Hemostatic sponge and preparation method thereof

Abstract

The invention discloses hemostatic sponge and a preparation method thereof. The hemostatic sponge is prepared from 2, 3-dihydroxybenzoic acid modified chitosan and phosphate, and the mass ratio of the 2, 3-dihydroxybenzoic acid modified chitosan to the phosphate is (10 to 1) to (40 to 1); the porosity of the hemostatic sponge is 70%-99%. The hemostatic sponge has an efficient hemostatic effect and an excellent capability of promoting wound healing, is excellent in biocompatibility, has multiple free radical scavenging capability, is remarkable in blood coagulation and hemostatic activity, and can be used for quickly stopping bleeding of various wounds.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology. More specifically, it relates to a hemostatic sponge and a preparation method thereof. Background Art

[0002] In clinical practice, uncontrollable bleeding of wounds has always been the primary factor leading to patient death in emergency surgery and daily injury processes. In the field of emergency surgery, the incidence of postoperative complications and the risk of transfer to the intensive care unit (ICU) of bleeding patients who require blood transfusion increase significantly by more than twice. In daily accidental injuries, deaths related to blood loss account for more than 50% of the total accidental deaths. Thus, it can be seen that wound bleeding has become a key factor leading to death globally, and achieving rapid hemostasis of wounds has great clinical significance for reducing hemorrhagic death.

[0003] In response to the clinical need for wound hemostasis, currently, a variety of means including hemostatic dressings, hemostatic bandages, hemostatic powders, hemostatic adhesives, and tourniquets are used clinically to control bleeding. Among them, the hemostatic sponge, as an important branch of hemostatic dressings, occupies a core position in the field of hemostasis. Thanks to the unique three-dimensional porous structure characteristics, the hemostatic sponge can not only accelerate the hemostasis process through mechanisms such as highly efficient water absorption to concentrate blood, physical adsorption of platelets, or chemical activation, but also form a physical barrier to protect the wound surface, and has extremely simple clinical operability. Based on the above characteristics, the hemostatic sponge dressing has become the preferred solution for achieving rapid hemostasis in clinical practice and plays an irreplaceable role in wound hemostasis.

[0004] Although the sponge dressing shows unique advantages in the field of wound hemostasis due to its excellent liquid absorption ability and clinical operation convenience endowed by its three-dimensional porous structure, the functions of traditional hemostatic sponge dressings are often limited to basic hemostasis needs. After completing the hemostasis function, they lack the ability to actively regulate the subsequent wound microenvironment, and thus cannot complete the whole-process management of the wound. For example, it cannot promote wound healing after achieving early hemostasis.

[0005] Therefore, how to ensure that the hemostatic sponge has excellent hemostatic effects while also having excellent effects in promoting wound healing is one of the relevant research directions. To sum up, constructing a multifunctional hemostatic sponge dressing with ROS scavenging function, by combining the dual functions of physical hemostasis and inhibition of wound oxidative stress injury, is a key treatment strategy for realizing the whole-process management of wounds. Summary of the Invention

[0006] Based on the above facts, the purpose of the present invention is to provide a hemostatic sponge and a preparation method thereof. The hemostatic sponge has both high-efficiency hemostatic effects and excellent ability to promote wound healing. The hemostatic sponge has excellent biocompatibility, multiple free radical scavenging abilities, significant coagulation and hemostatic activities, and can rapidly stop bleeding for various wounds.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] On the one hand, the present invention provides a hemostatic sponge, which contains: 2,3-dihydroxybenzoic acid modified chitosan and phosphate, and the mass ratio of the 2,3-dihydroxybenzoic acid modified chitosan to the phosphate is 10:1 - 40:1;

[0009] The porosity of the hemostatic sponge is 70 - 99%, preferably 70 - 85%.

[0010] Further, the phosphate is selected from one or more of sodium tripolyphosphate, sodium hexametaphosphate, trisodium phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium pyrophosphate, sodium acid pyrophosphate, calcium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, and β-glycerophosphate sodium.

[0011] Further, the 2,3-dihydroxybenzoic acid modified chitosan is obtained by coupling 2,3-dihydroxybenzoic acid with chitosan.

[0012] Further, the preparation of the 2,3-dihydroxybenzoic acid modified chitosan includes the following steps:

[0013] Completely dissolve chitosan, then add a catalyst and mix well to obtain reaction solution A;

[0014] Completely dissolve 2,3-dihydroxybenzoic acid to obtain reaction solution B;

[0015] Mix reaction solution A and reaction solution B and stir and react at room temperature;

[0016] Dialyze and freeze-dry the reaction solution after the reaction to obtain the 2,3-dihydroxybenzoic acid modified chitosan.

[0017] Further, the molecular weight of the chitosan is 50 - 200 kDa, and the degree of deacetylation is greater than 90%.

[0018] Further, in the process of forming reaction solution A, the catalyst is selected from 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

[0019] Further, the method for dissolving chitosan is: dissolve chitosan in MES buffer solution, then add hydrochloric acid and stir at room temperature to obtain a homogeneous solution;

[0020] Further, the dosage ratio of the chitosan, MES buffer solution and hydrochloric acid is 1 g:(50 - 100) mL:(0.5 - 1) mL.

[0021] Further, in the process of forming reaction solution B,

[0022] The method for dissolving 2,3-dihydroxybenzoic acid is to use a mixture of MES buffer and ethanol at a volume ratio of 1:1 to dissolve 2,3-dihydroxybenzoic acid.

[0023] Further, in the reaction solution A and the reaction solution B, the mass ratio of chitosan to 2,3-dihydroxybenzoic acid is 1:1 - 1:5.

[0024] In a second aspect, the present invention provides a method for preparing the hemostatic sponge as described above, and the preparation method includes the following steps:

[0025] Dissolve 2,3-dihydroxybenzoic acid-modified chitosan to obtain solution A;

[0026] Dissolve phosphate to obtain solution B;

[0027] Mix solution A and solution B evenly to obtain a mixed solution;

[0028] Perform freeze-thaw cycling treatment on the mixed solution to obtain a hydrogel;

[0029] Pre-freeze the hydrogel and then vacuum freeze-dry it to a constant weight to obtain the hemostatic sponge.

[0030] Further, in solution A, the concentration of 2,3-dihydroxybenzoic acid-modified chitosan is 10 - 40 mg / mL.

[0031] Further, in solution A, the solvent is acetic acid.

[0032] Further, in solution B, the concentration of phosphate is 5 - 20 mg / mL.

[0033] Further, in solution B, the solvent is deionized water.

[0034] Further, the mixing volume ratio of solution A to solution B is 5:1 - 20:1.

[0035] Further, the manner of the freeze-thaw cycling treatment is as follows:

[0036] Completely freeze at an environmental temperature of -16°C to -80°C and then completely melt at an environmental temperature of 4°C, and repeat the above steps 3 - 5 times.

[0037] Further, the temperature of the pre-freezing is -20°C and the time is 12 - 24 hours.

[0038] The beneficial effects of the present invention are as follows:

[0039] In the structure of the hemostatic sponge provided in the present invention, 2,3-dihydroxybenzoic acid is introduced into the chitosan backbone, realizing the functional upgrading of chitosan; further, the modified chitosan is combined with phosphate in a certain mass ratio, and the obtained hemostatic sponge not only has excellent hemostatic properties, but also exhibits excellent free radical scavenging ability, and can effectively inhibit oxidative stress damage of the wound surface. At the same time, the hemostatic sponge remodels the immune microenvironment of the wound surface, synergistically inhibits the inflammatory response and accelerates the tissue repair process. This multi-functional modification strategy of biomaterials significantly improves the comprehensive treatment effect of the hemostatic sponge dressing. In the preparation method of the hemostatic sponge provided in the present invention, through phosphate system , the hemostatic sponge with a double-network structure of electrostatic interaction and crystal region is prepared by a freeze-thaw and freeze-drying process. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The following further describes in detail the specific embodiments of the present invention with reference to the accompanying drawings.

[0041] Figure 1 A physical photograph of the hemostatic sponge prepared in Example 2 is shown.

[0042] Figure 2 A physical photograph of the hemostatic sponge prepared in Comparative Example 1 is shown.

[0043] Figure 3 A statistical chart of the survival rates of HaCaT and L929 cells treated with the hemostatic sponges prepared in Example 2 and Comparative Example 1 is shown.

[0044] Figure 4 A confocal live-dead staining photograph (scale bar is 200 μm) of L929 treated with the hemostatic sponges prepared in Example 2 and Comparative Example 1 is shown.

[0045] Figure 5 A statistical chart of the hemolysis rates of the hemostatic sponges prepared in Example 2 and Comparative Example 1 is shown.

[0046] Figure 6 A statistical chart of the coagulation indices of the hemostatic sponges prepared in Example 2 and Comparative Example 1 is shown.

[0047] Figure 7 A statistical chart of the DPPH free radical scavenging rates of the hemostatic sponges prepared in Example 2 and Comparative Example 1 is shown. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0048] To more clearly illustrate the present invention, the present invention will be further described below with reference to preferred embodiments and the accompanying drawings. Similar components in the drawings are denoted by the same reference numerals. Those skilled in the art should understand that the content specifically described below is illustrative rather than restrictive, and should not be used to limit the protection scope of the present invention.

[0049] Example 1

[0050] The synthesis of 2,3-dihydroxybenzoic acid modified chitosan comprises the following steps:

[0051] Weigh 0.5 g of chitosan with a molecular weight of 2×10 5 Da and a deacetylation degree of 93% and add it to 50 mL of 2-(N-morpholino)ethanesulfonic acid (MES) buffer solution (5 mM, pH = 4.80), and drop 0.5 mL of HCl and stir at room temperature for half an hour to completely dissolve the chitosan, thus obtaining a homogeneous solution with a mass-volume percentage concentration of 1%; then add 2.5 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to the above reaction solution and stir until completely dissolved to obtain reaction solution A;

[0052] Weigh 1 g of 2,3-dihydroxybenzoic acid and add it to a mixed solution of 75 mL of MES buffer solution (5 mM, pH = 4.80) and ethanol with a volume ratio of 1:1, and stir until completely dissolved to obtain reaction solution B;

[0053] Mix reaction solution A and reaction solution B, and continuously stir and react at room temperature for 24 hours, where the molar ratio of chitosan, 2,3-dihydroxybenzoic acid, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1:2:4; after the reaction is completed, transfer the reaction solution to a dialysis bag with a molecular weight cut-off of 8k - 14k Da, tie both ends of the dialysis bag tightly, place it in hydrochloric acid solution (pH = 5.00) for dialysis treatment, change the water once every 4 hours, and after changing the water 8 times, put the dialysis solution in -20°C and freeze overnight, and then put it into a vacuum freeze dryer until it is fully dried to a constant weight to obtain 2,3-dihydroxybenzoic acid modified chitosan (denoted as 2,3-CS).

[0054] Example 2

[0055] The preparation of a hemostatic sponge (2,3-dihydroxybenzoic acid modified chitosan / sodium tripolyphosphate hemostatic sponge) comprises the following steps:

[0056] Weigh an appropriate amount of 2,3-dihydroxybenzoic acid modified chitosan prepared in Example 1, dissolve it with 0.1 M acetic acid, and stir until completely dissolved to obtain a homogeneous solution A with a mass percentage concentration of 2% of the modified chitosan;

[0057] Weigh an appropriate amount of sodium tripolyphosphate (STPP), dissolve it with deionized water, and obtain a homogeneous solution B with a mass percentage concentration of 1% of STPP;

[0058] Mix the above solution A and solution B at a volume ratio of 10:1 (2,3-CS:STPP), and continuously stir until the precipitate is evenly dispersed; transfer the above mixture to a 12-well plate, place the plate in an environment of -20 °C, and take it out after the solution is completely frozen (about 12 h), place it in an environment of 4 °C until the solution is completely melted (about 2 h), and then put it back into the -20 °C environment for freezing; repeat this process at least 6 cycles to obtain a 2,3-dihydroxybenzoic acid modified chitosan / sodium tripolyphosphate precursor gel;

[0059] Put the 12-well plate in the freezer at -20 °C overnight, and then put it into a vacuum freeze dryer until it is fully dried to a constant weight to obtain a 2,3-dihydroxybenzoic acid modified chitosan sponge / sodium tripolyphosphate hemostatic sponge with a porosity of 70-85% (denoted as 2,3-CS / STPP). The physical photo of this hemostatic sponge is shown in Figure 1 , from Figure 1 it can be seen that this hemostatic sponge is a sheet-like sponge with a rough surface, soft touch, light texture, and presents a light yellow color.

[0060] Comparative Example 1

[0061] Preparation of chitosan / sodium tripolyphosphate hemostatic sponge:

[0062] Weigh an appropriate amount of chitosan, dissolve it with 0.1 M acetic acid, and stir until completely dissolved to obtain a uniform solution with a mass-volume percentage concentration of chitosan of 2%; weigh an appropriate amount of sodium tripolyphosphate (STPP), dissolve it with deionized water to obtain a uniform solution with a mass-volume percentage concentration of STPP of 1%; mix the above solutions at a volume ratio of 10:1 (CS:STPP), and continuously stir until the precipitate is evenly dispersed; transfer the above mixture to a 12-well plate, place the plate in an environment of -20 °C, and take it out after the solution is completely frozen (about 12 h), place it in an environment of 4 °C until the solution is completely melted (about 2 h), and then put it back into the -20 °C environment for freezing. Repeat this process at least 6 cycles to obtain a chitosan / sodium tripolyphosphate precursor gel. Put the 12-well plate in the freezer at -20 °C overnight, and then put it into a vacuum freeze dryer until it is fully dried to a constant weight to obtain a chitosan hemostatic sponge (denoted as CS / STPP). The physical photo of this sponge is shown in Figure 2 .

[0063] Performance test:

[0064] 1. Determination of the biocompatibility of the hemostatic sponge:

[0065] Digest the cells in the culture flask with trypsin solution, centrifuge, and redissolve with the culture medium solution; use a cell counting chamber to calculate the cell concentration in the solution and dilute it to 3×10 5 to 4×10 5cells / mL; Take a new 12-well plate, add the solution to the 12-well plate, 1.5 mL per well; Place the 12-well plate in an incubator at 37 °C with 5% CO2 for an appropriate time, and add fresh medium solution to one group as a negative control; Weigh an appropriate amount of the 2,3-dihydroxybenzoic acid-modified chitosan / sodium tripolyphosphate hemostatic sponge (2,3-CS / STPP) prepared in Example 2 and the chitosan / sodium tripolyphosphate hemostatic sponge (CS / STPP) prepared in Comparative Example 1; Take out the previous 12-well plate and aspirate the original medium solution; Add fresh medium solution to the 12-well plate, 750 μL per well, add a new transwell chamber (for 12-well plate, pore size 8 μm) to the 12 wells, add the corresponding hemostatic sponge of Example 2 or Comparative Example 1 and 750 μL of fresh medium solution to the upper chamber, and only add 750 μL of fresh medium solution to the upper chambers of the positive and negative controls. Each group has 6 parallel samples; Place the 12-well plate in an incubator at 37 °C with 5% CO2 for 24 h; Prepare a 10% (v / v) CCK-8 solution; Take out the previous 12-well plate, discard the transwell chamber, and aspirate the original solution; Wash 3 times with 1 mL of PBS; Transfer the CCK-8 solution to the 12-well plate, 1 mL per well; Place the 12-well plate in an incubator at 37 °C with 5% CO2 for 1 - 2 h; Take out the previous 12-well plate and a new 96-well plate, transfer the supernatant of the experimental group and the control group in the 12-well plate to the new 96-well plate, 100 μL per well; Read the absorbance with a microplate reader at a wavelength of 450 nm, and calculate H = (A 样品 - A 阴性 ) / (A 阳性 - A 阴性 ) as the cell survival rate, where A 样品 , A 阴性 and A 阳性 correspond to the readings of the sample, negative control, and positive control at a wavelength of 450 nm by the microplate reader respectively. The experimental results are as shown in Figure 3 . For mouse fibroblasts (L929 cells) and human immortalized epidermal keratinocytes (HaCaT), the cell proliferation rates of the hemostatic sponges obtained in Example 2 and Comparative Example 1 are both greater than 80%, and there is no cytotoxicity, indicating good cell compatibility.

[0066] 2. Cell viability staining experiment of the hemostatic sponge:

[0067] Digest the L929 cells in the culture flask with trypsin solution, centrifuge, and resuspend with the medium solution; Use a cell counting chamber to calculate the cell concentration in the solution and dilute it to 3×10 5 to 4×10 5cells / mL; Take a new 12-well plate, add the solution into the 12-well plate, 1.5 mL per well; Place the 12-well plate in an incubator with 5% CO2 at 37 °C for an appropriate time, and add fresh culture medium solution to one group as a negative control; Weigh an appropriate amount of the 2,3-dihydroxybenzoic acid modified chitosan / sodium tripolyphosphate hemostatic sponge (2,3-CS / STPP) prepared in Example 2 and the chitosan / sodium tripolyphosphate hemostatic sponge (CS / STPP) prepared in Comparative Example 1; Take out the previous 12-well plate and aspirate the original culture medium solution; Add fresh culture medium solution to the 12-well plate, 750 μL per well, add a new transwell chamber (for 12-well plate, pore size 8 μm) to the 12 wells, add the corresponding hemostatic sponge of Example 2 or Comparative Example 1 and 750 μL of fresh culture medium solution to the upper chamber, and only add 750 μL of fresh culture medium solution to the upper chambers of the positive and negative controls; Place the 12-well plate in an incubator with 5% CO2 at 37 °C for 24 h; Take out the previous 12-well plate, discard the transwell chamber, aspirate the original solution of the positive control group, add 75% ethanol and incubate at 37 °C for 30 min, then aspirate the original solution; Wash 3 times with 1×Assay Buffer, and then stain with Calcein-AM / PI fluorescence solution for 10 min; Wash with PBS; Use a confocal microscope (Nikon ARsi MP-LSM), observe the fluorescence of cell staining under a 10× lens with a laser wavelength of 488 / 561 nm, and take pictures. The experimental results are as Figure 4 shown. As can be seen from Figure 4 , for mouse fibroblasts (L929 cells), the cells treated with the hemostatic sponge obtained in Example 2 and the hemostatic sponge obtained in Comparative Example 1 were both stained with Calcein-AM, and the cell viability was good. The hemostatic sponge obtained in Example 2 and the hemostatic sponge obtained in Comparative Example 1 had good cell compatibility.

[0068] 3. Determination of blood compatibility of the hemostatic sponge:

[0069] 5 mL of fresh rabbit blood was centrifuged at 1000 g for 5 min to obtain red blood cells, which were washed 3 times with PBS, 30 mL of buffer each time. 2 mL of the red blood cell solution was added to 38 mL of PBS to resuspend the red blood cells. Approximately 7 mg of chitosan sample (CS), the hemostatic sponge of Comparative Example 1 (CS / STPP), 2,3-dihydroxybenzoic acid modified chitosan sample (2,3-CS sample), and the hemostatic sponge of Example 2 (2,3-CS / STPP) were weighed into centrifuge tubes respectively. 500 μL of PBS was added to the centrifuge tubes and incubated with shaking at 37 °C for 30 min. Among them, PBS was used as the negative control and PBS with 1‰ (v / v) Triton X-100 was used as the positive control. Each group had 3 parallel samples. 500 μL of rabbit blood suspension was added to each of the above centrifuge tubes and incubated at 37 °C for 1 h, then centrifuged at 1000 g at 4 °C for 10 min. The absorbance was measured with a microplate reader at a wavelength of 540 nm. Let H = (A 样品 -A 阴性 ) / (A 阳性 -A 阴性 ) be the hemolysis rate, where A 样品 , A 阴性 , and A 阳性 corresponded to the absorbance readings of the sample, negative control, and positive control with the microplate reader at a wavelength of 540 nm respectively. The experimental results are as shown in Figure 5 . It can be seen from Figure 5 that the samples of each experimental example did not cause red blood cell rupture, the hemolysis values were all less than 5%, and they did not have hemolytic toxicity. The hemostatic sponges obtained in Example 2 and Comparative Example 1 both had good blood compatibility.

[0070] 4. Coagulation effect experiment of the hemostatic sponge:

[0071] An appropriate amount of CS sample, 2,3-CS freeze-dried sample, CS / STPP hemostatic sponge in Comparative Example 1, and 2,3-CS / STPP hemostatic sponge in Example 2 were weighed into a 6-well plate, and the 6-well plate was incubated at 37 °C for 5 min; 3.75 μL of CaCl2 with a concentration of 0.2 M was added to 75 μL of rabbit whole blood for activation; 50 μL of activated whole blood was dropped onto the sample and incubated in an incubator at 37 °C for 10 / 30 / 60 s. Each group had 3 parallel samples; 2 mL of DDW was added to the wells, and the blood was gently rinsed, and the collected liquid was aspirated. Among them, 50 μL of whole blood plus 2 mL of DDW was used as the negative control, and DDW was used as the positive control; the absorbance was measured with a microplate reader at a wavelength of 540 nm. Let H = (A 样品 -A 阴性 ) / (A 阳性 -A 阴性 ) be the BCI, where A 样品 , A 阴性 , and A 阳性Corresponding to the readings of the sample, negative control, and positive control on the microplate reader at a wavelength of 540 nm. The experimental results are as Figure 6 shown. As can be seen from Figure 6 , compared with the corresponding sample raw materials, the hemostatic sponges obtained in Example 2 and Comparative Example 1 had a BCI lower than 15% at 10 / 30 / 60 s, and the activated whole blood was almost completely coagulated, showing excellent blood coagulation promoting ability.

[0072] 5. Determination of the antioxidant capacity (DPPH free radical scavenging rate) of the hemostatic sponge

[0073] Weigh an appropriate amount of 1,1-diphenyl-2-picrylhydrazyl (DPPH), dissolve it with ethanol, and prepare a solution with a DPPH molar concentration of 0.5 mM; weigh an appropriate amount of the hemostatic sponge prepared in Example 2 and the hemostatic sponge prepared in Comparative Example 1 into centrifuge tubes; add DPPH solution to the centrifuge tubes so that the concentration of the freeze-dried sponge is 2 mg / mL. For the negative control, add DPPH solution, and for the positive control, add ethanol. Each group has 3 parallel samples; react at room temperature in the dark for an appropriate time; read the microplate reader at a wavelength of 517 nm for the supernatant. Let H = 1 - (A 样品 -A 阴性 ) / (A 阳性 -A 阴性 ) be the DPPH free radical scavenging rate, where A 样品 , A 阴性 , and A 阳性 correspond to the readings of the sample, negative control, and positive control on the microplate reader at a wavelength of 517 nm, respectively. The experimental results are as Figure 7 shown. As can be seen from Figure 7 , compared with the hemostatic sponge in Comparative Example 1, the hemostatic sponge obtained in Example 2 showed good free radical scavenging ability, could effectively scavenge DPPH free radicals, and had good antioxidant capacity.

[0074] Obviously, the above-mentioned embodiments of the present invention are merely examples for clearly explaining the present invention, rather than limitations on the implementation manners of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the implementation manners here. Any obvious changes or modifications derived from the technical solutions of the present invention still fall within the protection scope of the present invention.

Claims

1. A hemostatic sponge, characterized in that, The hemostatic sponge contains: 2,3-dihydroxybenzoic acid modified chitosan and phosphate, and the mass ratio of the 2,3-dihydroxybenzoic acid modified chitosan to the phosphate is 10:1 - 40:1; The porosity of the hemostatic sponge is 70 - 99%.

2. The hemostatic sponge according to claim 1, wherein, The phosphate is selected from one or more of sodium tripolyphosphate, sodium hexametaphosphate, trisodium phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium pyrophosphate, sodium dihydrogen pyrophosphate, calcium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, and β-glycerophosphate sodium.

3. The hemostatic sponge according to claim 1, wherein, The 2,3-dihydroxybenzoic acid modified chitosan is obtained by coupling 2,3-dihydroxybenzoic acid with chitosan.

4. The hemostatic sponge according to claim 1 or 3, characterized in that, The preparation of the 2,3-dihydroxybenzoic acid modified chitosan includes the following steps: Completely dissolve chitosan, then add a catalyst and mix evenly to obtain reaction solution A; Completely dissolve 2,3-dihydroxybenzoic acid to obtain reaction solution B; Mix reaction solution A and reaction solution B and stir at room temperature for reaction; Dialyze and freeze-dry the reaction solution after the reaction to obtain the 2,3-dihydroxybenzoic acid modified chitosan.

5. The hemostatic sponge according to claim 4, wherein The molecular weight of the chitosan is 50 - 200 kD, and the degree of deacetylation is greater than 90%.

6. The hemostatic sponge according to claim 4, wherein, During the formation of reaction solution A, The catalyst is selected from 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; The method for dissolving chitosan is: dissolve chitosan in MES buffer solution, then add hydrochloric acid and stir at room temperature to obtain a homogeneous solution; Preferably, the dosage ratio of the chitosan, MES buffer solution, and hydrochloric acid is 1 g:(50 - 100) mL:(0.5 - 1) mL; and / or During the formation of reaction solution B, The method for dissolving 2,3-dihydroxybenzoic acid is to use a mixture of MES buffer solution and ethanol with a volume ratio of 1:1 to dissolve 2,3-dihydroxybenzoic acid.

7. The hemostatic sponge according to claim 4, wherein, In reaction solution A and reaction solution B, the mass ratio of chitosan to 2,3-dihydroxybenzoic acid is 1:1 - 1:

5.

8. The preparation method of the hemostatic sponge according to any one of claims 1-7, characterized in that, It includes the following steps: Dissolve 2,3-dihydroxybenzoic acid modified chitosan to obtain solution A; Dissolve phosphate to obtain solution B; Mix solution A and solution B evenly to obtain a mixed solution; Perform freeze-thaw cycle treatment on the mixed solution to obtain a hydrogel; Pre-freeze the hydrogel and then vacuum freeze-dry it to constant weight to obtain the hemostatic sponge.

9. The preparation method according to claim 8, characterized in that, In solution A, the concentration of 2,3-dihydroxybenzoic acid modified chitosan is 10 - 40 mg / mL; In solution B, the concentration of phosphate is 5 - 20 mg / mL; The mixing volume ratio of solution A and solution B is 5:1 - 20:

1.

10. The preparation method according to claim 8, wherein The method of the freeze-thaw cycle treatment is: Completely freeze at an environmental temperature of -16°C to -80°C and then completely melt at an environmental temperature of 4°C, and repeat the above steps 3 - 5 times; and / or The temperature of the pre-freezing is -20°C and the time is 12 - 24 hours.

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