Preparation method and application of a biodegradable polymer nanostop hemostatic dressing

Abstract

The application discloses a preparation method and application of a biodegradable polymer nanostop bleeding dressing. The bleeding dressing is prepared from chitosan, tranexamic acid and polylactic acid-glycolic acid copolymer as synthetic materials. The preparation method comprises the following steps: weighing PLGA, dissolving the PLGA in DMSO under nitrogen protection, adding DCC and NHS, magnetically stirring and reacting under nitrogen protection, adding CS and DMSO solution, and continuously reacting under nitrogen protection; weighing TXA, adding a methanol-acetic acid mixed solution, and refluxing and reacting; mixing the obtained reaction liquid, continuously refluxing and reacting, and obtaining a product solution; using a dialysis bag to dialyze, pre-freezing, freeze-drying and storing. The bleeding dressing is convenient to use, and when used for wound hemostasis, adverse reactions caused by the material do not have to be worried about, the material can be degraded in a short time after use, and the pain point of clinically using the bleeding dressing is ingeniously solved, and the health of a trauma patient is protected.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medical materials, and particularly relates to a preparation method of a biodegradable polymer nanostop bleeding dressing and application thereof. BACKGROUND

[0002] Traumatic bleeding occurs from time to time in daily life, in wars, traffic accidents, surgeries and natural disasters, and excessive blood loss is one of the main causes of death. Effective hemostasis is an important step in emergency medical treatment and is also a key to improving the survival rate of the injured. Using a hemostatic dressing is an effective way to quickly stop bleeding, accelerate wound healing and improve wound healing, and is also an important method to effectively avoid death caused by blood loss.

[0003] Traditional hemostatic dressings such as gauze, yarn, and cotton pads have strong water absorption and can effectively block the blood of bleeding vessel orifices, and have a significant hemostatic effect. In addition, they can absorb tissue fluid exuded from the wound surface during wound healing and maintain the cleanliness of the wound site. However, traditional hemostatic dressings have many drawbacks in actual operation. For example, gauze cannot be degraded by itself and needs to be removed by a second operation after use, which may cause a risk of secondary bleeding. The surface permeability of gauze is relatively large, which causes the wound surface to dry and easily form a scab, which is not conducive to wound healing. The pores of gauze are large, and dust and bacteria in the air can easily invade the wound, causing infection of the wound surface. Gauze is easily adhered to the wound, and replacement can cause the shedding of new tissue, causing secondary damage to the wound surface, prolonging the wound healing time, and increasing the stress on the patient. Therefore, the research and development of fast, safe and effective hemostatic dressings are imperative.

[0004] Chitosan (CS) is a product of N-deacetylation of chitin, and its chemical name is β-(1, 4)-2-amino-2-deoxy-D-glucan. It is a new type of hemostatic material and belongs to a linear biological macromolecular compound. Chitosan has strong plasticity and can form various forms of hemostatic materials. Chitosan carries a large number of positive charges and can combine with the negative charges on the surface of red blood cell membranes and platelets. It has good affinity, adsorption, film formation, permeability, fiber formation, moisture absorption and moisture retention for human cells. Chitosan is gradually widely used in clinical applications due to its excellent properties such as good biocompatibility, biodegradability, antibacterial property, ability to promote wound healing and prevent film adhesion. Chitosan contains hydroxyl and amino groups on the molecular chain. Under specific conditions, chitosan can be acylated, etherified, alkylated, quaternized and grafted copolymerized to generate various chitosan derivatives with different properties. However, the hemostatic effect of single chitosan is limited, and it is often necessary to combine chitosan with other hemostatic components to improve the hemostatic effect and generate various chitosan derivatives with different properties to expand the application range of chitosan.

[0005] Tranexamic acid, also known as TXA, is a synthetic lysine derivative that is effective in controlling bleeding from trauma or surgery in organs rich in plasminogen activators. It has high affinity for the lysine binding site of plasminogen, which can block the lysine binding site and prevent the binding of fibrinolysin to fibrin molecules to achieve hemostatic effect, protect the formation of mature blood clots, and achieve the purpose of rapid hemostasis. It can safely and effectively reduce the mortality rate of patients with traumatic bleeding in clinical practice, and can be used for bleeding caused by acute, chronic, localized or systemic hyperfibrinolysis, and is widely used in craniocerebral trauma, excessive bleeding, postpartum hemorrhage, surgery and other fields. Because of its low price, it belongs to non-patent drugs, and has been included in the list of essential drugs of WHO, and will be widely used in countries around the world.

[0006] In view of the above problems, the present application provides a preparation and application of a biodegradable polymer nanostop bleeding dressing. The amount of the dressing can be taken as needed, and the size and amount of the dressing can be adjusted by itself. The dressing can be quickly adhered to the wound surface, effectively absorb the wound blood and achieve the purpose of rapid hemostasis, efficiently promote wound healing, and will not cause wound infection. The dressing is convenient to use, and does not need to worry about adverse reactions caused by the material during surgery. The material can be degraded in a short time after surgery, which ingeniously solves the pain points of clinical use of the dressing and provides protection for the health of the trauma patients.

[0007] The present application selects a new bio-based high molecular hemostatic material with wide sources and low price to follow the basic principles of "rapid hemostasis", "rapid wound healing" and "non-toxic and harmless", and uses chitosan, tranexamic acid and polylactic acid-glycolic acid copolymer as raw materials, and N-hydroxysuccinimide (NHS), dicyclohexyl carbodiimide (DCC) and the like as chemical reaction condensate to prepare a new porous sponge-like composite hemostatic dressing with rapid hemostasis, air permeability and healing promotion and biodegradability. SUMMARY

[0008] The present application provides a preparation method of a biodegradable polymer nanostop bleeding dressing.

[0009] Another object of the present application is to provide the application of the biodegradable polymer nanostop bleeding dressing in the preparation of hemostatic materials or hemostatic medical products.

[0010] In order to achieve the object of the present application, the present application adopts the following technical solutions and steps:

[0011] The preparation method of the biodegradable polymer nanostop bleeding dressing provided by the present application comprises the following steps:

[0012] (1) take PLGA, dissolve in DMSO (anhydrous grade) under nitrogen protection, add DCC and NHS, magnetically stir under nitrogen protection, then add CS and DMSO (anhydrous grade) solution, continue to react under nitrogen protection, to obtain a first reaction solution;

[0013] (2) take TXA, add methanol-acetic acid mixed solution, reflux to react, to obtain a second reaction solution;

[0014] (3) mix the reaction solutions obtained in steps (1) and (2), continue to reflux to react, to obtain a product solution;

[0015] (4) use a dialysis bag to dialyze the product solution obtained in step (3), pre-freeze, freeze-dry and store, to obtain the product.

[0016] The preparation method described in the application specifically comprises the following steps:

[0017] (1) precisely take 0.1g PLGA, dissolve in 8mL DMSO (anhydrous grade) under nitrogen protection, add 0.021g DCC and 0.012g NHS, magnetically stir under nitrogen protection for 10-15h, then add 1g CS and 2mL DMSO (anhydrous grade) solution, continue to react under nitrogen protection for 8h, to obtain a first reaction solution;

[0018] (2) take 0.27g TXA, add methanol-acetic acid mixed solution, reflux to react for 1h, to obtain a second reaction solution;

[0019] (3) mix the reaction solutions obtained in steps (1) and (2), continue to reflux to react for 8h, to obtain a product solution;

[0020] (4) use a dialysis bag to dialyze the product solution obtained in step (3) for 48h, pre-freeze at-20℃ for 48h, freeze-dry and store, to obtain the product.

[0021] The magnetic stirring condition described in the application is: temperature 40℃, rotation speed 400rpm.

[0022] The ratio of chitosan: tranexamic acid described in the application is m(TXA):m(CS)=1:1.85-7.41.

[0023] Preferably, the ratio of chitosan: tranexamic acid described in the application is m(TXA):m(CS)=1:3.7.

[0024] The ratio of chitosan: polylactic acid-glycolic acid copolymer described in the application is m(PLGA):m(CS)=1:5-10.

[0025] Preferably, the ratio of chitosan: polylactic acid-glycolic acid copolymer described in the application is m(PLGA):m(CS)=1:10.

[0026] The volume ratio of the methanol-acetic acid mixed solution is methanol: acetic acid = 1:1.6, and the reflux temperature is 60 DEG C.

[0027] The molecular weight cut-off of the dialysis bag is 10000.

[0028] The hemostatic dressing is prepared by using chitosan, tranexamic acid and polylactic acid-glycolic acid copolymer as high polymer materials and using N-hydroxysuccinimide (NHS), dicyclohexyl carbodiimide (DCC) and the like as chemical reaction condensate.

[0029] The present application has the following advantages:

[0030] 1. The raw materials used in the present application are widely available, low in price, safe and non-toxic, have strong plasticity and wide application, and the hemostatic dressing prepared by the preparation method of the present application is stable, convenient to use, does not cause adverse reactions when used for surgical hemostasis, and can be degraded in vivo in a short time after surgery, thus skillfully solving the pain points of clinical use of hemostatic dressings, achieving rapid hemostasis, being more strongly adhesive and having higher blood absorption capacity than commercially available dressings, effectively accelerating wound healing, greatly improving patient trust in medical dressings, and providing a new idea for the research and development of similar products in the future.

[0031] 2. The hemostatic material is subjected to screening test, and CS / TXA, CS / PEG / TXA, CS / PVA / SA and CS / TXA / PLGA are selected for investigation, and the results show that only CS / TXA / PLGA is used as the hemostatic material, the generated reaction solution is a yellow uniform transparent liquid, slightly sticky, and after freeze-drying, it is a yellow sponge-like nanomaterial, slightly soluble in water, has good stability, appears as a transparent gel in water, has good water absorption and speed, and the hemostatic effect is obviously improved, and has good adhesion, and finally, CS / TXA / PLGA is selected as the hemostatic material.

[0032] 3. The ratio of the selected materials is screened, and when the ratio of CS:TXA is investigated, the properties of the generated nanohemostatic sponge are determined by rat tail cutting and mouse liver hemostasis experiments, and the ratio of TXA:CS = 1:3.7 is determined to be the best; when the ratio of PLGA:CS is investigated, the functions of the generated nanohemostatic sponge are determined by porosity and solution stability experiments, and the ratio of PLGA:CS = 1:10 is determined to be the best, and the material is the most stable.

[0033] 4. The hemostatic performance of the prepared hemostatic dressing is tested, and the results show that:

[0034] (1) In the experimental group, the mean hemostasis times of the chitosan-tranexamic acid hemostatic sponges with three different grafting degrees were 89.61s, 73.88s, and 107.52s, respectively, while the mean hemostasis time of the positive control group was 101.55s. The blank control group continued to bleed slowly after 180s. In the experimental group, the hemostasis times of the hemostatic sponges with grafting ratios ① and ② were not significantly different, but were significantly shorter than those of the other groups.

[0035] (2) Seven days later, the healing degree of the tail wounds of rats was observed. The blank control group still showed inflammation, while the positive control group showed no significant inflammation but the wounds were not completely healed. No inflammation was observed in the experimental groups and the wounds were scab-forming, indicating that the PLGA-CS-TXA hemostatic sponge is beneficial in protecting the wound surface, avoiding further damage to newly formed tissue, and effectively reducing the wound infection rate.

[0036] (3) PLGA-CS-TXA-1 (sample) had a shorter in vitro whole blood absorption time than PLGA-CS-TXA-2 (control), and a small amount of material could absorb all the blood without serious coagulation. Although the average whole blood absorption time of the material was longer than that of sterile cotton, sterile cotton was prone to shedding cotton fibers, which could cause wound infection. Overall, the whole blood absorption effect of PLGA-CS-TXA material was better than that of sterile cotton.

[0037] (4) In the PLGA-CS-TXA-1 group, the blood stopped flowing, the material absorbed all the blood, and the volume increased.

[0038] 5. The hemostatic dressing prepared in this invention was subjected to cytotoxicity assay. The MTT assay was used to detect the cytotoxicity of PLGA-CS-TXA hemostatic dressing on L929 mouse fibroblasts. The results showed that the cell viability was greater than 100% and the material had no cytotoxicity. Attached Figure Description

[0039] Figure 1 Chitosan / tranexamic acid polymer dressing

[0040] Figure 2 CS / PEG / TXA dissolved in PBS buffer (where 1: TXA + PBS; 2: PEG + PBS; 3: CS + PBS)

[0041] Figure 3 : Reaction products generated using CS / PVA / SA as materials (where, Figure a: gel-like mixture of hemostatic powder, SA, and PVA; Figure b: freeze-dried product of CS+SA+PVA; Figure c: freeze-dried product of hemostatic powder+SA+PVA)

[0042] Figure 4 PLGA-CS-TXA Polymer Hemostatic Dressing

[0043] Figure 5 : Hemostasis after the tail of rats was cut off and treated with sterile cotton, chitosan freeze-dried product (CS), CS-TXA hemostatic sponge (ratio 1), CS-TXA hemostatic sponge (ratio 2) and CS-TXA hemostatic sponge (ratio 3)

[0044] Figure 6 : Hemostasis after the liver of mice was bled and treated with sterile cotton, chitosan freeze-dried product (CS), CS-TXA hemostatic sponge (ratio 1), CS-TXA hemostatic sponge (ratio 2) and CS-TXA hemostatic sponge (ratio 3)

[0045] Figure 7 : Reaction and product generated when dichloromethane was used as solvent (wherein, a: reaction process phenomenon; b: product self-water phenomenon after 3h without timely treatment; c: freeze-dried product (m(PLGA):m(CS)=1:5))

[0046] Figure 8 : m(PLGA):m(CS):m(TXA)=1:10:37 product figure

[0047] Figure 9 : Reaction and product generated when purified water was used as solvent (wherein, a: reaction process phenomenon; b: product room temperature state; c: freeze-dried product)

[0048] Figure 10 : Rat tail amputation model hemostasis experiment: a. bleeding model; b. PLGA-CS-TXA-1; c. PLGA-CS-TXA-2; d. CS (chitosan); e. gauze

[0049] Figure 11 : Wound healing after the tail of rats was cut off and treated with different materials (wherein, a. untreated group (Blank); b. PLGA-CS-TXA-1; c. PLGA-CS-TXA-2; d. CS (chitosan); e. gauze)

[0050] Figure 12 : Mouse liver hemostasis experiment (wherein, a. bleeding model; b. PLGA-CS-TXA-1; c. PLGA-CS-TXA-2; d. CS (chitosan); e. gauze)

[0051] Figure 13 : In vitro whole blood absorption experiment (wherein, a. PLGA-CS-TXA-1; b. PLGA-CS-TXA-2; c. sterile cotton)

[0052] Figure 14SEM images of CS-TXA (scale bar ②) (a, scale bar 10 μm; b, scale bar 20 μm; c, scale bar 100 μm; d, scale bar 100 μm)

[0053] Figure 15 SEM images of PLGA-CS-TXA (a, b scale bar 500 μm)

[0054] Figure 16 Fourier infrared spectrum of the nanostop bleeding sponge

[0055] Figure 17 NMR hydrogen spectrum of the nanostop bleeding sponge

[0056] Figure 18 Biodegradation experiment of the nanostop bleeding sponge in a 37°C thermostat

[0057] Figure 19 Mass change of the nanostop bleeding sponge in a 37°C thermostat for one week

[0058] Figure 20 In vitro degradation of sterile cotton

[0059] Figure 21 In vitro degradation of CS

[0060] Figure 22 In vitro degradation of TXA

[0061] Figure 23 In vitro degradation of PLGA

[0062] Figure 24 In vitro degradation of the nanostop bleeding sponge

[0063] Figure 25 In vitro biodegradation experiment of the nanostop bleeding sponge (30d)

[0064] Figure 26 Water absorption capacity evaluation results of the nanostop bleeding sponge (a. gauze; b. physically synthesized CS-PLGA-TXA hemostatic material; c. chemically synthesized CS-PLGA-TXA hemostatic material)

[0065] Figure 27 Solution stability test results at each time period (1 min, 1 h, 1 d) (left: PLGA-CS-TXA-2 hemostatic material (physically synthesized); middle: gauze; right: PLGA-CS-TXA-1 hemostatic material (chemically modified)

[0066] Figure 28 Determination of the porosity of the nanostop bleeding sponge by the pycnometer method

[0067] Figure 29 Figure 1 is a schematic diagram of water contact angle of a nanostop hemostatic dressing and a control thereof DETAILED DESCRIPTION

[0068] Example 1

[0069] (1) precisely weigh 0.1 g of PLGA, dissolve in 8 mL of DMSO (anhydrous grade) under nitrogen protection, add 0.021 g of DCC and 0.012 g of NHS, react under nitrogen protection at 40°C with magnetic stirring at 400 rpm for 10-15 h, then add 1 g of CS and 2 mL of DMSO (anhydrous grade) solution, continue to react under nitrogen for 8 h to obtain a first reaction solution;

[0070] (2) weigh 0.27 g of TXA, add a methanol-acetic acid (1:1.6) mixed solution, reflux at 60°C for 1 h to obtain a second reaction solution;

[0071] The mass ratio of chitosan: tranexamic acid is m(TXA): m(CS) = 1:3.7;

[0072] The ratio of chitosan: polylactic acid-glycolic acid copolymer is m(PLGA): m(CS) = 1:10. (m(PLGA): m(DCC): m(NHS) = 8.33: 1.75: 1)

[0073] (3) mix the reaction solutions obtained in steps (1) and (2), continue to reflux at 60°C for 8 h to obtain a product solution;

[0074] (4) dialyze the product solution obtained in step (3) using a dialysis bag with a molecular weight cut-off of 10000 for 48 h, pre-freeze at -20°C for 48 h, freeze-dry and store, to obtain the product.

[0075] Example 2

[0076] (1) precisely weigh 0.2 g of PLGA, dissolve in 8 mL of DMSO (anhydrous grade) under nitrogen protection, add 0.042 g of DCC and 0.024 g of NHS, react under nitrogen protection with magnetic stirring for 10-15 h, then add 1 g of CS and 2 mL of DMSO (anhydrous grade) solution, continue to react under nitrogen for 8 h to obtain a first reaction solution;

[0077] (2) weigh 0.54 g of TXA, add a methanol-acetic acid mixed solution, reflux for 1 h to obtain a second reaction solution;

[0078] The mass ratio of chitosan: tranexamic acid is m(TXA): m(CS) = 1:1.85;

[0079] The ratio of chitosan: poly-lactic-glycolic acid copolymer is m(PLGA):m(CS)=1:5.

[0080] (3) The reaction solutions obtained in steps (1) and (2) are mixed, and reflux reaction is continued for 8 h to obtain a product solution.

[0081] (4) The product solution obtained in step (3) is dialyzed using a dialysis bag for 48 h, pre-frozen at -20℃ for 48 h, and freeze-dried for storage, thereby obtaining the product.

[0082] Example 3

[0083] (1) 0.1 g of PLGA is precisely weighed and dissolved in 8 mL of DMSO (anhydrous grade) under nitrogen protection, 0.021 g of DCC and 0.012 g of NHS are added, and magnetic stirring reaction is carried out for 10-15 h under nitrogen protection, then 1 g of CS and 2 mL of DMSO (anhydrous grade) solution are added, and nitrogen protection reaction is continued for 8 h, thereby obtaining a first reaction solution;

[0084] (2) 0.135 g of TXA is weighed and added into a methanol-acetic acid mixed solution, and reflux reaction is carried out for 1 h to obtain a second reaction solution;

[0085] The mass ratio of chitosan: tranexamic acid is m(TXA):m(CS)=1:7.41.

[0086] The ratio of chitosan: poly-lactic-glycolic acid copolymer is m(PLGA):m(CS)=1:10.

[0087] (3) The reaction solutions obtained in steps (1) and (2) are mixed, and reflux reaction is continued for 8 h to obtain a product solution.

[0088] (4) The product solution obtained in step (3) is dialyzed using a dialysis bag for 48 h, pre-frozen at -20℃ for 48 h, and freeze-dried for storage, thereby obtaining the product.

[0089] In order to further verify the feasibility and effectiveness of the present application and screen out the best scheme, the inventors have carried out a series of tests, which are as follows:

[0090] I. Experimental method

[0091] 1. Preparation of hemostatic dressing

[0092] 1.1 Selection of hemostatic material

[0093] 1.1.1 Chitosan / tranexamic acid (CS / TXA)

[0094] Experimental method: with carbodiimide as a reaction coupling agent, synthesis of chitosan tranexamic acid polymer with amphiphilic. Chitosan is dissolved in water, tranexamic acid, EDC (activated carboxyl of tranexamic acid) is dissolved in methanol-acetic acid homogeneous reaction solvent, 60 ℃ continuous magnetic stirring reflux 8 h, using the molecular weight of 10000 dialysis bag dialysis 48 h, freeze drying.

[0095] Experimental results: chitosan-tranexamic acid copolymer formed amide bond grafting on the chain end of chitosan molecules, through non-covalent bond assembly and form ordered nanostructure, can form nano barrier in vivo to help the repair of surrounding tissue, but in water, easy to dissolve, poor water absorption, not conducive to wound repair and better degradation in vivo, see Figure 1 .

[0096] 1.1.2 chitosan / polyethylene glycol / tranexamic acid (CS / PEG / TXA)

[0097] Precise amount of CS, TXA, PEG were placed in beaker (m(CS):m(TXA):m(PEG)=1:1:5), each added equal amount of PBS buffer solution, mixed with equal volume, room temperature for 5 min, observe the state. TXA, PEG dissolved in PBS buffer, CS in PBS buffer, slightly soluble, mixed with each other, white turbid liquid, almost no viscosity, can not be used for hemostasis, see Figure 2 .

[0098] 1.1.3 chitosan / polyethylene glycol / sodium alginate (CS / PVA / SA)

[0099] A certain amount of SA, PVA were placed in beaker, 5% PVA solution, 2% SA solution were prepared, each two parts, mixed, one part added hemostatic powder synthesized in scheme three, the other part added equal amount of CS, mixed, -18 ℃ cooling 12 h, room temperature thawing 6 h, repeated three times; after taking out, immersed in 2% calcium chloride for 4 h, washed with purified water for 3-5 times. Pre-freezing 48 h at-20 ℃, freeze-drying, 4 ℃ constant temperature preservation, two groups of comparative experimental results: the formation of smaller pore size, more soft texture of sponge-like nanobiomaterials, other related indicators need further investigation, see Figure 3 .

[0100] 1.1.4 chitosan / tranexamic acid / polylactic acid-glycolic acid copolymer (CS / TXA / PLGA)

[0101] Precisely take 0.1 g of the prescription amount of PLGA, dissolve in 8 mL of DMSO (anhydrous grade) under nitrogen protection, add 0.021 g of DCC and 0.012 g of NHS under nitrogen protection, 40°C, 400 rpm magnetic stirring reaction for 10-15 h, then add 1 g of CS and 2 mL of DMSO (anhydrous grade) solution, continue to react under nitrogen for 8 h, and then take 0.27 g of TXA and add it into a methanol-acetic acid mixed solution, reflux at 60°C for 1 h; mix the reaction solutions obtained in the two steps, continue to reflux at 60°C for 8 h to obtain a product solution. Use a dialysis bag with a molecular weight cutoff of 10000 to dialyze for 48 h, pre-freeze at -20°C for 48 h, and freeze-dry for storage.

[0102] The generated reaction solution is a yellow homogeneous transparent liquid, slightly sticky, and after freeze-drying it is a yellow sponge-like nanomaterial, slightly soluble in water, with good stability, appearing as a transparent gel in water, with good water absorption and speed, and significantly improved hemostatic effect, and good adhesion, which is the final determined hemostatic material, see Figure 4 .

[0103] 1.2 Material ratio screening (chitosan / tranexamic acid / polylactic acid-glycolic acid copolymer)

[0104] 1.2.1 Chitosan:tranexamic acid molar ratio

[0105] (1) m(TXA):m(CS)=1:1.85

[0106] (2) m(TXA):m(CS)=1:3.7

[0107] (3) m(TXA):m(CS)=1:7.41

[0108] Table 1. TXA-CS (1:1.85)

[0109]

[0110] Table 2. TXA-CS (1:3.7)

[0111]

[0112] Table 3. TXA-CS (1:7.41)

[0113]

[0114] Precisely take 1 g of chitosan dissolved in water, add 30 mL of pure water to make a suspension, preheat in a 60°C water bath for standby. Then precisely take the prescribed amount of tranexamic acid (m(TXA): m(CS) = 1:1.85-7.41) and 1.22 g of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) dissolved in a mixed solution of 5 mL of methanol and 8 mL of acetic acid, placed in a 60°C water bath, refluxed at 400 rpm for 1 h. The solution is isothermally added to the chitosan suspension using a rubber dropper, continuously stirred at 60°C, and refluxed for 8 h. After the reaction is completed, the reaction solution is transferred to a dialysis bag with a molecular weight cut-off of 10000, and dialyzed for 48 h to obtain a colorless transparent liquid. Freeze-drying to obtain a white sponge-like sample. The generated nanostop bleeding sponge is used to determine its related performance by rat tail cutting and mouse liver hemostasis experiment, and the comprehensive ratio 2, i.e. m(TXA): m(CS) = 1:3.7, is the best, see Figure 5 、 Figure 6 .

[0115] 1.2.2 Chitosan: Polylactic acid-glycolic acid copolymer ratio

[0116] (1) m(PLGA) : m(CS) = 1:5

[0117] (2) m(PLGA) : m(CS) = 1:10

[0118] Precisely take the prescribed amount of 0.1 g of PLGA, dissolve it in 8 mL of DMSO (anhydrous grade) under nitrogen protection, add 0.021 g of DCC and 0.012 g of NHS, and react at 40°C under nitrogen protection with magnetic stirring at 400 rpm for 10-15 h, then add CS (1:5 or 1:10) and 2 mL of DMSO (anhydrous grade) solution, continue to react under nitrogen protection for 8 h, and then add m(TXA): m(CS) = 1:3.7) to a mixed solution of methanol-acetic acid (1:1.6) and reflux at 60°C for 1 h; mix the reaction solutions obtained in the two steps, continue to reflux at 60°C for 8 h to obtain a product solution. Use a dialysis bag with a molecular weight cut-off of 10000 to dialyze for 48 h, pre-freeze at -20°C for 48 h, and freeze-dry for storage.

[0119] The generated reaction solution is a yellow, uniform and transparent liquid with slight stickiness, and after freeze-drying, it is a yellow sponge-like nanomaterial. Comparing the performance of the two, the (m(PLGA): m(CS) = 1:5) group dissolves in water immediately, has poor stability, and has poor water absorption, while the (m(PLGA): m(CS) = 1:10) group is stable in water and can quickly absorb a large amount of solution and swell. Therefore, the m(PLGA): m(CS) is selected as 1:10 in the subsequent experiment. See Figure 7 、 Figure 8 .

[0120] 1.2.3 Reaction solvent screening

[0121] 1.2.3.1 Dichloromethane

[0122] Precisely weigh PLGA, dissolve in appropriate amount of dichloromethane under nitrogen protection, add DCC and NHS, and react under magnetic stirring at 40°C under nitrogen protection (m(PLGA):m(DCC):m(NHS)=8.33:1.75:1). Then add a certain amount of CS and 4% dilute acetic acid solution, continue to react under nitrogen protection for 8h, weigh TXA, add appropriate amount of 4% dilute acetic acid, and reflux for 1h (n(CS):n(TXA)=1:5 or 1:10). After mixing, continue to reflux at 60°C for 8h to obtain a reaction solution. Use a dialysis bag with a molecular weight cut-off of 10000 to dialyze for 48h, and freeze-dry to obtain a material with irregular sponge-like structure and a pungent smell. Therefore, dichloromethane is not used as a reaction solvent in subsequent experiments.

[0123] 1.2.3.2 Dimethyl sulfoxide (anhydrous grade)

[0124] Precisely weigh 0.1g of PLGA, dissolve in 8mL of DMSO (anhydrous grade) under nitrogen protection, add 0.021g of DCC and 0.012g of NHS, and react under magnetic stirring at 40°C and 400rpm for 10-15h. Then add 1g of CS and 2mL of DMSO (anhydrous grade) solution, continue to react under nitrogen protection for 8h, and weigh 0.27g of TXA, add it into a mixture of methanol-acetic acid (1:1.6), and reflux at 60°C for 1h. Mix the reaction solutions obtained in the two steps, continue to reflux at 60°C for 8h to obtain a product solution. Use a dialysis bag with a molecular weight cut-off of 10000 to dialyze for 48h, pre-freeze at -20°C for 48h, and freeze-dry for storage.

[0125] The generated reaction solution is uniform, and the performance of the generated product is good. DMSO is used as a reaction solvent in subsequent experiments.

[0126] 1.2.3.3 Purified water (laboratory water)

[0127] Precisely weigh PLGA, dissolve in appropriate amount of DMSO (anhydrous grade), add DCC and NHS, and react under magnetic stirring at room temperature under nitrogen protection (m(PLGA):m(DCC):m(NHS)=8.33:1.75:1). Then add CS, continue to react under nitrogen protection for 8h, weigh TXA, add appropriate amount of distilled water, and reflux for 1h (n(CS):n(TXA)=1:5 or 1:10). After mixing, continue to reflux at 60°C for 8h, use a dialysis bag with a molecular weight cut-off of 10000 to dialyze for 48h, and freeze-dry, see Figure 9 .

[0128] The reaction generated was yellow fine particulate precipitate, insoluble in water, and had poor hemostatic effect.

[0129] 1.3 Hemostatic performance test

[0130] 1.3.1 Rat tail amputation hemostasis

[0131] The rat tail amputation model was used as the experimental object, and the surgical procedure for rat tail amputation was as follows Figure 10 The hemostatic effect of the three different grafting ratios of PLGA-CS-TXA hemostatic sponge and CS freeze-dried sponge and sterile cotton was compared, and the statistical results showed that in the experimental group, the mean hemostatic time of the three different grafting ratios of chitosan-aminomethylcyclohexane hemostatic sponge was 89.61 s, 73.88 s, and 107.52 s, respectively, and the mean hemostatic time of the positive control group was 101.55 s. The blank control group still had slow bleeding after 180 s. In the experimental group, the hemostatic time of the hemostatic sponge with grafting ratio of ratio ① and ratio ② had no significant difference, and the hemostatic time was significantly reduced compared with other groups.

[0132] As shown in Figure 11 After seven days, the wound healing degree of the rat tail was observed, and the blank control group still had inflammation, the positive control group had no significant inflammation but the wound healing degree was not complete. The experimental groups had no inflammation and the wound was scabbed, indicating that the PLGA-CS-TXA hemostatic sponge was beneficial to protect the wound, avoid damage to the newly formed tissue, effectively reduce the wound infection rate, and accelerate wound healing.

[0133] The data recorded and measured are shown in Table 4. The tail amputation model of 14 rats was successfully modeled, and the three groups of dressings could successfully stop bleeding. The hemostatic time of PLGA-CS-TXA-1 and PLGA-CS-TXA-2 was 73.14±1.84 s and 86.58±2.24 s, respectively, and the hemostatic time of the positive control group CS was 94.82±1.73 s. The blank experimental gauze group still had continuous bleeding after 180 s; the blood absorption amount of PLGA-CS-TXA-1, PLGA-CS-TXA-2, and CS group was 318.10±25.13, 386±201.45, and 161±120.02 mg, respectively; thus, it can be shown that the hemostatic performance of the chemically modified PLGA-CS-TXA-1 is the best, and it has strong blood absorption capacity and good adhesion to the wound.

[0134] One week after the operation, the tail healing phenomenon of the rats was observed, and after comparing the four groups, it can be seen that the tails of the rats in the PLGA-CS-TXA-1 and PLGA-CS-TXA-2 groups have scabbed, and there is no pressing bleeding phenomenon, no inflammation and allergic reaction, and the wound healing effect is good. It can also be seen from the side that the PLGA-CS-TXA composite dressing has a certain antibacterial and anti-infection effect.

[0135] Table 4 Rat tail hemostasis data

[0136]

[0137] 1.3.2 Mouse liver injury hemostasis

[0138] Experimental procedure:

[0139] ① Preparation: surgical instruments were placed in medical alcohol for disinfection, three portions of gauze, physical synthetic PLGA-CS-TXA-2 hemostatic dressing, and chemically modified PLGA-CS-TXA-1 were taken (similar mass and volume), respectively, weighed and recorded. ② Grouping. The mice were randomly divided into groups, weighed, labeled, and recorded. ③ Anesthesia. The mice were anesthetized with sodium pentobarbital, and the abdomen was shaved. ④ Liver bleeding model was established. The mouse was placed on a surgical sheet, and the incision site was disinfected with medical alcohol. The abdominal cavity was carefully opened, and the liver was removed from the abdominal cavity and placed on gauze. The liver was disinfected with iodophor, and a 0.5 cm long incision was made. The blood was wiped off with gauze after 5 seconds of free bleeding. Figure 12 .

[0140] Hemostatic time was recorded. The material was applied to the wound, and the time was started. The wound hemostasis was observed, and the time required for wound hemostasis was recorded. The hemostatic product was weighed, and the records were made. The bleeding amount was calculated after the experiment. See Table 5. All 14 mouse liver injury models were successfully established. About 5 mg of PLGA-CS-TXA-1 could completely stop bleeding. The hemostatic time of PLGA-CS-TXA-1 and PLGA-CS-TXA-2 was 26.84 ± 1.84 s and 39.19 ± 17.41 s, respectively. The hemostatic time of the positive control group CS was 43.78 ± 10.36 s. The blank experiment gauze group still bled after 60 s. PLGA-CS-TXA-1 had better hemostatic effect in the liver bleeding model.

[0141] Table 5. Mouse liver injury hemostasis

[0142]

[0143]

[0144] 1.3.3 In vitro whole blood absorption

[0145] 1 mL of blood was taken and added to a plastic vial, which was preheated at 37°C for 5 min. Then 100 mg of compressed sponge was added to the vial to contact the blood, and the time from the sponge contacting the blood to absorbing all the blood was recorded.

[0146] The experimental results: through the experimental phenomenon and data can be obtained, the PLGA-CS-TXA-1 (sample) is shorter than the PLGA-CS-TXA-2 (control) in vitro whole blood absorption time, and a small amount of material can absorb all the blood, and there is no serious blood clotting phenomenon; although the average whole blood absorption time of the material is longer than that of the sterile cotton, the sterile cotton is easy to fall off the cotton flock, which can cause wound infection, and the comprehensive evaluation of the whole blood absorption effect of the PLGA-CS-TXA material is better than that of the sterile cotton. See Figure 13 Table 6. In vitro whole blood absorption experiment record

[0147] The experimental results: through the experimental phenomenon and data can be obtained, the PLGA-CS-TXA-1 (sample) is shorter than the PLGA-CS-TXA-2 (control) in vitro whole blood absorption time, and a small amount of material can absorb all the blood, and there is no serious blood clotting phenomenon; although the average whole blood absorption time of the material is longer than that of the sterile cotton, the sterile cotton is easy to fall off the cotton flock, which can cause wound infection, and the comprehensive evaluation of the whole blood absorption effect of the PLGA-CS-TXA material is better than that of the sterile cotton. See

[0148] Table 6. In vitro whole blood absorption experiment record

[0149]

[0150] 1.3.4 In vitro blood clotting time (WBCT)

[0151] 5mg of sample was added to 5mL of glass test tube, incubated in 37℃ constant temperature water bath for 3min, 100μL of anticoagulant whole blood was added and continued to incubate for 3min, then 50μL of 0.025mol / L CaCl2 solution was added in the test tube, and the test tube was tilted every 15s and observed whether the blood flowed or not under the environment of 37℃, until the blood did not flow when the test tube was tilted 90°, which was blood clotting, and the clotting time was recorded. The anticoagulant whole blood without sample was used as a blank control group, and each sample was measured 3 times. The results are shown in Table 7.

[0152] The WBCT values of PLGA-CS-TXA-1, PLGA-CS-TXA-2 and positive control CS with different synthesis methods are as follows Figure 14As shown in the figure, compared with the blank group, the WBCT values of PLGA-CS-TXA-1 and PLGA-CS-TXA-2 were significantly reduced (P<001), and the WBCT value of PLGA-CS-TXA-1 was greater than that of PLGA-CS-TXA-2 when the degree of substitution was the same. Therefore, the hemostatic ability of PLGA-CS-TXA is closely related to the length and degree of substitution of the grafted chain. When the degree of substitution of the grafted carbon chain is the same, the longer the carbon chain, the better the coagulation effect, which may be because the longer the carbon chain, the easier it is to embed into the blood cell membrane, thereby accelerating the formation of three-dimensional gel network and causing coagulation; on the contrary, the shorter the carbon chain, the more difficult it is for the carbon chain to insert into the blood cell membrane, and the worse the coagulation effect. In summary, compared with chitosan, the coagulation ability of PLGA-CS-TXA-1 is significantly improved, and the coagulation ability is stronger with the increase of alkyl chain length and degree of substitution.

[0153] Table 7. In vitro coagulation time experiment record

[0154]

[0155] 1.4 SEM characterization

[0156] As Figure 15 PLGA-CS-TXA-1 and PLGA-CS-TXA-2 composite hemostatic sponge, we can make this gel sponge according to different wound size to make corresponding size and shape. PLGA-CS-TXA composite hemostatic sponge presents a yellowish white color, the surface is complete and regular. The SEM images of PLGA-CS-TXA composite hemostatic sponge at 120x, 500x and 2000x respectively can be seen that the porous structure is fine, uniform and highly connected, which lays a certain foundation for rapid liquid absorption; among them, PLGA-CS-TXA-1 is observed at 4000x to be fibrous structure, which can quickly swell to block the wound when encountering blood and enhance the coagulation performance of the material.

[0157] 1.5 Infrared spectrum structure characterization

[0158] 1000~1500cm -1 Two weak peaks of ether bond C-O and amide bond C-N stretching vibration, 1000~1250cm -1 The peak of C-N stretching vibration is strong, which shows that TXA and CS are successfully grafted and polymerized together, 1500~1750cm -1 is the stretching vibration of non-cyclic lactone group, 900-650cm -1 N-H is the outer bending deformation vibration, which shows that PLGA is successfully modified on the chain end of CS. See Figure 16 .

[0159] The peak positions of the sample and the reference standard are basically the same at each wavelength, but the height of their positions is different, with the reference standard being slightly higher, indicating that the transmittance of the two is different.

[0160] 1.6 Structural characterization by proton nuclear magnetic resonance (NMR) spectrum

[0161] The chemical structure of PLGA-CS-TXA was confirmed by proton nuclear magnetic resonance spectroscopy.

[0162] like Figure 17 As shown in the PLGA-CS-TXA proton NMR spectrum, -CH2O- may be present at 5.1 ppm, -OH may be present at 4.3 ppm, -NH2 may be present at 2.6 ppm, -CH2NR2 may be present at 2.2 ppm, and -CH2-CH may be present in the range of 1.2–1.4 ppm. 2- -CH3 may be present at 1.0 ppm.

[0163] A doublet was observed at a chemical shift of 1.58 ppm, indicating the presence of a methylene group with an amide bond, and a sharp peak was observed at 1.24 ppm, indicating the presence of a methyl group.

[0164] 1.7 Biodegradation Experiment in a 37℃ Constant Temperature Chamber

[0165] Take a piece of the synthesized hemostatic sponge, cut it twice to divide it into four relatively equal pieces. Each piece is precisely weighed, recorded, and labeled. The pieces are placed in four transparent plastic petri dishes and incubated at 37°C (similar to body temperature) for one week (7 days). Weighing and recording the results at fixed times each day, and observing the observed phenomena, see... Figure 18 , Figure 19 As shown in the line graph, after the material was placed in a constant temperature chamber at 37℃ for one week, the mass decreased at fixed times and locations every day. The PLGA-CS-TXA composite hemostatic sponge showed a faster mass change and a greater decrease in mass over the same period of time compared to the CS sponge, indicating that the material has good in vitro biodegradability.

[0166] 1.8 Evaluation of in vitro biodegradability

[0167] Under room temperature and standard atmospheric pressure conditions, accurately measure six 5 mL aliquots each of PBS buffer and physiological saline solution, and place them in separate capped glass bottles for later use, labeling them accordingly. Accurately weigh two portions each of sterile cotton, CS, TXA, PLGA, and hemostatic sponge, each 0.05 g, and take two equal portions of each. Add these 10 portions of materials to the aforementioned glass bottles, noting the labeling and timing. Observe and record the solubility of the samples at 0, 1, 2, 5, 10, and 30 min. Additionally, record the degradation of the hemostatic sponge in PBS and physiological saline for 30 days. See [link to relevant documentation]. Figures 20-25 The results are shown in Table 8.

[0168] Table 8. Experimental Records for In Vitro Biodegradability Evaluation

[0169]

[0170] 1.9 Evaluation of water absorption capacity

[0171] Composite hemostatic materials of equal volume (1.5 cm in diameter, 1 cm in height) and similar mass were pre-weighed and labeled as m1. The samples were immersed in 0.1 M PBS buffer solution at 37°C. An equal mass of gauze was used as a control group. The samples were shaken in a shaking water bath (37°C, 100 rpm) for 30 seconds, and the surface moisture was absorbed with filter paper. Immediately afterward, the samples were weighed and labeled as m2. Three replicates were prepared for each group of samples. The water absorption ratio (%) was calculated using the formula:

[0172]

[0173] Depend on Figure 26 It is evident that the self-made composite sponges have a higher water absorption capacity than gauze, especially the chemically modified CS-PLGA-TXA composite hemostatic sponge, which can absorb 75.0 ± 6.5 times its own weight in water within 60 seconds, far exceeding the 20.1 ± 2.4 times that of gauze. This is mainly due to its fluffy porous structure and the presence of unreduced hydrophilic -COOH groups. The sponge's ability to rapidly and efficiently absorb water helps to quickly concentrate blood components in a short time, increasing the contact area between blood and the material. This facilitates interfacial stimulation, allowing the material to more quickly activate platelets and clotting factors in the blood, thereby accelerating coagulation.

[0174] Meanwhile, the CS-PLGA-TXA composite hemostatic sponge with the best water absorption was added to PBS, and its state changes were observed. This composite sponge can rapidly absorb liquid and expand from its compressed state back to its original cylindrical shape within seconds, which provides a basis for the preparation of injectable composite sponges for rapid hemostasis when the wound is not visible, showing great development potential.

[0175] 1.10 Solution stability test

[0176] Under room temperature and standard atmospheric pressure conditions, accurately measure 5 mL of 0.1 M PBS buffer (12 portions) and place them in 12 capped glass bottles for later use, labeling them accordingly. Accurately weigh gauze and take 3 portions each of PLGA-CS-TXA composite hemostatic sponge (structurally modified, physically synthesized) with similar volume and mass. Add the above 12 portions of materials to the above glass bottles respectively, and take pictures to record the changes in state after 1 min and 1 h respectively.

[0177] In addition to good porous structure and compressibility, it is also important for the composite sponge to maintain integrity during use in contact with body fluids. In this study, the composite sponge was added to PBS to observe whether it could maintain good morphology.

[0178] As shown in Figure 27 , there was no obvious difference in the appearance of the composite sponges prepared by different methods, but as can be seen from the figure, after being added to PBS aqueous solution, the chemically modified composite sponge could maintain its original morphology well after 1 min or 1 h, because the presence of PLGA allowed the combination of -NH2 and -COOH in CS, and the chemical combination was more compact and firm, which improved the usability of the composite sponge and avoided the problem of sponge breaking due to the presence of water, thus making it difficult to use and clean later. Therefore, the chemical method was selected for further evaluation and research in the subsequent study.

[0179] 1.11 Porosity determination

[0180] First, the mass m of the hemostatic sponge was accurately weighed s (g); then the total mass of the volumetric flask was measured as m1(g) after filling it with anhydrous ethanol, the sample to be tested was immersed in the volumetric flask, and ultrasonic was used to expel the air in the sponge, then sufficient anhydrous ethanol was added to the volumetric flask until it was full, and the total mass was measured again as m2(g); after taking out the sponge-like material filled with anhydrous ethanol, the mass of the volumetric flask was measured as m3(g). See Figure 28 .

[0181] Each material was tested in triplicate, and the porosity (P) was calculated according to the following formula:

[0182]

[0183]

[0184] p e = 0.7894 g / mL (density of anhydrous ethanol at 20°C).

[0185] The measured data and records are shown in Table 9.

[0186] Table 9. Experimental data records for porosity determination

[0187]

[0188] Experimental results: The porosity of the hemostatic material is an important factor in determining the material's morphology, structure, and mechanical properties. High porosity can increase the specific surface area of the material, thus positively affecting the water absorption rate of the material. The porosity of the hemostatic material was characterized by the volumetric flask method, and the results are shown in the figure.

[0189] From the table, it can be obtained that the porosity of CS-PLGA-TXA is 83±2.221%, and the porosity of the material can be effectively improved by grafting modification of CS, because stable ester bonds are generated in the modified CS, which can prevent the material from dissolving rapidly in water, and the crosslinking rate of the material may be improved during the preparation of the sponge structure material due to the crosslinking and modification of the material itself; for the same reason, the carboxyl groups on the TXA after compounding will also increase the crosslinking rate of the material by reacting with the amino groups on the CS, so grafting modification of CS or modification with TXA will increase the porosity of the sponge.

[0190] 1.12 Cytotoxicity test

[0191] Experimental principle: CCK-8 method was used to detect the cytotoxicity of PLGA-CS-TXA hemostatic dressing (physical synthesis, chemical structure modification) on 3T3 mouse embryonic fibroblasts. The specific method was in accordance with the requirements of the People's Republic of China national standard GB / T16886.5-2003 "Medical devices-biological evaluation-part 5: in vitro cytotoxicity tests".

[0192] Experimental method:

[0193] (1) Preparation of material extract

[0194] After the material was placed under the ultraviolet lamp for 12 h, the sample was soaked in 10 mL of DMEM medium containing 10% serum, and was placed in a 37°C carbon dioxide incubator for 24 h. The extract was filtered through a microporous filter, and the filtrate was collected for use.

[0195] (2) Toxicity test of material

[0196] The 3T3 cells in logarithmic growth phase were digested with 1 mL of 0.2% trypsin for 3 min, and then an equal amount of DMEM medium was added to terminate the digestion. After centrifugation at 1000 rpm for 4 min, the supernatant was discarded. Fresh medium was added and the cells were gently blown to suspend evenly in the medium. The concentration of the cells was adjusted to about 5.0×10 4 The cells were inoculated in 96-well plates at 100 μL per well, and a blank group was set around the well plate. PBS solution was added to the negative control group, and only culture medium was added without cells. After incubation at 37°C in a 5% CO2 cell incubator for 24 h, the original culture medium was discarded, and 100 μL of extract was added for further incubation for 24 h and 48 h. Then the original culture medium was aspirated, and fresh culture medium was added at 100 μL per well, and 10 μL of CCK-8 reagent was added. The plates were returned to the incubator for another 4 h, and the absorbance was measured at 490 nm using a microplate reader. The relative growth rate (RGR) was calculated according to the following formula:

[0197] RGR=OD(实验组) / OD (对照组) x 100%

[0198] The evaluation and measured data are shown in Table 10 and Table 11.

[0199] Table 10. Cell toxicity grade evaluation

[0200]

[0201] Table 11. Cell toxicity experimental data record

[0202]

[0203]

[0204] Experimental results: RCR is greater than 100%, the material has no cytotoxicity.

[0205] 1.13 Water contact angle measurement

[0206] The contact angle refers to the angle formed by the air-liquid interface and the solid-liquid interface at the three-phase junction point on the solid surface when a liquid droplet is dropped on a solid horizontal plane. The contact angle measuring instrument is mainly used for measuring the contact angle of liquid on solid, i.e. the wettability of liquid on solid. The instrument can measure the contact angle of various liquids on various materials. Equal amounts of materials and control samples with relatively flat surfaces are taken and measured respectively. See Figure 29 .

[0207] The smaller the contact angle, the better the wettability of water on the material, which reflects that the product has better water absorption.

[0208] Although the present application has been described in detail in the foregoing general description, specific embodiments and experiments, some modifications or improvements can be made on the basis of the present application, which is obvious to those skilled in the art or technicians. Therefore, these modifications or improvements made on the basis of not deviating from the spirit of the present application, all belong to the scope of protection required by the present application.

Claims

1. A method of preparing a biodegradable polymeric nanostop hemostatic dressing, characterized by, The preparation method comprises the following steps: (1) 0.1 g of PLGA is weighed and dissolved in 8 ml of anhydrous DMSO under nitrogen protection; 0.021 g of DCC and 0.012 g of NHS are added, and the reaction is carried out under nitrogen protection at a temperature of 40 DEG C and a rotation speed of 400 rpm for 10-15 h by magnetic stirring; then 1 g of CS and 2 ml of anhydrous DMSO solution are added, and the reaction is continued under nitrogen protection for 8 h to obtain a first reaction solution; The mass ratio of CS to PLGA is PLGA:CS = 1:10; (2) 0.27 g of TXA is weighed and added into a methanol-acetic acid mixed solution, and the reaction is carried out under reflux for 1 h to obtain a second reaction solution; The volume ratio of the methanol-acetic acid mixed solution is methanol:acetic acid = 1:1.6; the reflux temperature is 60 DEG C; The mass ratio of CS to TXA is TXA:CS = 1:3.7; (3) the reaction solutions obtained in steps (1) and (2) are mixed, and the reaction is continued under reflux for 8 h at a reflux temperature of 60 DEG C to obtain a product solution; (4) the product solution obtained in step (3) is dialyzed by using a dialysis bag for 48 h, pre-frozen at -20 DEG C for 48 h, and freeze-dried and stored; the molecular weight cut-off of the dialysis bag is 10000.

2. Use of the biodegradable polymer nanostop bleeding dressing prepared by the preparation method of claim 1 in the preparation of a hemostatic material.

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