5mL/h, and the receiver rotation speed was 150rpm. And obtaining the electrostatic spinning film based on carboxymethylated zymosan after the electric spinning solution is used.

(3) Freezing the electrospun film obtained in the step (2) by using liquid nitrogen, and grinding the frozen electrospun film into electrospun powder of 200 meshes. And then 1g of electrospinning powder is put into 200mL of anhydrous tert-butyl alcohol, the mixture is homogenized at the speed of 30000rpm, the homogenate is poured into a cylindrical mold, the cylindrical mold is frozen at the temperature of minus 80 ℃, and the cylindrical mold is freeze-dried in a freeze-drying machine for 48 hours, so that the carboxymethylated zymosan aerogel sponge in the embodiment is obtained.

Example 4

(1) 1g of zymosan was dispersed in 20mL of isopropanol and stirred at 3000 rpm, followed by slow dropwise addition of 30wt% sodium hydroxide solution (3 g of sodium hydroxide in 10mL of deionized water), pH adjusted to 12.0 and stirring continued for 30min. After the major part of the zymosan was dissolved, 0.25g of monochloroacetic acid was added to the reaction solution, and the reaction temperature was adjusted to 50 ℃ at the same time, and the reaction was terminated for 5 hours. Then placing the reaction solution into a dialysis membrane (molecular weight cut-off 8000 DA), dialyzing in 5wt% sodium bicarbonate water solution and deionized water for 48 hours respectively, and freeze-drying to obtain carboxymethylated zymosan, which is recorded as CMYG-4.

(2) 0.5g carboxymethylated zymosan (CMYG-4) and 1g polyethylene oxide (PEO) were added to 10mL deionized water and stirred overnight to give a clear PEO/CMYG-4 electro-spun solution, which was then electro-spun using a high pressure electro-spinning machine under the conditions: the positive voltage range was 26.0eV, the injection rate was 0.5mL/h, and the receiver rotation rate was 150rpm. And obtaining the electrostatic spinning film based on carboxymethylated zymosan after the electric spinning solution is used.

The carboxymethylated zymosan aerogel sponges prepared in examples 1, 3-4 had substantially the same performance as example 2, and therefore the present invention was tested for performance using the carboxymethylated zymosan aerogel sponges prepared in example 2.

Comparative example 1

The carboxymethylated zymosan sponge in the embodiment is prepared by the following preparation method, which specifically comprises the following steps:

(1) 1g of zymosan was dispersed in 20mL of isopropanol and stirred at 3000 rpm, followed by slow dropwise addition of 30wt% sodium hydroxide solution (3 g of sodium hydroxide in 10mL of deionized water), pH adjusted to 12.0 and stirring continued for 30min. After most of the zymosan is dissolved, 0.75g of monochloroacetic acid is added into the reaction solution, the reaction temperature is adjusted to 50 ℃, and the reaction is finished for 5 hours. Then putting the reaction solution into a dialysis membrane (molecular weight cut-off 8000 DA), dialyzing in 5wt% sodium bicarbonate water solution and deionized water for 48 hours respectively, and freeze-drying to obtain carboxymethylated zymosan: CMYG-2.

(2) Adding 1g carboxymethylated zymosan (CMYG-2) into 200mL of anhydrous tertiary butanol, homogenizing at 30000rpm, pouring the homogenate into a cylindrical mold, freezing at-80 ℃, and freeze-drying in a freeze-drying machine for 48h to obtain the carboxymethylated zymosan gel sponge in the embodiment.

And (3) performance testing:

(1) Shape testing

Comparing the shape of the carboxymethylated zymosan aerogel sponge in example 2 with the shape of the carboxymethylated zymosan aerogel sponge in comparative example 1, as shown in fig. 1, fig. 1 (a) is a diagram of the carboxymethylated zymosan aerogel sponge in example 2; fig. 1 (b) is an outline diagram of the carboxymethylated zymosan gel sponge in comparative example 1, and it can be seen from fig. 1 that the carboxymethylated zymosan gel sponge prepared in example 2 of the present invention is softer.

(2) Microstructure testing

The micro-structures of the carboxymethylated zymosan aerogel sponge in example 2 and the carboxymethylated zymosan aerogel sponge in comparative example 1 were respectively tested, as shown in fig. 2, wherein fig. 2 (a) and fig. 2 (b) are the micro-topography maps of the carboxymethylated zymosan aerogel sponge in example 2; FIG. 2 (b) is an enlarged view of the surface in FIG. 2 (a); FIGS. 2 (c) and 2 (d) are micrographic images of the carboxymethylated zymosan gel sponge of comparative example 1 at different positions. Analysis of the pore microstructures of the two sponges obtained in fig. 2 shows that the carboxymethylated zymosan aerogel sponge in the invention example 2 shows a fibrous micro-nano structure, while the carboxymethylated zymosan aerogel sponge in the comparative example 1 shows a common porous structure, and the micro-topography of the sponge products obtained in the invention example 2 and the comparative example 1 is greatly different.

(3) Adhesive Properties

The adhesion performance of the carboxymethylated zymosan aerogel sponge in example 2 and the carboxymethylated zymosan aerogel sponge in comparative example 1 were tested separately, and the specific test method was: the sponges prepared in example 2 and comparative example 1 were placed on the surface of the pigskin, respectively, and their tensile strengths were measured using a universal mechanical tester, which indicated the adhesive properties of the sponges, and the specific test results are shown in fig. 3. As can be seen from fig. 3, the adhesive capacity of the carboxymethylated zymosan aerogel sponge in example 2 of the present invention on the skin surface is higher than 3.42MPa, and is much higher than 0.68MPa of the carboxymethylated zymosan aerogel sponge in comparative example 1, which indicates that the aerogel sponge prepared in the present invention has excellent adhesive performance, and has a certain stretch-resistant capability, which is helpful for controlling the massive hemorrhage in vivo.

(4) Hemostatic properties

The sponge and Celox of comparative example 1 and example 2 were tested separately ^TM (hemostatic commercial Celox ^TM The main component of the composition is carboxymethylated chitosan), the specific test method comprises the following steps: the hemostatic properties of the samples were studied using a rat liver bleeding model. 100mg of carboxymethylated zymosan aerogel sponge of example 1, 100mg of carboxymethylated zymosan aerogel sponge of comparative example 1, and 100mg of hemostatic commercial Celox were separately mixed ^TM Respectively applied to the bleeding positions of the liver of the rat, and then the hemostatic effects are compared, and the specific test results are shown in fig. 4, wherein: FIGS. 4 (a), 4 (b), and 4 (c) are schematic diagrams of models of bleeding from the liver of rats before, during, and after treatment with the aerogel sponge of example 2; FIG. 4 (d) is an enlarged partial view of the wound site of FIG. 4 (c); FIGS. 4 (e), 4 (f) and 4 (g) are schematic diagrams of models of bleeding from the liver of rats before, during and after treatment with the sponge of comparative example 1; FIG. 4 (h) is an enlarged partial view of the wound site of FIG. 4 (g); FIG. 4 (i), FIG. 4 (j), and FIG. 4 (k) are schematic views of a hemostatic commercial product Celox ^TM Model graphs of rat liver bleeding before, during and after treatment; FIG. 4 (l) is a partial enlarged view of the wound site of FIG. 4 (k). As can be seen from FIG. 4, celox is a commercially available hemostatic product ^TM And the sponge in the comparative example 1, the carboxymethylated zymosan aerogel sponge of the invention can rapidly stop tissue bleeding, can rapidly scar at a wound and is beneficial to wound healing.

(5) Biocompatibility Properties

The carboxymethylated zymosan aerogel sponge in example 2 was tested for biocompatibility by the following specific test methods: the blood compatibility is measured by measuring the absorbance of hemoglobin released after lysis of the red blood cells. In this experiment, fresh whole blood as an anticoagulant was collected from the upper arm of a healthy adult and used within 2 hours after blood collection. 20mL of PBS buffer was added to 10mL of whole blood, gently mixed, and then centrifuged at 5000rpm for 5 minutes, and then red blood cells were collected from the bottom of the centrifuge tube. The obtained red blood cells were mixed with a double volume of PBS buffer, centrifuged and collected, and the blood was removed of other substances, and the same protocol was repeated 5 times. Then, erythrocytes with a concentration of 5% v/v were prepared by dilution with fresh PBS buffer and stored at 4 ℃ for subsequent experiments. Carboxymethylated zymosan aerogel sponges were dispersed in PBS buffer to prepare carboxymethylated zymosan aerogel sponge suspensions. Then gently mixing carboxymethylated zymosan aerogel sponge and red blood cell suspension in a 1.5mL test tube at a ratio of 1; control 2 group was prepared with water and red blood cells in a 1.5mL tube at a ratio of 1; control 3 groups were formulated with zymosan and red blood cells in a 1.5mL tube at a ratio of 1. Culturing at 25 deg.C for 10min, 45 min, 90 min, 180 min, 360 min and 720min, respectively, and the profile of the culture is shown in FIG. 5, wherein FIG. 5 (a), FIG. 5 (b) and FIG. 5 (c) are the profile of the culture at 0min, 10min and 12h (i.e., 720 min), respectively; the samples were centrifuged at 5000rpm for 5 minutes. Thereafter, the supernatant was transferred to a 96-well plate, and the absorbance of the sample was recorded at 540nm using a microplate reader.

The hemolysis rate is calculated as: hemolysis rate (%) = (As-An)/(Ap-An) × 100

Wherein As represents the absorbance of the test sample, an is the absorbance of a negative control (PBS buffer), and Ap is the absorbance of a positive control (deionized water).

Then, the hemolysis ratio calculated according to the different incubation times is plotted, specifically as shown in FIG. 6, wherein the abscissa in FIG. 6 is the incubation time and the ordinate in FIG. 6 is the non-hemolysis ratio value. As can be seen from FIG. 6, the carboxymethylated zymosan aerogel sponge of the present invention has a non-hemolytic rate of more than 95% in 12h, i.e., a hemolytic rate of less than 5%, and does not cause rupture of erythrocytes.

To further evaluate the morphology of the erythrocytes after 12h of treatment, the erythrocytes remaining at the bottom of the test tube after centrifugation were removed and fixed with 4% formaldehyde at 25 ℃ for 2 hours, and then the treated erythrocytes were soaked with ethanol at concentrations of 50%, 60%, 70%, 80%, 90% and 99.7% for 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes and 30 minutes, respectively. After dehydration of the sample, the cell suspension was spread on a glass coverslip, dried overnight at 25 ℃ and gold plated. Finally, the sample is observed under a scanning electron microscope, the specific test result is shown in fig. 7, and fig. 7 (a) is an SEM image of the red blood cells treated by PBS; FIG. 7 (b) is an SEM image of red blood cells after treatment with 800. Mu.g/mL carboxymethylated zymosan aerogel sponge. According to the fig. 5-7, the carboxymethylated zymosan aerogel sponge of the invention can not cause the rupture of red blood cells within 12h, has good biocompatibility and better safety to organisms.

(6) Anti-bacterial infection properties

The carboxymethylated zymosan in example 2 was tested for its antibacterial properties by the following specific test methods: respectively incubating carboxymethylated zymosan and zymosan with macrophages for 24h to activate phagocytosis performance of the macrophages, adding staphylococcus aureus (stained by FITC), and testing the bacterial phagocytosis capacity of the macrophages within 2h, wherein the specific test result is shown in figure 8, wherein figure 8 (a) is a fluorescence graph of the bacterial phagocytosis capacity of untreated macrophages; FIG. 8 (b) is a bright field plot of the ability of untreated macrophages to phagocytose bacteria; FIG. 8 (c) is a merged view of FIGS. 8 (a) and 8 (b); FIG. 8 (d) is a fluorescent plot of the ability of macrophages treated with zymosan (YG) to phagocytose bacteria; FIG. 8 (e) is a bright field graph of the ability of macrophages treated with zymosan (YG) to phagocytose bacteria; FIG. 8 (f) is a merged view of FIGS. 8 (d) and 8 (e); FIG. 8 (g) is a fluorescence plot of the ability of macrophages treated with CMYG-2 to phagocytose bacteria; FIG. 8 (h) is a bright field plot of the ability of macrophages treated with CMYG-2 to phagocytose bacteria; fig. 8 (i) is a combined view of fig. 8 (h) and 8 (g). As can be seen from fig. 8, the carboxymethylated zymosan aerogel sponge of the present invention can activate phagocytic ability of macrophages, and has excellent antibacterial property.

(7) In vitro experiment for accelerating defect healing

The wound repair mainly depends on the migration and proliferation of fibroblasts, and the wound repair acceleration after hemostasis is crucial, the invention tests the migration acceleration capability of the carboxymethylated zymosan in the embodiment 2, and the specific test method comprises the following steps: respectively incubating zymosan and carboxymethylated zymosan with fibroblasts for 0h, 12h and 24h, and then testing the migration capacity of the fibroblasts, wherein the specific test result is shown in fig. 9, wherein fig. 9 (a), 9 (d) and 9 (g) are migration schematic diagrams of fibroblast solution incubation for 0h, 12h and 24h respectively; FIGS. 9 (b), 9 (e) and 9 (h) are migration profiles of fibroblasts incubated with zymosan for 0h, 12h and 24h, respectively; FIGS. 9 (c), 9 (f) and 9 (i) are migration schematic diagrams of fibroblasts incubated with carboxymethylated zymosan for 0h, 12h and 24h, respectively. As can be seen from fig. 9, the carboxymethylation of the carboxymethylated zymosan in the present invention can significantly enhance the migration of fibroblasts relative to zymosan; further shows that the carboxymethylated zymosan aerogel sponge provided by the invention has the potential of accelerating the repair of defective tissues.

(8) Ultraviolet and infrared testing

The ultraviolet absorption spectrum and the infrared absorption spectrum of the zymosan, the carboxymethylated zymosan in examples 1 to 4 were respectively tested, wherein the ultraviolet absorption spectrum of the zymosan and the carboxymethylated zymosan in examples 1 to 4 is shown in FIG. 10, and the infrared absorption spectrum of the monochloroacetic acid, the zymosan and the carboxymethylated zymosan in examples 1 to 4 is shown in FIG. 11. As can be seen from FIG. 10, the UV absorption profiles of carboxymethylated zymosan in examples 1 to 4 clearly shift in the short wavelength direction compared to zymosan. As can be seen from FIG. 11, the carboxymethylation peaks in the infrared absorption profiles of the carboxymethylated zymosan in examples 1 to 4 are significantly enhanced as compared with the zymosan, i.e., the carboxymethylation modification of the zymosan by the production method of the present invention was successfully carried out as can be seen from FIGS. 10 and 11.

While the embodiments of the present invention have been described in detail, the present invention is not limited to the embodiments, and various changes can be made without departing from the spirit of the present invention within the knowledge of those skilled in the art. Furthermore, the embodiments of the present invention and the features of the embodiments may be combined with each other without conflict.

Claims

1. A preparation method of carboxymethylated zymosan sponge is characterized by comprising the following steps: the method comprises the following steps:

2. The method of preparing a carboxymethylated zymosan sponge according to claim 1, wherein: the high polymer material comprises at least one of polyvinyl alcohol, polyethylene oxide and polycaprolactone.

3. The method of preparing a carboxymethylated zymosan sponge according to claim 1, wherein: the electrostatic spinning conditions are as follows: the voltage is 18.0-26.0 eV; the injection speed is 0.1-0.5 mL/h; the rotating speed of the receiver is as follows: 100-300 rpm.

4. The method of preparing a carboxymethylated zymosan sponge according to claim 1, wherein: the freeze drying is liquid nitrogen freeze drying.

5. The method of preparing a carboxymethylated zymosan sponge according to claim 1, wherein: the mass ratio of the carboxymethylated zymosan to the high molecular material is 1 (1-4).

6. The method of preparing a carboxymethylated zymosan sponge according to any one of claims 1 to 5, which is characterized in that: the preparation method of the carboxymethylated zymosan comprises the following steps: mixing zymosan and chloroacetic acid to react under alkaline condition to obtain carboxymethylated zymosan.

7. The method of preparing a carboxymethylated zymosan sponge according to claim 6, wherein: the mass ratio of the chloroacetic acid to the zymosan is (1-4): 1.

8. the method of preparing a carboxymethylated zymosan sponge according to claim 6, wherein: the temperature of the mixing reaction is 45-65 ℃; the mixing reaction time is 2-8 h.

9. A carboxymethylated zymosan sponge, characterized by: the preparation method of any one of claims 1 to 8.

10. Use of a carboxymethylated zymosan sponge according to claim 9 in a haemostatic product, an anti-infective drug or a tissue repair product.