CN117143385A

CN117143385A - Preparation method and application of multifunctional sponge

Info

Publication number: CN117143385A
Application number: CN202311129084.4A
Authority: CN
Inventors: 赵九龙; 王世革; 张馨元; 李兆申
Original assignee: First Affiliated Hospital of Naval Military Medical University of PLA; University of Shanghai for Science and Technology
Current assignee: First Affiliated Hospital of Naval Military Medical University of PLA; University of Shanghai for Science and Technology
Priority date: 2023-09-01
Filing date: 2023-09-01
Publication date: 2023-12-01

Abstract

The invention discloses a preparation method and application of a multifunctional sponge. The preparation method comprises the following steps: dissolving gamma-polyglutamic acid in a solvent, and stirring to completely dissolve the gamma-polyglutamic acid; dissolving carboxymethyl chitosan in a solvent, and stirring to completely dissolve the carboxymethyl chitosan; uniformly mixing the two solutions to obtain a mixed solution; then directly adding a carboxyl activating agent into the mixed solution, or adding a high polymer and then adding the carboxyl activating agent, or sequentially adding an antibacterial agent, the high polymer and the carboxyl activating agent, and obtaining the multifunctional sponge after the reaction is completed. The multifunctional sponge prepared by the invention has good biocompatibility and can be applied to safe and efficient drug delivery and drug slow release.

Description

Preparation method and application of multifunctional sponge

Technical Field

The invention relates to a gel compound, in particular to a preparation method and application of a multifunctional sponge, and belongs to the technical field of biological nano materials.

Background

Carboxymethyl chitosan (CMCS) is a natural polymer compound and is a product obtained by the carboxymethyl reaction of chitosan. The chemical structure of the water-soluble polymer contains carboxymethyl functional groups, so that the water-soluble polymer has good water solubility and biocompatibility. In addition, the carboxymethyl chitosan can be further functionalized in chemical modification, self-assembly and other modes, so that different physicochemical properties and functions are realized, and the requirements of different application fields are met. Therefore, the carboxymethyl chitosan has been widely applied to the fields of medicine slow release, tissue engineering, food preservation and the like, and has good application prospect.

Polyglutamic acid (gamma-PGA) is a biodegradable polymer material with good biocompatibility. The natural amino acid glutamic acid is polymerized to form the natural amino acid glutamic acid which can be degraded and metabolized into carbon dioxide and water through microorganisms. The physicochemical properties of the polyglutamic acid can be regulated and controlled by changing the polymerization degree, substituent groups, crosslinking and the like, so that the polyglutamic acid has wide application prospect. Currently, polyglutamic acid is applied to the fields of medicine, food, agriculture and the like, and becomes an important biodegradable polymer material.

Polyvinylpyrrolidone (PVP) is a common polymer material and has excellent biocompatibility, biodegradability and chemical stability. Polyvinylpyrrolidone is soluble in various solvents such as water, ethanol, acetone, etc., has good stability and transparency, and can be used as a cosolvent, a dispersing agent, a stabilizer or a thickener, etc., so that the polyvinylpyrrolidone is widely applied to the fields of medicines, cosmetics, foods, etc. In addition, polyvinylpyrrolidone can be modified by crosslinking, copolymerization and the like to obtain different functions and properties. Along with the continuous and deep research on polyvinylpyrrolidone functionalization, the polyvinylpyrrolidone has a wider application prospect in various fields.

Hydrogels are substances composed of high molecular substances and having a three-dimensional network structure, which form gel states in water. The hydrogel has excellent biocompatibility and biodegradability, and is suitable for the fields of tissue engineering, drug slow release, biosensing and the like. The physical and chemical properties of the hydrogel can be regulated and controlled by the types, crosslinking degree, pore size and other modes of high molecular substances, so that the mechanical property, water absorption property, drug slow release property and other aspects of the gel are controlled. In addition, the hydrogel can be prepared by various methods, such as self-assembly, a template method, a microfluidic technology and the like, so as to meet the requirements of different application fields. Because of the unique structure and excellent performance, the hydrogel becomes an important biological material and has wide application prospect in the biomedical field.

Disclosure of Invention

The technical problems to be solved by the invention are as follows: the existing hydrogel material has low utilization efficiency and can not be reused.

In order to solve the problems, the invention provides a preparation method of a multifunctional sponge, which is characterized by comprising the following steps:

step 1): dissolving gamma-polyglutamic acid in a solvent, and stirring to completely dissolve the gamma-polyglutamic acid;

step 2): dissolving carboxymethyl chitosan in a solvent, and stirring to completely dissolve the carboxymethyl chitosan;

step 3): uniformly mixing the two solutions obtained in the step 1) and the step 2) to obtain a mixed solution; then directly adding a carboxyl activating agent into the mixed solution, or adding a high polymer and then adding the carboxyl activating agent, or sequentially adding an antibacterial agent, the high polymer and the carboxyl activating agent, and obtaining the multifunctional sponge after the reaction is completed.

Preferably, the solvent in the step 1) is any one of distilled water, phosphate buffer solution (ph=7.4), physiological saline (w/v=0.9%); the mass concentration of the dissolved gamma-polyglutamic acid is 0.5-15%; the dissolution temperature of the gamma-polyglutamic acid is 30-80 ℃.

Preferably, in the step 2), the mass concentration of the dissolved carboxymethyl chitosan is 0.5-20%; the dissolution temperature of the carboxymethyl chitosan is 30-80 ℃.

Preferably, the carboxyl activating agent in the step 3) is any one or two of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide; the mass concentration of the carboxyl activating agent is 1-30%; the mass concentration or the volume concentration of the carboxyl activating agent (any one or two of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide) in the mixed solution is 0.5-30%; the mass ratio of the gamma-polyglutamic acid to the carboxymethyl chitosan is 0.4-0.6: 1.

preferably, the temperature of the reaction in the step 3) is 10 to 30 ℃.

Preferably, the polymer in the step 3) is any one of polyvinylpyrrolidone and polyvinyl alcohol; the mass concentration of the polymer is 10-40%; the mass concentration or the volume concentration of the high polymer (any one of polyvinylpyrrolidone and polyvinyl alcohol) in the mixed solution is 40-80%.

Preferably, the antibacterial and anti-inflammatory agent in the step 3) is various water-soluble or oil-soluble agents. For example, amoxicillin, escitalopram, resveratrol, etc.; the concentration of the antibacterial drug in the mixed solution is 0.5-10 mg/mL.

The invention also provides application of the multifunctional sponge prepared by the preparation method in drug delivery or drug slow release antibacterial anti-inflammatory biological materials.

The invention adds co-crosslinking polymer and carboxyl activator into gamma-polyglutamic acid and carboxymethyl chitosan solution. Taking polyvinylpyrrolidone as a co-crosslinking polymer and taking 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride-N-hydroxysuccinimide (EDC-NHS) as a carboxyl activator as an example, two gel preparation methods are provided: (1) dissolving gamma-polyglutamic acid and carboxymethyl chitosan in a solvent, uniformly stirring, and then adding EDC-NHS to enable the sponge to be glued; (2) dissolving gamma-polyglutamic acid and carboxymethyl chitosan in a solvent, uniformly stirring, adding a polyvinylpyrrolidone solution, and then adding EDC-NHS to enable the sponge to form gel. Both methods can obtain a multifunctional sponge with a certain network structure.

The invention provides a preparation method for realizing crosslinking and gelling of gamma-polyglutamic acid, carboxymethyl chitosan and polyvinylpyrrolidone under the activation of EDC-NHS by utilizing the property that a carboxyl group in gamma-polyglutamic acid can be activated by a carboxyl activating agent so that the carboxyl group can be subjected to crosslinking reaction with carboxymethyl chitosan and polyvinylpyrrolidone.

The invention has simple process and short preparation time of the product, and the obtained product has good biocompatibility in-vivo and in-vitro mold experiments. According to the invention, gamma-polyglutamic acid and carboxymethyl chitosan are selected as matrixes, polyvinylpyrrolidone is used as a co-crosslinked polymer, and EDC-NHS is utilized to activate carboxyl groups in gamma-polyglutamic acid, so that the gamma-polyglutamic acid can be crosslinked with carboxymethyl chitosan and can also undergo a crosslinking reaction with polyvinylpyrrolidone to prepare the multifunctional sponge. Experiments show that the sponge prepared by the invention has good flexibility and elasticity. After the medicine is loaded, the sponge can enter the body through various ways such as oral administration, patches and the like to gradually release the medicine, so that the bioavailability of the medicine is improved, the stability and the persistence of the medicine are improved, the side effect of the medicine is reduced, and the damage influence on normal tissues and organs is reduced.

Compared with the prior art, the invention has obvious technical progress. The sponge prepared by the invention has simple process, easily obtained product, good flexibility and elasticity, and can adapt to different shapes and surfaces. The preparation method can make up the defects of low utilization efficiency, incapability of being reused and the like of the conventional hydrogel material, is hopeful to be applied to the fields of drug delivery, drug slow release and the like, and has a certain clinical application value.

The invention also provides application of the sponge as a safe and efficient drug delivery, drug slow-release antibacterial and anti-inflammatory biological material. The multifunctional sponge prepared by the invention has excellent flexibility and elasticity and good biocompatibility, and can be applied to safe and efficient drug delivery and drug slow release.

Drawings

FIG. 1 is a FESEM picture of a multifunctional sponge; wherein a and b are the multifunctional sponge prepared in example 1, and c and d are the multifunctional sponge prepared in example 2;

FIG. 2 is a dynamic time-scanning rheological analysis of a multifunctional sponge; wherein a is the multifunctional sponge prepared in example 1 and b is the multifunctional sponge prepared in example 2;

FIG. 3 is a mechanical property test result of the multifunctional sponge in example 8; wherein a is a compressive strain-stress curve, b is a compressive modulus, and c is an average compressive stress;

FIG. 4 is the in vitro biosafety of the multifunctional sponge prepared in example 2; wherein a is a hemolysis test result, b is the survival rate of gastric epithelial cells after the co-culture of the multifunctional sponge, and c-f is a DEAD/LIVE staining result: untreated material (c) and treated with multifunctional sponge 2.5mg/mL (d), 5mg/mL (e), 10mg/mL (f);

FIG. 5 shows the swelling ratio, swelling kinetics and in vitro degradation curves of the multifunctional sponges of examples 11 and 12, wherein a, b, e are deionized water and c, d, f are phosphate buffered solutions;

fig. 6 shows the in vitro release analysis results of the multifunctional drug-loaded sponges of examples 13 and 14, wherein a is the standard curve of amoxicillin, b is the in vitro release result of amoxicillin, c is the standard curve of escitalopram, and d is the in vitro release result of escitalopram;

FIG. 7 shows the results of in vitro antibacterial experiments on the multifunctional sponge in example 15, wherein a is the antibacterial rate of the multifunctional sponge against helicobacter pylori, b is the plate count result, and c is the proliferation of bacterial colonies on an agar plate.

Detailed Description

In order to make the invention more comprehensible, preferred embodiments accompanied with figures are described in detail below.

Example 1

Dissolving 0.5g of carboxymethyl chitosan and 0.25g of gamma-polyglutamic acid in 10mL of deionized water, and uniformly stirring at 60 ℃ to obtain carboxymethyl chitosan-gamma-polyglutamic acid mixed solution. Then, 1mL of carboxymethyl chitosan-gamma-polyglutamic acid mixed solution is taken in a glass bottle, 200 mu L of EDC-NHS mixed solution (with the concentration of 0.1 g/mL) is added into the glass bottle, and the mixture is stirred uniformly, thus obtaining the multifunctional sponge.

Example 2

Dissolving 0.5g of carboxymethyl chitosan and 0.25g of gamma-polyglutamic acid in 10mL of deionized water, and uniformly stirring at 60 ℃ to obtain carboxymethyl chitosan-gamma-polyglutamic acid mixed solution. 0.25g of polyvinylpyrrolidone was dissolved in 10mL of deionized water to obtain a polyvinylpyrrolidone solution. Then, 1mL of carboxymethyl chitosan-gamma-polyglutamic acid mixed solution and 600 mu L of polyvinylpyrrolidone solution are taken in a glass bottle, and 200 mu LEDC-NHS mixed solution (with the concentration of 0.1 g/mL) is added into the glass bottle and stirred uniformly, thus obtaining the multifunctional sponge.

Example 3

Dissolving 0.5g of carboxymethyl chitosan and 0.25g of gamma-polyglutamic acid in 10mL of deionized water, and uniformly stirring at 60 ℃ to obtain carboxymethyl chitosan-gamma-polyglutamic acid mixed solution. 0.25g of polyvinylpyrrolidone was dissolved in 10mL of deionized water to obtain a polyvinylpyrrolidone solution. Then, 4mg of amoxicillin is taken to be dissolved in 1mL of carboxymethyl chitosan-gamma-polyglutamic acid mixed solution, then the amoxicillin and 600 mu L of polyvinylpyrrolidone solution are mixed uniformly in a glass bottle, 200 mu L of EDC-NHS mixed solution (with the concentration of 0.1 g/mL) is added into the glass bottle, and the amoxicillin-carrying multifunctional sponge is obtained after uniform stirring.

Example 4

Dissolving 0.5g of carboxymethyl chitosan and 0.25g of gamma-polyglutamic acid in 10mL of deionized water, and uniformly stirring at 60 ℃ to obtain carboxymethyl chitosan-gamma-polyglutamic acid mixed solution. 0.25g of polyvinylpyrrolidone was dissolved in 10mL of deionized water to obtain a polyvinylpyrrolidone solution. Then, 2.5mg of the ebselen is dissolved in 1mL of carboxymethyl chitosan-gamma-polyglutamic acid mixed solution, then evenly mixed with 600 mu L of polyvinylpyrrolidone solution in a glass bottle, 200 mu L of EDC-NHS mixed solution (with the concentration of 0.1 g/mL) is added into the glass bottle, and evenly stirred, thus obtaining the ebselen-carrying multifunctional sponge.

Example 5

Dissolving 0.5g of carboxymethyl chitosan and 0.25g of gamma-polyglutamic acid in 10mL of deionized water, and uniformly stirring at 60 ℃ to obtain carboxymethyl chitosan-gamma-polyglutamic acid mixed solution. 0.25g of polyvinylpyrrolidone was dissolved in 10mL of deionized water to obtain a polyvinylpyrrolidone solution. Then, 4mg of amoxicillin and 2.5mg of ebselenam are dissolved in 1mL of carboxymethyl chitosan-gamma-polyglutamic acid mixed solution, then are uniformly mixed with 600 mu L of polyvinylpyrrolidone solution in a glass bottle, 200 mu L of EDC-NHS mixed solution (with the concentration of 0.1 g/mL) is added into the glass bottle, and the amoxicillin-ebselenam-carrying multifunctional sponge is obtained after uniform stirring.

Example 6

The multifunctional sponges of example 1 and example 2 were freeze-dried and analyzed for morphology. The samples were analyzed on a FEI Magellan 400 field emission scanning electron microscope.

As can be seen from the electron microscope scanning pictures, the multifunctional sponges all show three-dimensional pore structures (fig. 1). However, the size and distribution of the pores varies with the degree of crosslinking of the sponge. The lower degree of crosslinking CMCS/gamma-PGA sponge prepared in example 1 had larger pore sizes (a, b in FIG. 1), and the higher degree of crosslinking CMCS/gamma-PGA/PVP sponge prepared in example 2 had a dense pore distribution and smaller pore sizes (c, d in FIG. 1).

Example 7

The gelation process of the multifunctional sponge was studied by observing the final storage modulus (G') and the final loss modulus (G ") in a dynamic time-sweep rheological test. The dynamic rheological study was performed using a plate geometry (P20 TiL, diameter 20 mm) with a rotational rheometer (MARIII HAAKE). The multifunctional sponges in examples 1 and 2 were subjected to time-sweep oscillation test at a frequency of 1Hz, a gap of 1mm and a strain of 10%. Injecting corresponding sponge precursor solution on the flat plate, and adjusting the gap to be 1mm. Frequency sweep measurements of hydrogels are denoted as G' and G ". When G 'exceeds G', the gel point is determined. The G' of the sponge increases rapidly due to intermolecular crosslinking, indicating that the sponge formation efficiency is high (a, b in FIG. 2).

Example 8

The multifunctional sponges prepared in examples 1 and 2 were mechanically evaluated on a Zwick Roell Z2.5 TH universal material tester using a 2.5kN sensor. The compression properties of the multifunctional sponge were studied using the modified american society for testing and materials method. In the compression test, the multifunctional sponge is molded in a cylinder with the diameter of 10mm and the thickness of 3mm, and the compression strain rate is 1mm/min. The compressive modulus was recorded by a linear fit of the stress-strain curve over a strain range of 10-20% (a in fig. 3). The maximum compressive stress and compressive modulus of the multifunctional sponge prepared in example 1 were 857.21kPa and 498.06kPa, respectively, and the maximum compressive stress and compressive modulus of the multifunctional sponge prepared in example 2 were 1745.95kPa and 325.99kPa, respectively (b, c in fig. 3). In addition, the repeated compression performance of the multifunctional sponge prepared in example 2 was evaluated. As shown in fig. 3 d, the multifunctional sponge can be quickly and completely recovered in the cyclic compression process, and shows excellent anti-fatigue capability.

Example 9

The blood compatibility of the multifunctional sponge was studied. Red blood cells were obtained after centrifugation (5000 rpm,5 minutes) of 2mL whole blood and three washes with phosphate buffer solution. The resulting erythrocytes were stored in 50mL of phosphate buffer solution for further use. In the hemolysis test, 0.5mL of the above-mentioned mouse erythrocytes were placed in a 5.0mL centrifuge tube, and the multifunctional sponge (1.25, 2.5, 5, 10mg/mL, in 2.5mL of phosphate buffer solution) prepared in example 2 of different qualities from (1) 2.5mL of phosphate buffer solution (negative control), (2) 2.5mL of deionized water (positive control) and (3) were co-incubated in a 37 ℃ water bath for 2 hours. The absorbance of the supernatant at 541nm (Shimadzu UV-3600 ultraviolet visible near infrared spectrometer) was collected, and the hemolysis rate of erythrocytes was calculated. As shown in FIG. 4 a, the calculated sponge hemolysis rates were all less than 5%. From the supernatant photographs, it can be seen that the red cell supernatant incubated with the multifunctional sponge and phosphate buffer solution is transparent. However, blood treated with deionized water appears significantly red due to hemolysis positivity. The results show that the multifunctional sponge has good blood compatibility.

Example 10

Gastric epithelial cells were seeded in 96-well plates and cultured overnight with 100 μl of cell culture medium. The above medium was discarded, and the multifunctional sponge (2.5, 5, 10 mg/mL) obtained in example 2 and 100. Mu.L of new cell culture medium were added in different weights, and the control group was added with only 100. Mu.L of cell culture medium (survival rate was set to 100%). Placing the above cells in CO ₂ Incubation was performed for 24 hours in a constant temperature incubator, and the survival of the cells was quantitatively and qualitatively detected using CCK-8 and LIVE/DEAD cell Activity detection kit. As shown in fig. 4 b, the multifunctional sponge does not affect the survival of cells. Similar to the control group (c in FIG. 4), the multifunctional sponge-treated cells could be stained green with LIVE/DEAD reagent (living cells were stained green), and almost no cells were stained red (DEAD cells were stained red) (d, e, and f in FIG. 4). The results of CCK-8 and LIVE/DEAD cell staining indicate that the prepared multifunctional sponges have good cell compatibility.

Example 11

To investigate the liquid absorbing ability of the sponges, the multifunctional sponges obtained in examples 1 and 2 were added to deionized water, respectively, and immersed at 37℃for 24 hours. The sponge was then gently wiped to remove surface moisture and weighed. Finally, the sponge was freeze-dried and the initial mass was weighed to determine the expansion ratio (a in fig. 5). In addition, the swelling kinetics of the sponge in deionized water (b in FIG. 5) was studied, demonstrating that the sponge absorbs fluid very rapidly, and swelling rates reached 166.6396g/g and 116.7119g/g, respectively, after 20 minutes of storage in deionized water. Swelling of the multifunctional sponge in phosphate buffer solution at ph=7.4 was the same as the experimental procedure in deionized water described above. After 20 minutes of storage in phosphate buffer, the swelling ratios of the sponges reached 129.5653g/g and 85.7991g/g, respectively. (c and d in FIG. 5)

Example 12

Dissolving lysozyme in deionized water and phosphate buffer solution respectively at a concentration of 1×10 ⁴ U/mL. The multifunctional sponge obtained in freeze-dried examples 1 and 2 was then weighed, incubated with 10mL of lysozyme-containing deionized water or phosphate buffer solution, and continuously stirred at 37 ℃ for 28 days. The medium was changed every other day. At each time point, the multifunctional sponge was removed from the culture medium, gently rinsed with deionized water or phosphate buffer solution, and lyophilized. The mass of the sponge after lyophilization was weighed to calculate the degradation rate. It was found that after 28 days of degradation of the sponge in lysozyme-containing deionized water, the degradation rates were similar, maintaining about 37.12% and 51.39% of the original quality, respectively (e in fig. 5). In contrast, the sponge degraded slightly faster in the phosphate buffer solution containing lysozyme, maintaining about 26.24% and 42.21% of the original mass after 28 days of degradation, respectively (f in fig. 5). The sponge is completely degraded after 56 days, thereby ensuring the long-term biocompatibility of the sponge.

Example 13

The absorbance of amoxicillin solutions at 272nm wavelength was measured using an ultraviolet-visible spectrophotometer (UV-vis) to obtain a standard curve (fig. 6 a). To evaluate the drug release, 8mg of sponge (prepared in example 3) was filled into a dialysis bag having a cutoff molecular weight of 10000kDa, and then the dialysis bag was placed into a 50mL centrifuge tube containing 10mL of deionized water. Subsequently, the centrifuge tube was incubated in a steam bath shaker at 37 ℃. At each particular time point, 1mL of deionized water was taken and 1mL of fresh deionized water was added. Finally, the concentration of amoxicillin released by the sponge was determined using a UV-vis spectrophotometer. The release of amoxicillin in phosphate buffer solution at ph=7.4 was the same as the experimental procedure in deionized water described above.

As shown in fig. 6 b, the sponge showed a more pronounced sustained release in the case of deionized water or phosphate buffer solution. Then, the release tends to a stable state, which shows that the prepared multifunctional sponge has good application potential in the aspect of drug slow release.

Example 14

To 40mL of acetic acid-sodium acetate buffer (pH=4) were added 10mL of 3,3', 5' -Tetramethylbenzidine (TMB) solution (10 mM) and 30. Mu.L of AgCl solution (100 mM), to give TMBox solution. Next, 5. Mu.L of a reducing glutathione solution (3 mg/mL) and 50. Mu.L of an ebselenam solution (0.5 mg/mL) were added to a centrifuge tube containing 245. Mu.L of acetic acid-sodium acetate buffer solution, and incubated at 37℃for 1 hour. Then, 700. Mu.L of TMBox solution was added to the centrifuge tube, and the reaction was carried out at room temperature for 10 minutes. The absorbance of the solution at 652nm wavelength was measured using a UV-vis spectrophotometer to give a standard curve (c in fig. 6).

To evaluate the drug release, 8mg of sponge (prepared in example 4) was filled into a dialysis bag having a cutoff molecular weight of 10000kDa, and then the dialysis bag was placed into a 50mL centrifuge tube containing 10mL of deionized water. Subsequently, the centrifuge tube was incubated in a steam bath shaker at 37 ℃. At each particular time point, 1mL of deionized water was taken and 1mL of fresh deionized water was added. Next, 5. Mu.L of a reduced glutathione solution (3 mg/mL) and 50. Mu.L of a test solution were added to a centrifuge tube containing 245. Mu.L of acetic acid-sodium acetate buffer solution, and incubated at 37℃for 1 hour. Then, 700. Mu.L of TMBox solution was added to the centrifuge tube, and the reaction was carried out at room temperature for 10 minutes. Finally, the concentration of the released esceleam was determined using a UV-vis spectrophotometer. The release of escitalopram in phosphate buffer solution at ph=7.4 was the same as the experimental procedure in deionized water described above.

As shown in fig. 6 d, the sponge showed a more pronounced sustained release in the case of deionized water or phosphate buffer solution. Then, the release tends to a stable state, which shows that the prepared multifunctional sponge has good application potential in the aspect of drug slow release.

Example 15

Helicobacter pylori was activated in BHI broth and incubated in a three-gas incubator at 37℃for 24 hours at 120 rpm. Then respectively adding the bacterial suspension into sterile BHI liquid culture medium, and regulating the OD value of the bacterial suspension at 600nm to about 0.1. Multifunctional sponges (prepared in examples 2-5) were placed in test tubes containing 5mL of bacterial suspension, respectively, and incubated in a three-gas incubator at 37℃for 24 hours. Under the same conditions, liquid culture medium without strain is taken as negative control, and pure bacterial suspension is taken as positive control. The absorption of the bacterial suspension at 600nm was measured using a UV-vis spectrophotometer and the bacteriostatic rate was calculated. Subsequently, the resuscitator suspension (10. Mu.L) was diluted and spread on an agar plate, and incubated at 37℃for 3 to 4 days in a three-gas incubator. The number of bacteria grown on the agar plates was counted.

As shown in FIG. 7 a, after 24 hours of incubation, the bacteria inhibition rates of the sponges against H.pylori were 26.01%, 76.92%, 69.78% and 89.19%, respectively. After the incubation, we spread the bacterial suspension on agar plates and incubate for 3-4 days. The growth of the bacterial colony proves that all the sponges containing the medicines have certain antibacterial effect. The antibacterial effect of the sponge carrying amoxicillin and ebselenam is particularly remarkable (b, c in fig. 7).

Claims

1. The preparation method of the multifunctional sponge is characterized by comprising the following steps of:

2. The method for preparing a multifunctional sponge according to claim 1, wherein the solvent in the step 1) is any one of distilled water, phosphate buffer solution and physiological saline; the mass concentration of the dissolved gamma-polyglutamic acid is 0.5-15%; the dissolution temperature of the gamma-polyglutamic acid is 30-80 ℃.

3. The method for preparing a multifunctional sponge according to claim 1, wherein in the step 2), the mass concentration of the dissolved carboxymethyl chitosan is 0.5-20%; the dissolution temperature of the carboxymethyl chitosan is 30-80 ℃.

4. The method for preparing a multifunctional sponge according to claim 1, wherein the carboxyl activating agent in the step 3) is one or two of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide; the mass concentration of the carboxyl activating agent is 1-30%; the mass concentration or the volume concentration of the carboxyl activating agent (any one or two of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide) in the mixed solution is 0.5-30%; the mass ratio of the gamma-polyglutamic acid to the carboxymethyl chitosan is 0.4-0.6: 1.

5. the method for preparing a multifunctional sponge according to claim 1, wherein the temperature of the reaction in the step 3) is 10-30 ℃.

6. The method for preparing a multifunctional sponge according to claim 1, wherein the polymer in the step 3) is any one of polyvinylpyrrolidone and polyvinyl alcohol; the mass concentration of the polymer is 10-40%; the mass concentration or the volume concentration of the high polymer (any one of polyvinylpyrrolidone and polyvinyl alcohol) in the mixed solution is 40-80%.

7. The method for preparing a multifunctional sponge according to claim 1, wherein the antibacterial agent in the step 3) is amoxicillin or escitalopram; the concentration of the antibacterial drug in the mixed solution is 0.5-10 mg/mL.

8. Use of a multifunctional sponge prepared by the method of any one of claims 1-7 in drug delivery or drug sustained release antimicrobial anti-inflammatory biomaterials at a wound site.