An extracellular polysaccharide riclin / graphene oxide composite hemostatic sponge and its application

Abstract

The invention provides an extracellular polysaccharide riclin / graphene oxide composite hemostatic sponge and an application thereof. The extracellular polysaccharide riclin / graphene oxide composite hemostatic sponge is prepared by aldehyde-forming the extracellular polysaccharide riclin, cross-linking with graphene oxide, and then freeze-drying. The invention provides an extracellular polysaccharide riclin / graphene oxide composite hemostatic sponge and an application thereof. The hemostatic sponge has strong swelling property, good hemostatic effect, rapid absorption, and low irritation to the body.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical materials, and in particular relates to an extracellular polysaccharide riclin / graphene oxide composite hemostatic sponge and application thereof. Background Art

[0002] In medical practice, hemostatic sponges are widely used as hemostatic materials in surgery and surgical operations. Traditional hemostatic sponges are usually made of natural cellulose or other synthetic materials, and their purpose is to promote the hemostasis process by increasing the coagulation ability of blood. However, traditional hemostatic sponges have some shortcomings, such as easy to cause red blood cell rupture, poor biocompatibility, easy to induce allergic reactions, and too single function, which limits their application in specific clinical scenarios.

[0003] In order to overcome the limitations of traditional hemostatic sponges, a series of important progress has been made in the research and development of multifunctional hemostatic sponges in recent years. By introducing new multifunctional materials, improving preparation processes and applying biotechnology, the new generation of multifunctional hemostatic sponges has achieved significant improvements in hemostatic effect, biocompatibility and material absorption performance.

[0004] Previous studies have reported on the extracellular polysaccharide riclin and its preparation method, and pointed out that the extracellular polysaccharide riclin has anti-tumor activity and anti-inflammatory activity (Y. Yang, X. Sun, Y. Zhao, et al., Anti tumor activity and immunogenicity of a succinoglycan riclin, Carbohydr. Polym. 2021 Mar 1; 255: 117370.; R. Cheng, L. Wang, J. Li., et al., In vitro and in vivo antiinflammatory activity of a succinoglycan Riclin from Agrobacterium sp. ZCC3656, J. Appl. Microbiol. 2019 Dec; 127 (6): 1716-1726.), but there are no reports on the use of extracellular polysaccharide riclin as a hemostatic material. This is partly because the hemostatic effect of the extracellular polysaccharide riclin has not yet been discovered, and partly because of the high hydrophilicity of the extracellular polysaccharide riclin. Graphene has a two-dimensional sheet structure and its surface is easy to functionalize, which makes it possible to realize multifunctional composites, which is conducive to the synergistic hemostasis effect. However, the poor biocompatibility and loose structure of pure graphene limit its application in hemostasis. Therefore, if the extracellular polysaccharide riclin can be composited with graphene to prepare a hemostatic sponge, it will be expected to obtain an efficient and multifunctional hemostatic material. Summary of the invention

[0005] Based on the above technical problems, the present invention provides an extracellular polysaccharide riclin / graphene oxide composite hemostatic sponge and its application. The hemostatic sponge has strong swelling ability, good hemostatic effect, rapid absorption and low irritation to the body.

[0006] The invention provides an extracellular polysaccharide riclin / graphene oxide composite hemostatic sponge, which is prepared by aldehyde-forming the extracellular polysaccharide riclin, cross-linking with graphene oxide, and then freeze-drying.

[0007] In the present invention, the extracellular polysaccharide riclin is selected as the substrate and is compounded with graphene oxide to form the hemostatic sponge. On the one hand, the extracellular polysaccharide riclin is an optimized modified polysaccharide-based material, and after being compounded with graphene oxide, it can further enhance the water absorption and stability of the hemostatic sponge; on the other hand, it can also reduce the toxicity of graphene oxide to achieve a non-toxic effect, and when it contacts blood, it can quickly coagulate blood in a short time, thereby more effectively achieving a hemostatic effect.

[0008] Preferably, the exopolysaccharide riclin is produced by fermentation of Agrobacterium ZCC3656;

[0009] Preferably, the deposit number of the Agrobacterium ZCC3656 is CCTCCNO:M2018797.

[0010] In the present invention, the structural formula of the extracellular polysaccharide riclin is as follows:

[0011]

[0012] Wherein, R is -OCCH2CH2COOH or -H, and n is an integer greater than 0.

[0013] In the present invention, the extracellular polysaccharide riclin is specifically prepared by sequentially activating Agrobacterium sp. ZCC3656 (with the deposit number of CCTCC NO: M 2018797), subjecting the crude riclin polysaccharide to seed culture, fermentation culture and alcohol precipitation to obtain a crude riclin polysaccharide, and then removing the protein in the crude riclin polysaccharide by an alkali purification method to obtain the extracellular polysaccharide riclin with R being -OCCH2CH2COOH, or purifying and removing the protein in the crude riclin polysaccharide by a Sevag method, and simultaneously removing the succinyl group in the riclin polysaccharide to obtain the extracellular polysaccharide riclin with R being -H.

[0014] In the present invention, the extracellular polysaccharide riclin molecular chain contains a large number of hydroxyl groups and carboxyl groups, has good biocompatibility and biodegradability, and has a molecular weight of about 2.0×106 -3.0×10 6 Da, average molecular weight is 2.5×10 6 Da.

[0015] Preferably, the “aldehyde-forming the exopolysaccharide riclin” specifically comprises: dissolving the exopolysaccharide riclin in water, adding an oxidant to carry out an oxidation reaction, and obtaining the aldehyde-formyl exopolysaccharide riclin;

[0016] Preferably, the oxidant is at least one of periodate, hypochlorite or hydrogen peroxide;

[0017] Preferably, the mass ratio of the oxidant to the extracellular polysaccharide riclin is 0.2-0.5:1;

[0018] Preferably, the oxidation reaction temperature is 15-30°C, the time is 2-12h, and the pH is 7-9.

[0019] Preferably, the aldehyde rate of the aldehyde-ylated exopolysaccharide riclin is 25-50%.

[0020] In the present invention, the hydrochloric acid hydroxylamine titration method is used to determine the degree of product formaldehyde.

[0021] Preferably, the “cross-linking with graphene oxide” specifically comprises: stirring and mixing an aqueous solution of aldehyde-modified extracellular polysaccharide riclin with an aqueous solution of graphene oxide to obtain an extracellular polysaccharide riclin / graphene oxide composite hydrogel;

[0022] Preferably, the concentration of the aldehyded exopolysaccharide riclin aqueous solution is 5-30 mg / mL, and the concentration of the graphene oxide aqueous solution is 1-20 mg / mL;

[0023] Preferably, the weight ratio of graphene oxide to extracellular polysaccharide riclin is 1:1-15;

[0024] Preferably, the stirring and mixing reaction is carried out at a temperature of 4-25°C and for a time of 4-8 hours;

[0025] Preferably, the stirring rate is 1000-3000 rpm.

[0026] Preferably, the graphene oxide aqueous solution is prepared by adding graphene oxide to water and uniformly dispersing it by ultrasonication;

[0027] Preferably, the temperature of the ultrasonic dispersion is 0-10°C, the time is 0.5-1h, and the ultrasonic amplitude is 10-30%.

[0028] In the present invention, a stable monolayer graphene oxide solution can be prepared by selecting a suitable preparation method and process parameters, and the monolayer graphene oxide solution can be better blended with the aldehyde-modified extracellular polysaccharide riclin, thereby preparing a hemostatic sponge with excellent effect.

[0029] Preferably, the “freeze drying” specifically includes: dialyzing the extracellular polysaccharide riclin / graphene oxide composite hydrogel and then freeze drying it to obtain the extracellular polysaccharide riclin / graphene oxide composite hemostatic sponge;

[0030] Preferably, the dialysis bag used for dialysis has a molecular weight cutoff of 500-2000Da, the dialysis time is 0.5-2 days, and the water change frequency is 3-8h / time;

[0031] Preferably, the freeze-drying temperature is -(70-80)°C and the time is 24-48h.

[0032] The present invention also provides a method for preparing the above-mentioned extracellular polysaccharide riclin / graphene oxide composite hemostatic sponge, comprising:

[0033] (1) dissolving the exopolysaccharide riclin in water, adding an oxidant to carry out an oxidation reaction, and obtaining the aldehyde-modified exopolysaccharide riclin;

[0034] (2) stirring and mixing the aldehyde-modified exopolysaccharide riclin aqueous solution and the graphene oxide aqueous solution to react, thereby obtaining an exopolysaccharide riclin / graphene oxide composite hydrogel;

[0035] (3) The extracellular polysaccharide riclin / graphene oxide composite hydrogel is dialyzed and then freeze-dried to obtain the extracellular polysaccharide riclin / graphene oxide composite hemostatic sponge.

[0036] In the present invention, aldehyde-modified extracellular polysaccharide riclin and graphene oxide cross-linked composite are used as hemostatic sponge. The obtained hemostatic sponge overcomes the defects of poor hemostatic ability, low safety and weak antibacterial ability of traditional hemostatic sponges, and can reduce the inflammatory response of wounds and reduce irritation to skin. It also has good biocompatibility and superior hemostatic and coagulation ability, providing a safer and more superior choice for the treatment of acute massive bleeding.

[0037] The present invention also proposes an application of the extracellular polysaccharide riclin / graphene oxide composite hemostatic sponge in tissue trauma hemostasis or acute wound heavy bleeding products.

[0038] In the present invention, the inventors found that when the extracellular polysaccharide riclin / graphene oxide composite is used as a hemostatic sponge, it has a unique three-dimensional mesh structure, which gives it a large surface area and a high ability to absorb liquid. When the hemostatic sponge is applied to the bleeding site, it can quickly absorb a large amount of blood, which helps to quickly reduce the amount of bleeding and reduce the risk of blood loss in patients. In addition, combined with the unique rapid recoverability of the sponge, it can quickly fill the wound in a very small volume and achieve efficient hemostasis. Because the hemostatic sponge contains graphene oxide, this polysaccharide composite sponge can interact with the blood at the bleeding site, promote binding with coagulation factors, accelerate the blood coagulation process, form thrombi, thereby helping to seal the wound and prevent further bleeding. This makes the hemostasis process simpler and faster, and can be effectively used even in emergency situations such as first aid. In addition, since its shape and size can be adjusted as needed, they can be used for wounds of different sizes and positions. And it has biocompatibility, memory recoverability, and no adverse effects on the environment; in general, the structural advantages of the obtained hemostatic sponge make it a commonly used treatment tool in the medical field. It can quickly stop bleeding, promote coagulation, prevent infection, and play a key role in emergency and surgical scenarios, helping to improve patients' survival rate and recovery effect. However, despite these advantages of hemostatic sponges, professional medical assistance is still needed for severe or uncontrollable bleeding.

[0039] Compared with the prior art, the present invention has the following technical effects:

[0040] (1) The present invention provides an extracellular polysaccharide riclin / graphene oxide composite hemostatic sponge. The extracellular polysaccharide riclin containing adjacent hydroxyl groups is subjected to an oxidation reaction, and the extracellular polysaccharide riclin containing adjacent hydroxyl groups is oxidized to form adjacent free aldehyde groups. The free aldehyde groups reduce the hydroxyl groups in the extracellular polysaccharide riclin, thereby reducing the water solubility of the extracellular polysaccharide riclin, which is helpful for the subsequent formation of a hemostatic sponge. At the same time, it can also form a cross-linked mixture with a graphene oxide solution to obtain an extracellular polysaccharide riclin / graphene oxide composite hydrogel, and then the water molecules are sublimated and removed by vacuum freeze drying, and finally a hemostatic sponge with a loose porous honeycomb-like three-dimensional physical structure is formed. On the one hand, the present invention abandons a large number of toxic chemical cross-linking agents and only uses polysaccharides and graphene oxide, and finally obtains a cheap and easily available hemostatic sponge; on the other hand, the whole process is easy to produce on a large scale, and the prepared polysaccharide-based composite graphene oxide hemostatic sponge is inexpensive.

[0041] (2) The present invention provides an extracellular polysaccharide riclin / graphene oxide composite hemostatic sponge, which has good mechanical properties and liquid adsorption properties. While giving the hemostatic sponge product rapid shape recovery performance and high-efficiency hemostatic performance, it can also adapt to non-compressible wounds and can be applied to special wounds in emergency situations, effectively solving the shortcomings of existing hemostatic sponge products in these aspects.

[0042] (3) The present invention provides an extracellular polysaccharide riclin / graphene oxide composite hemostatic sponge, which is modified to make the polysaccharide riclin have rich aldehyde groups, and is cross-linked with graphene oxide in a specific ratio to prevent graphene oxide from escaping, thereby improving the toxicity problem of high-dose and high-concentration graphene oxide in the past, having a strong adhesion ability to red blood cells, platelets and other blood cells, and forming a coagulation protein network, having excellent coagulation performance, and animal hemostasis experiments show that the extracellular polysaccharide riclin / graphene oxide composite hemostatic sponge can quickly and effectively stop bleeding in hemostasis models of different parts of large animals, and the hemostatic effect is significantly better than commercial medical hemostatic gauze. In vitro blood compatibility experiments show that the present invention can also not destroy blood cells during the use time, and has good biocompatibility. Therefore, the hemostatic sponge of the present invention is an ideal actual clinical wound first aid hemostatic material, which can play a role in visceral bleeding and arterial bleeding.

[0043] (4) The present invention provides an extracellular polysaccharide riclin / graphene oxide composite hemostatic sponge, which is unique in that the hemostatic sponge is mainly composed of aldehyde-treated extracellular polysaccharide riclin and graphene oxide, which have complementary advantages and have strong market application prospects. In addition, the preparation process is simple and fast, with significant cost-effectiveness, and is suitable for use in emergency medical situations. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is a scanning electron micrograph of the surface pore structure of the extracellular polysaccharide riclin / graphene oxide composite hemostatic sponge described in Example 1;

[0045] Figure 2 This is a scanning electron microscope image of the extracellular polysaccharide riclin / graphene oxide composite hemostatic sponge described in Example 1 after adhering to platelets, red blood cells and whole blood;

[0046] Figure 3 This is a compression recovery state diagram of the extracellular polysaccharide riclin / graphene oxide composite hemostatic sponge described in Example 1;

[0047] Figure 4 This is a graph showing the hemolysis rate of the extracellular polysaccharide riclin / graphene oxide composite hemostatic sponge described in Example 1;

[0048] Figure 5 This is a diagram showing the application of the extracellular polysaccharide riclin / graphene oxide composite hemostatic sponge described in Example 1 in a pig liver perforation bleeding model and a pig femoral artery rupture bleeding model;

[0049] Figure 6 This is a test graph of the absorption performance of the extracellular polysaccharide riclin / graphene oxide composite hemostatic sponge described in Example 1. DETAILED DESCRIPTION

[0050] Hereinafter, the present invention will describe the technical solution in detail through specific embodiments, but it should be clearly stated that these embodiments are for illustration only and are not to be construed as limiting the scope of the present invention.

[0051] The sodium periodate and ethylene glycol used below are from Shanghai, China. Reagent Co., Ltd.; graphene oxide comes from Carbon Leakage Technology (Shenzhen) Co., Ltd.; all the water used below is deionized water.

[0052] Example 1

[0053] This embodiment provides an extracellular polysaccharide riclin / graphene oxide composite hemostatic sponge, and the preparation method thereof includes:

[0054] (1) A single colony of Agrobacterium sp. ZCC3656 (deposit number: CCTCCNO: M2018797) was inoculated into a seed medium (LB liquid medium) for seed culture, and then inoculated into a fermentation medium (M9 medium, carbon source: 3% sucrose) at a ratio of 2% after 16 hours, and fermented at 30° C. for 2 days, and 3 volumes of pre-cooled 95% (v / v) ethanol aqueous solution were added to the obtained fermentation broth for alcohol precipitation, and the precipitate was collected by centrifugation (6000 g, 5 min), and dried at 40° C. to obtain a crude exopolysaccharide riclin;

[0055] The crude exopolysaccharide riclin was added into water to prepare an aqueous solution with a concentration of 10 mg / mL, and a NaOH aqueous solution was added to adjust the pH to 9.0, and the solution was sterilized under high pressure at 105°C for 20 min, and centrifuged (10000 g, 10 min) to obtain a supernatant, and 3 times the volume of pre-cooled 95% (v / v) ethanol aqueous solution was added for alcohol precipitation, and the precipitate was collected by centrifugation (6000 g, 10 min) to obtain an exopolysaccharide riclin (shown in the above structural formula, and R is -H);

[0056] (2) The above-mentioned extracellular polysaccharide riclin was added to water to prepare a polysaccharide solution with a concentration of 2% (w / v), and a 5wt% sodium periodate solution was added according to a mass ratio of extracellular polysaccharide riclin to sodium periodate of 1:0.33. After stirring and reacting for 24 hours at room temperature in the dark, ethylene glycol was added to terminate the reaction. The reaction solution was dialyzed in deionized water using a dialysis bag (Mw=1000Da) for 24 hours, and the water was changed every 4 hours to obtain a 20 mg / mL aldehyde-formyl extracellular polysaccharide riclin aqueous solution;

[0057] (3) adding graphene oxide (represented by GO) to water, and ultrasonically dispersing it at 5°C with an ultrasonic amplitude of 20% for 45 minutes to obtain an aqueous solution of graphene oxide with a concentration of 8 mg / mL; adding the aqueous solution of graphene oxide to the aqueous solution of aldehyde-modified exopolysaccharide riclin according to (the mass ratio of graphene oxide to exopolysaccharide riclin is 1:8), stirring the reaction at room temperature at a stirring rate of 2000 rpm for 6 hours, and obtaining an exopolysaccharide riclin / graphene oxide composite hydrogel;

[0058] (4) The extracellular polysaccharide riclin / graphene oxide composite hydrogel was dialyzed in deionized water using a dialysis bag (Mw=1000Da) for 24 hours, the water was changed every 4 hours, and then poured into the corresponding mold, placed in a freeze dryer for freeze drying, first cooled to -70°C, and then reduced to 0 kPa. At this temperature and pressure, freeze-dried for 48 hours, washed with ethanol for 10 hours, and dried to obtain the extracellular polysaccharide riclin / graphene oxide composite hemostatic sponge (expressed as ARGS).

[0059] Comparative Example 1

[0060] This comparative example proposes a hemostatic sponge, the preparation method of which refers to the method described in Example 1, except that step (2) is omitted and in step (3), the aldehyde-modified extracellular polysaccharide riclin aqueous solution is replaced with an extracellular polysaccharide riclin aqueous solution with a concentration of 20 mg / mL, thereby preparing the hemostatic sponge.

[0061] The hemostatic sponge is easily soluble in water and cannot exert a hemostatic effect.

[0062] Comparative Example 2

[0063] This comparative example proposes a hemostatic sponge, the preparation method of which refers to the method described in Example 1, except that step (2) is omitted, and in step (3), a 20 mg / mL extracellular polysaccharide riclin aqueous solution is used instead of the aldehyde-containing extracellular polysaccharide riclin aqueous solution, and a 20 wt % concentration of glutaraldehyde solution is added (the mass ratio of glutaraldehyde to extracellular polysaccharide riclin is 0.2:1).

[0064] The hemostatic sponge is also easily soluble in water and cannot exert a hemostatic effect.

[0065] Comparative Example 3

[0066] This comparative example proposes a hemostatic sponge, the preparation method of which refers to the method described in Example 1, except that step (3) is omitted and in step (4), a 20 mg / mL aldehyde-modified extracellular polysaccharide riclin aqueous solution is used instead of the extracellular polysaccharide riclin composite graphene oxide hydrogel, thereby preparing the hemostatic sponge (represented by AR).

[0067] Internal structure test of the extracellular polysaccharide riclin / graphene oxide composite hemostatic sponge described in Example 1

[0068] The extracellular polysaccharide riclin / graphene oxide composite hemostatic sponge described in Example 1 was subjected to gold spraying and then scanned under a scanning electron microscope. The results are as follows: Figure 1 shown. Figure 1 This is a scanning electron microscope image of the pore structure of the extracellular polysaccharide riclin / graphene oxide composite hemostatic sponge described in Example 1. Figure 1 It can be seen that the hemostatic sponge described in Example 1 has a mutually interpenetrating pore structure, which enables the hemostatic sponge to have a large surface area, thereby increasing its ability to absorb blood. When the hemostatic sponge is placed on a bleeding site, the pores can quickly absorb the surrounding blood, thereby quickly forming a clot and helping to promote the aggregation of platelets. Platelets are an important component of the blood coagulation process. They aggregate at the wound and release platelet-derived growth factor to promote blood coagulation.

[0069] Aggregation morphology test of the extracellular polysaccharide riclin / graphene oxide composite hemostatic sponge adhering to platelets, red blood cells and whole blood described in Example 1

[0070] Since blood components are mainly composed of platelets (PLTs), red blood cells (RBCs) and coagulation factors, it is very important to explore the interaction between blood components and the extracellular polysaccharide riclin / graphene oxide composite hemostatic sponge described in Example 1. The aggregation morphology of the extracellular polysaccharide riclin / graphene oxide composite hemostatic sponge described in Example 1 after adhesion to red blood cells (RBCs), platelets (PLTS) and whole blood (WB) was scanned under a scanning electron microscope. The results are as follows Figure 2 shown. Figure 2 This is a scanning electron micrograph of the extracellular polysaccharide riclin / graphene oxide composite hemostatic sponge described in Example 1 after adhering to platelets, red blood cells and whole blood, referring to Figure 2It can be seen that a large number of platelets (PLTS), red blood cells (RBCs) and whole blood (WB) adhere to and aggregate on the hemostatic sponge described in Example 1, which shows that the hemostatic sponge has strong adsorption, aggregation and adhesion effects on PLTS, RBCs and WB.

[0071] Rapid recoverability test of the extracellular polysaccharide riclin / graphene oxide composite hemostatic sponge described in Example 1

[0072] The extracellular polysaccharide riclin / graphene oxide composite hemostatic sponge described in Example 1 was made into a cylindrical shape with a length*height*width of 1cm*1cm*1cm, and the initial length was recorded as L1, and the deformation was recorded as L2 when it was two-thirds deformed. When the water was completely squeezed out of the absorbed hemostatic sponge, the compression gauge length was recorded as L3. When the load was maintained at the lowest end of the hemostatic sponge, the hand began to be raised. When the hemostatic sponge was deformed by two-thirds again, the compression gauge length was recorded as L4. When the pressure on the hemostatic sponge was completely released, the recovery gauge length was measured and recorded as L5. The sizes of L1-L5 were tested respectively, and the results are as follows: Figure 3 shown. Figure 3 This is the compression recovery state diagram of the extracellular polysaccharide riclin / graphene oxide composite hemostatic sponge described in Example 1, refer to Figure 3 It can be seen that the lengths of L1 and L5 are equal, and the lengths of L2 and L4 are equal, which indicates that the hemostatic sponge described in Example 1 has significant shape memory and rapid recoverability.

[0073] Hemolysis rate test of the extracellular polysaccharide riclin / graphene oxide composite hemostatic sponge described in Example 1

[0074] Fresh anticoagulated blood (5 mL) from healthy volunteers was added to 10 mL of phosphate buffered saline (PBS, pH 7.2-7.4) and centrifuged at 500 g for 10 minutes to separate red blood cells from serum. The purification step was repeated four times, and the collected red blood cells were diluted to 50 mL in PBS. 0.2 mL of diluted red blood cells were added to 0.8 mL of a suspension solution containing test samples (including ARGS, GO, AR) ranging from 7.8 to 500 μg / mL. In order to obtain a suspension solution containing test samples, the test sample powder was added to PBS and sonicated for 2 h, the OD value was (ODexperiment), and the addition of PBS was used as a negative control (ODnegative), and 2% of Triton X-100 (Triton X-100) was added as a positive control. All samples were placed in a 37 ° C shaker for 3 h. After incubation, the samples were centrifuged at 1000 g for 10 minutes. The absorbance of hemoglobin was measured at 540 nm using a microplate reader (Bio-Tek US).

[0075] The hemolysis percentage (hemolysis rate) was calculated by the following formula:

[0076]

[0077] According to the hemolysis rate of different concentrations, a hemolysis rate diagram was drawn. The results are as follows Figure 4 As shown, Figure 4 This is a graph showing the hemolysis rate of the extracellular polysaccharide riclin / graphene oxide composite hemostatic sponge described in Example 1. Figure 4 It can be seen that the extracellular polysaccharide riclin / graphene oxide composite hemostatic sponge described in Example 1 has good blood compatibility, a hemolysis rate of less than 1%, and almost no harmful effect on red blood cells.

[0078] Test of the hemostatic ability of the extracellular polysaccharide riclin / graphene oxide composite hemostatic sponge described in Example 1

[0079] (1) Animal liver perforation hemostasis test: Male healthy piglets (10-15 kg) were treated with sodium pentobarbital (3% concentration, 1 mL kg -1 ) Pigs were anesthetized by intraperitoneal injection. After complete anesthesia, an incision was made near the xiphoid process in the upper abdomen to expose the largest lobe of the liver on the filter paper. In some animals, the blood vessels were clamped with hemostatic forceps. At the same time, gauze was placed just below the liver-carrying filter paper to record the amount of bleeding out of the filter paper. At the same time, a plastic film was placed on the bottom layer to prevent the leakage of peritoneal fluid and remove the interference of non-liver bleeding. A hole was made with a puncher (diameter 20 mm) and the visceral block was taken from the perforated site until bleeding; in order to close the top of the hole, ARGS (~0.3 g) was immediately pressed for 5 seconds; the hemostasis time was determined by the blood stopping continuous oozing on the material and the blood on the filter paper no longer flowing and dripping, and the hemostasis time was recorded; after bleeding for 5-10 minutes, the material was removed from the wound and the blood loss was recorded. The hemostasis process was recorded by a camera;

[0080] (2) Animal femoral artery hemostasis test: Male healthy piglets (10-15 kg) were treated with sodium pentobarbital (3% concentration, 1 mL kg -1 ) Pigs were anesthetized by intraperitoneal injection and completely anesthetized. Hair was removed before the operation. The overlying muscle layer was incised in the inguinal area to expose the femoral artery, and the femoral artery was cut to cause arterial bleeding. After natural bleeding for 10 seconds, the blood that overflowed in the first 10 seconds was wiped with gauze. ARGS (~1g) was manually pressed on the wound. After successful hemostasis, the compression was ended. This period of pressing was the hemostasis time of the material. The hand pressure was removed and then observed, and the time was recorded to observe the wound condition; after bleeding for 5-10 minutes, the material was removed from the wound, the condition of the femoral artery rupture was observed, and the blood loss was recorded at the same time. The hemostasis process was recorded by a camera, and the rest was treated with medical hemostatic gauze (Gauze, Sinopharm Group, ~8g).

[0081] Test results such as Figure 5 As shown, Figure 5This is a diagram showing the application of the extracellular polysaccharide riclin / graphene oxide composite hemostatic sponge described in Example 1 in a pig liver perforation bleeding model and a pig femoral artery rupture bleeding model. Figure 5 It can be seen that in the pig liver perforation bleeding model, the average blood loss of liver perforation of the hemostatic sponge described in Example 1 was less than 5g at a relatively low dosage, and the non-compression hemostasis time was reduced to less than 100s; Figure 5 It can be seen that in the pig femoral artery rupture bleeding model, the average blood loss of the hemostatic sponge described in Example 1 was within 10 g, and the non-compression hemostasis time was reduced to within 100 s.

[0082] Absorption performance test of the extracellular polysaccharide riclin / graphene oxide composite hemostatic sponge described in Example 1

[0083] ARGS, Gauze, Celox (chitosan, SAM Medical, USA), Chito SAM (chitosan, SAM Medical, USA), Surgicel (oxidized cellulose, Johnson & Johnson (Shenzhen Agent) Medical Company, USA) and gelatin (hemostatic gelatin sponge, Xiangen Medical Technology Development Company, Jiangxi Province, China) were weighed (W0, g), immersed in blood, simulated body fluid SBF, and normal saline at room temperature for 60 seconds, placed on filter paper to remove excess liquid on the surface, and then weighed (W, g). The adsorption rate of different liquids was calculated according to the following formula:

[0084] LAR(%)=[(W-W0) / W0]×100%

[0085] The results are as follows Figure 6 As shown, Figure 6 This is a test diagram of the absorption performance of the extracellular polysaccharide riclin / graphene oxide composite hemostatic sponge described in Example 1. Figure 6 It can be seen from Table 1 that, compared with Gauze, Celox, Chito SAM, Surgicel and gelatin, ARGS (i.e. the hemostatic sponge described in Example 1) has the highest liquid adsorption rate in blood, simulated body fluid SBF or physiological saline, and its hemostatic effect will also be the best.

[0086] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. An extracellular polysaccharide riclin / graphene oxide composite hemostatic sponge, characterized in that: It is prepared by aldehyde-forming the extracellular polysaccharide riclin, cross-linking it with graphene oxide, and then freeze-drying it. The exopolysaccharide riclin is produced by fermentation of Agrobacterium ZCC3656; the deposit number of Agrobacterium ZCC3656 is CCTCCNO:M2018797; The aldehyde rate of the aldehyde-formyl exopolysaccharide riclin is 25-50%; The "cross-linking with graphene oxide" specifically includes: stirring and mixing the aldehyde-modified extracellular polysaccharide riclin aqueous solution with the graphene oxide aqueous solution to obtain the extracellular polysaccharide riclin / graphene oxide composite hydrogel; the mass ratio of the graphene oxide to the extracellular polysaccharide riclin is 1:1-15.

2. The extracellular polysaccharide riclin / graphene oxide composite hemostatic sponge according to claim 1, characterized in that: The structural formula of the extracellular polysaccharide riclin is as follows: Wherein, R is -OCCH2CH2COOH or -H, and n is an integer greater than 0.

3. The extracellular polysaccharide riclin / graphene oxide composite hemostatic sponge according to claim 1 or 2, characterized in that: The "aldehyde-forming the exopolysaccharide riclin" specifically includes: dissolving the exopolysaccharide riclin in water, adding an oxidant to carry out an oxidation reaction, and obtaining the aldehyde-formylated exopolysaccharide riclin.

4. The extracellular polysaccharide riclin / graphene oxide composite hemostatic sponge according to claim 3, characterized in that: The oxidant is at least one of periodate, hypochlorite or hydrogen peroxide; the mass ratio of the oxidant to the extracellular polysaccharide riclin is 0.2-0.5:1; the oxidation reaction temperature is 15-30°C, the time is 2-12h, and the pH is 7-9.

5. The extracellular polysaccharide riclin / graphene oxide composite hemostatic sponge according to claim 1, characterized in that: The concentration of the aldehyded extracellular polysaccharide riclin aqueous solution is 5-30 mg / mL, and the concentration of the graphene oxide aqueous solution is 1-20 mg / mL; the temperature of the stirring and mixing reaction is 4-25° C., and the time is 4-8 hours; and the stirring rate is 1000-3000 rpm.

6. The extracellular polysaccharide riclin / graphene oxide composite hemostatic sponge according to claim 1, characterized in that: The graphene oxide aqueous solution is prepared by adding graphene oxide into water and uniformly dispersing it by ultrasonication; the temperature of the ultrasonic dispersion is 0-10°C, the time is 0.5-1h, and the ultrasonic amplitude is 10-30%.

7. The extracellular polysaccharide riclin / graphene oxide composite hemostatic sponge according to claim 1, characterized in that: The "freeze drying" specifically includes: dialyzing the extracellular polysaccharide riclin / graphene oxide composite hydrogel and then freeze drying it to obtain the extracellular polysaccharide riclin / graphene oxide composite hemostatic sponge.

8. The extracellular polysaccharide riclin / graphene oxide composite hemostatic sponge according to claim 7, characterized in that: The molecular weight cutoff of the dialysis bag used in the dialysis is 500-2000Da, the dialysis time is 0.5-2 days, and the water change frequency is 3-8h / time; the freeze-drying temperature is -(70-80)℃, and the time is 24-48h.

9. A method for preparing the extracellular polysaccharide riclin / graphene oxide composite hemostatic sponge according to any one of claims 1 to 8, characterized in that: include: (1) dissolving the exopolysaccharide riclin in water, adding an oxidant to carry out an oxidation reaction, and obtaining the aldehyde-modified exopolysaccharide riclin; (2) stirring and mixing the aldehyde-modified exopolysaccharide riclin aqueous solution and the graphene oxide aqueous solution to react, thereby obtaining an exopolysaccharide riclin / graphene oxide composite hydrogel; (3) The extracellular polysaccharide riclin / graphene oxide composite hydrogel is dialyzed and then freeze-dried to obtain the extracellular polysaccharide riclin / graphene oxide composite hemostatic sponge.

10. Use of the extracellular polysaccharide riclin / graphene oxide composite hemostatic sponge according to any one of claims 1 to 9 in tissue trauma hemostasis or acute wound hemorrhage products.

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