A polysaccharide-based hemostatic sponge with both antibacterial and highly efficient hemostatic properties and its application

Abstract

A polysaccharide-based hemostatic sponge with both antibacterial and highly efficient hemostatic properties and its application provided by the present invention are obtained by removing the succinyl groups from Riclin polysaccharide and then oxidizing it to form a hydrogel, and then freeze-drying it to form a porous structure. The polysaccharide-based hemostatic sponge is modified after removing the succinyl groups from Riclin polysaccharide, and a three-dimensional porous hemostatic sponge with the Riclin polysaccharide without succinyl groups as the skeleton is obtained. It can not only play an efficient hemostatic effect in internal organ bleeding, vascular injury, etc., but also has excellent antibacterial properties and biodegradability, and has good biocompatibility, is non-toxic and has no side effects on the human body, and can prevent further damage to the wound by the outside world without affecting wound healing.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and particularly relates to a polysaccharide-based hemostatic sponge with both antibacterial and highly efficient hemostatic properties and its application. Background Art

[0002] Massive bleeding and wound infection after tissue trauma have always been one of the important factors leading to death in wars, natural disasters, and traffic accident injuries. This requires that the hemostatic material can not only stop bleeding efficiently but also prevent infection, which is of great significance for saving the lives of the wounded, reducing the disability rate and fatality rate. For an ideal hemostatic material, it is not enough to simply achieve rapid hemostasis, but also needs to have characteristics such as biological safety, convenient use at the wound, anti-bacterial infection in a short time, and low price.

[0003] Currently, the raw materials of commonly used external hemostatic materials on the market include cotton fiber, zeolite, gelatin, oxidized cellulose, chitosan, etc. These external hemostatic materials can be made into various forms, including tourniquets, colloids, sponges, and powders. Among them, the sponge, as a new type of compressible and excellent effective wound hemostatic material, is more attractive due to its shape recovery performance and liquid absorption ability, which may provide advantages for dealing with certain special wounds, and can achieve rapid expansion while stopping bleeding and be in full contact with irregular bleeding wounds. However, an obvious disadvantage of most hemostatic sponges is their low hemostatic efficiency.

[0004] To solve the problem of low hemostatic efficiency of hemostatic sponges, polysaccharide-based hemostatic sponges have been widely developed. A variety of hemostatic sponges represented by chitosan have been commercialized. However, the single chitosan has weak hemostatic effect, unstable curative effect, and needs to add many other toxic cross-linking agents or more hemostatic and blood-activating substances to play a certain hemostatic role. In addition, chitosan is mainly derived from marine crustaceans, with high extraction cost, low yield, and high price, which is not suitable for wide social applications. There are many types of polysaccharides. Among them, microbial extracellular polysaccharides have attracted wide attention due to their significant advantages such as rich sources, low price, good stability, low immunogenicity, and good biocompatibility / biodegradability. However, at present, most polysaccharide-based hemostatic materials generally have poor hemostatic effects and do not have antibacterial infection characteristics, and they cannot ensure the safety of the wound after hemostasis during the first aid process. Therefore, developing a polysaccharide hemostatic sponge material with high hemostatic efficiency, antibacterial infection, biological safety, good biocompatibility, simple production process, and low cost is an urgent task in this field.

[0005] Riclin polysaccharide is an extracellular polysaccharide derived from Agrobacterium, which exhibits antioxidant and anti-inflammatory properties, antitumor activity, and immunomodulatory effects (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 anti-inflammatory activity of a succinoglycan Riclin from Agrobacterium sp. ZCC3656, J. Appl. Microbiol. 2019 Dec; 127(6):1716-1726.). Although the strain producing this polysaccharide shows a high extracellular polysaccharide yield and low fermentation cost, with great commercial value and application prospects, there is no report on whether Riclin polysaccharide can be used in hemostatic materials. Due to the high hydrophilicity of Riclin polysaccharide, it is impossible to achieve effective hemostasis when directly prepared into materials such as hemostatic sponges. Therefore, how to obtain a hemostatic sponge with high hemostatic properties, excellent antibacterial properties, biosecurity, and low cost using Riclin polysaccharide is of great significance for the innovative development of biomedical materials. Summary of the Invention

[0006] Based on the above technical problems, the present invention provides a polysaccharide-based hemostatic sponge with both antibacterial and highly efficient hemostatic functions and its application. The polysaccharide-based hemostatic sponge is obtained by removing the succinyl groups from Riclin polysaccharide and then modifying it to obtain a three-dimensional porous hemostatic sponge with the de-succinylated Riclin polysaccharide as the backbone. It can not only play an efficient hemostatic effect in internal organ bleeding, vascular injury, etc., but also has excellent antibacterial properties, good biocompatibility, no toxic and side effects on the human body, and can prevent further damage to the wound by the outside world without affecting wound healing.

[0007] A polysaccharide-based hemostatic sponge with both antibacterial and highly efficient hemostatic properties proposed by the present invention is obtained by removing the succinyl groups from Riclin polysaccharide, oxidizing it to form a hydrogel, and then freeze-drying it to form a porous structure.

[0008] In the present invention, the polysaccharide-based hemostatic sponge uses Riclin polysaccharide with succinyl groups removed as the backbone, thereby making a compressible hemostatic sponge that not only has good mechanical properties, a strong and rapid liquid absorption capacity, and the characteristics of liquid absorption and compression recovery of the sponge itself, but also has excellent tissue adhesion and blood compatibility; after performing physical and chemical property characterization, animal hemostasis experiments, antibacterial experiments, and in vitro hemolysis experiments on this polysaccharide-based hemostatic sponge, it was found that:

[0009] (1) Chemical characterization experiments showed that the polysaccharide-based hemostatic sponge containing free aldehyde groups was successfully prepared in the present invention. Multiple hemiacetal characteristic peaks appeared at a chemical shift between 5 - 6.5 ppm, and free aldehyde characteristic peaks appeared at a chemical shift between 9 - 10 ppm. And it was shown by scanning electron microscopy that it formed a honeycomb-like pore network structure.

[0010] (2) Physical characterization experiments showed that the polysaccharide-based hemostatic sponge provided in the present invention had good mechanical properties after being soaked. It could absorb liquid and quickly return to its original shape after being compressed to 20% of its original length, which was beneficial for application in in vivo compression hemostasis.

[0011] (3) Animal hemostasis experiments showed that compared with various commercial hemostatic materials, the polysaccharide-based hemostatic sponge provided in the present invention had strong hemostatic performance. It could basically control the hemostasis time of a severed porcine femoral artery within 100 s with a relatively low dosage, and reduce the arterial blood loss to 1 - 8 g; the hemostasis time for liver injury was controlled within 150 s, and the blood loss was reduced to 2 - 8 g, having high hemostatic effects and practical application value.

[0012] (4) In vitro blood compatibility experiments showed that the polysaccharide-based hemostatic sponge provided in the present invention could keep the hemolysis rate below 0.5%, having excellent biocompatibility.

[0013] (5) Antibacterial experiments showed that the polysaccharide-based hemostatic sponge provided in the present invention significantly inhibited the growth and development process and survival ability of Escherichia coli and Staphylococcus aureus, and could resist the infection of Escherichia coli and Staphylococcus aureus.

[0014] Based on the above experimental results, it can be seen that the polysaccharide-based hemostatic sponge provided in the present invention has both high hemostatic and antibacterial infection performance, and can be applied in the preparation of medical hemostatic materials for first aid, portable, compressible, anti-infective, medical materials that can carry drugs and other bioactive substances.

[0015] Preferably, the Riclin polysaccharide is purified by an alkali purification method from the crude Riclin polysaccharide to remove succinyl groups;

[0016] Preferably, the crude Riclin polysaccharide is produced by fermenting Agrobacterium sp. ZCC3656 with the preservation number of CCTCC NO: M2018797.

[0017] Preferably, the step of "purifying the crude Riclin polysaccharide by alkaline purification to remove succinyl groups" specifically includes: dissolving the crude Riclin polysaccharide in water, adding an inorganic base for hydrolysis reaction, after centrifugal separation, performing alcohol precipitation on the obtained supernatant, and the obtained precipitate is the Riclin polysaccharide with succinyl groups removed;

[0018] Preferably, the hydrolysis reaction temperature is 100 - 110 °C, the time is 15 - 30 min, the pH is 8.0 - 9.5, and the pressure is 5 - 45 kPa;

[0019] Preferably, the protein content of the Riclin polysaccharide with succinyl groups removed is 1 - 5 wt%.

[0020] In the present invention, the Riclin polysaccharide is a natural extracellular polysaccharide extracted from Agrobacterium sp. ZCC3656, and its molecular chain contains a large number of hydroxyl groups, having good biocompatibility and biodegradability; it has been found through research that the Riclin polysaccharide has biological functional activities such as inhibiting tumor growth, antioxidant, and improving hypercholesterolemia; in the present invention, the protein in the extracellular polysaccharide Riclin produced by fermentation can be effectively removed by alkaline purification, and at the same time, the succinyl groups in the extracellular polysaccharide Riclin can be removed, so as to quickly extract and obtain the Riclin polysaccharide with succinyl groups removed, which is suitable for industrial production.

[0021] Preferably, the structural formula of the Riclin polysaccharide after removing succinyl groups is as follows:

[0022]

[0023] Among them, n is an integer greater than 0.

[0024] In the present invention, after removing the succinyl groups from the Riclin polysaccharide, a more stable structure and higher biological activity will be obtained. Subsequently, using the Riclin polysaccharide with succinyl groups removed as a raw material, after oxidation, dialysis, and freeze-drying, a hemostatic sponge is prepared, and it will have more excellent antibacterial and hemostatic properties than the hemostatic sponge prepared solely from the Riclin polysaccharide as a raw material.

[0025] Preferably, the step of "oxidizing to form a hydrogel" specifically includes: dissolving the Riclin polysaccharide with succinyl groups removed in water, adding an oxidant for oxidation reaction to obtain an aldehyde group-containing polysaccharide, and after cross-linking reaction of the aldehyde group-containing polysaccharide in water, a hydrogel is formed;

[0026] Preferably, the oxidant is at least one of sodium periodate, sodium hypochlorite or hydrogen peroxide;

[0027] Preferably, the mass ratio of the oxidant to the Riclin polysaccharide with succinyl groups removed is 0.175 - 1.25:1;

[0028] Preferably, the oxidation reaction temperature is 15 - 25 °C, the time is 2 - 5 h, and the pH is 7 - 9.

[0029] In the present invention, when the Riclin polysaccharide with succinyl groups removed is subjected to an oxidation reaction with an oxidant (such as sodium periodate), the Riclin polysaccharide with succinyl groups removed needs to be prepared into an aqueous solution (concentration 10 - 50 mg / mL), and then mixed with powdered crystalline sodium periodate for oxidation. After crosslinking, the hydrogel is obtained.

[0030] Preferably, the "freeze-drying to form a porous structure" specifically includes: first subjecting the hydrogel to freeze-thaw to form an aqueous solution with uniformly dispersed substances, and then performing vacuum freeze-drying to form a porous structure, thereby obtaining the polysaccharide-based hemostatic sponge;

[0031] Preferably, the number of freeze-thaw cycles is 1 - 4 times, the freezing temperature is -(10 - 20) °C, and the melting temperature is 4 - 25 °C;

[0032] Preferably, the vacuum freeze-drying temperature is -(60 - 80) °C, the pressure is 0 - 10 kPa, and the time is 24 - 60 h.

[0033] Preferably, before the "freeze-drying to form a porous structure", the hydrogel is also subjected to dialysis;

[0034] Preferably, the dialysis bag used for dialysis has a cut-off molecular weight Mw of 500 - 14000 da, the replacement frequency of the dialysis water is 3 - 8 h / time, and the dialysis time is 12 - 24 h.

[0035] The present invention also provides a method for preparing the above-mentioned polysaccharide-based hemostatic sponge with both antibacterial and highly efficient hemostatic properties, including: removing the succinyl groups from Riclin polysaccharide, and performing an oxidation reaction with an oxidant to obtain an aldehyde group-containing polysaccharide; subjecting the aldehyde group-containing polysaccharide to a crosslinking reaction in water to obtain a hydrogel; and performing freeze-drying on the hydrogel to obtain the polysaccharide-based hemostatic sponge.

[0036] The present invention also provides an application of the above-mentioned polysaccharide-based hemostatic sponge in tissue trauma hemostasis, tissue trauma repair or tissue trauma antibacterial products.

[0037] The present invention further provides an application of the Riclin polysaccharide with succinyl groups removed in tissue trauma hemostasis, tissue trauma repair or tissue trauma antibacterial products.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] (1) The present invention provides a polysaccharide-based hemostatic sponge with both antibacterial and efficient hemostatic properties. The method comprises the following steps: removing the succinyl group from Riclin polysaccharide and subjecting the polysaccharide to an oxidation reaction with an oxidant, allowing the aldehyde formation reaction to fully occur under dark conditions, oxidizing the Riclin polysaccharide containing the succinyl group removed and containing adjacent hydroxyl groups to form adjacent free aldehyde groups. After molding, the solvent molecules in the mixed liquid form ice nuclei of different sizes with water molecules according to different freezing / thawing times and the number of cycles. The water molecules are removed by sublimation through vacuum freeze drying, and finally a hemostatic sponge with a loose, porous, honeycomb-like three-dimensional physical structure is formed, which greatly improves the liquid adsorption performance. The whole process is easy to produce on a large scale, and the prepared polysaccharide-based hemostatic sponge is inexpensive. A large amount of toxic chemical cross-linking agents are discarded, and only the finally obtained cheap and readily available extracellular polysaccharides are used.

[0040] (2) The present invention provides a polysaccharide-based hemostatic sponge with both antibacterial and high-efficiency hemostatic properties, which has good liquid adsorption and shape recovery properties, so that the polysaccharide-based hemostatic sponge can adapt to non-compressible wounds and can be used for local vascular massive bleeding, effectively solving the shortcomings of existing hemostatic sponge products in these aspects.

[0041] (3) The polysaccharide-based hemostatic sponge provided by the present invention has both antibacterial and efficient hemostatic properties, and has a unique hemostatic mechanism; wound adsorption effect: the polysaccharide-based hemostatic sponge can quickly adhere to the wound and adsorb the liquid components in the blood, enriching blood cells on the interface. The red blood cells and platelets in the blood adhere to the contact surface in large quantities in a short time, thereby promoting the hemostasis process; interface adsorption effect, coagulation cascade stimulation: the sponge spatial structure and aldehyde group stimulation of the polysaccharide-based hemostatic sponge can enhance the aggregation function of blood cells and red blood cells. Red blood cells are the key component of primary blood clots. Therefore, more red blood cells are aggregated to form larger clots, shortening the bleeding time, Reduce blood loss. The hemostatic sponge of the present invention can quickly aggregate highly concentrated and tightly adsorb red blood cells to form a typical biconcave structure and a polyhedral structure, and finally form a clot of a certain strength at the macroscopic level. In addition, the polysaccharide-based hemostatic sponge of the present invention has the function of adsorbing platelets. Platelet adhesion will activate the signal pathway, leading to the formation of thromboxane A2 and the secretion of platelet granule contents. These substances can cause integrins and glycoprotein IIb / IIIa to form fibrinogen receptors, leading to platelet aggregation and activation. Then, on the surface of activated platelets, coagulation is accelerated and thrombin is generated, thereby promoting the hemostatic process. The activated platelets can release Ca 2+ Promotes thrombosis, Ca 2+It can trigger the conversion of G-PⅡb / -Ⅲa to fibrinogen, promote the release of active substances such as ADP and TXA2, accelerate the clustering and aggregation of red blood cells, promote the formation of fibrin in a short time through the common pathway, enhance the strength of the newly formed blood clot, and ultimately achieve excellent hemostatic effects.

[0042] (4) The polysaccharide-based hemostatic sponge with both antibacterial and highly efficient hemostatic properties provided by the present invention has the function of resisting bacterial infection; due to the presence of free aldehyde groups in the polysaccharide-based hemostatic sponge, it can effectively damage the outer membrane and cell wall of bacteria. In addition, the three-dimensional honeycomb pore structure provides a larger area of binding sites, significantly inhibiting the growth and development process and survival ability of Gram-negative bacteria represented by Escherichia coli and Gram-positive bacteria represented by Staphylococcus aureus.

[0043] (5) The polysaccharide-based hemostatic sponge with both antibacterial and highly efficient hemostatic properties provided by the present invention has excellent hemostatic function, plays a role in resisting external bacterial infection after hemostasis, and has good biocompatibility; the present invention has a strong adhesion ability to red blood cells, platelets and other blood cells, and forms a coagulation protein network, with excellent coagulation performance. Animal hemostasis experiments show that the polysaccharide-based hemostatic sponge can quickly and effectively stop bleeding in different parts of large animals. The hemostatic effect is significantly better than that of commercial medical hemostatic gauze. Especially in the non-compressible porcine liver penetrating bleeding model, the blood loss of the polysaccharide-based sponge is only about 25% of that of the gauze. In the porcine femoral artery bleeding model, the blood loss of the modified sponge is only about 23% of that of the gauze. In vitro blood compatibility experiments show that the present invention does not damage blood cells during the use time and has good biocompatibility. Therefore, the polysaccharide-based hemostatic sponge is an ideal material for actual clinical wound first aid hemostasis and can play a role in internal organ bleeding and arterial bleeding.

[0044] (6) The polysaccharide-based hemostatic sponge with both antibacterial and highly efficient hemostatic properties provided by the present invention is developed by removing the succinyl group from Riclin polysaccharide and then performing simple process modification; the main component of the polysaccharide-based hemostatic sponge is only the polysaccharide Riclin with the succinyl group removed by aldehyde group modification, and the hemostatic sponge is formed without adding any extra toxic cross-linking agent. The raw material is prepared by microbial fermentation, with high output, fast production, low cost, almost no pollution in the production process, simple and fast preparation process, low cost, and the finished product can be applied in actual first aid scenarios. At the same time, various other hemostatic and blood-activating substances can be added in its sponge-like pore structure, with a wide range of clinical applications and potential great commercial value. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 11H-NMR spectra of the polysaccharide-based hemostatic sponge and Riclin polysaccharide with succinyl groups removed described in the examples; 1 1H-NMR

[0046] Figure 2 Scanning electron microscope images of the surface pore structure of the polysaccharide-based hemostatic sponge described in the examples;

[0047] Figure 3 Compression 80% recovery diagrams and compression curves of the polysaccharide-based hemostatic sponges described in the examples and comparative examples;

[0048] Figure 4 Diagrams of inhibition zones and dilution coating cultures after the polysaccharide-based hemostatic sponges described in the examples and comparative examples, as well as Riclin polysaccharide with succinyl groups removed and Riclin polysaccharide without succinyl groups acting on bacteria;

[0049] Figure 5 Application diagrams of the polysaccharide-based hemostatic sponges described in the examples and comparative examples in a porcine liver perforation bleeding model and a porcine femoral artery transection bleeding model;

[0050] Figure 6 Blood compatibility test diagrams of the polysaccharide-based hemostatic sponge and Riclin polysaccharide with succinyl groups removed described in the examples. Detailed implementation manners

[0051] Next, the present invention will be described in detail with specific examples for the technical solutions, but it should be clearly stated that these examples are for illustrative purposes only and are not construed as limiting the scope of the present invention.

[0052] Examples

[0053] This example presents a polysaccharide-based hemostatic sponge with both antibacterial and highly efficient hemostatic properties, and its preparation method includes:

[0054] (1) Inoculate a single colony of Agrobacterium sp. ZCC3656 (preservation number: CCTCC NO: M2018797) into LB medium for seed culture. After 16 h, inoculate the obtained seed culture solution into M9 fermentation medium (with 3% sucrose as the carbon source) at a ratio of 2%, and ferment at 30 °C for 2 days. Add 3 volumes of isopropanol to the obtained fermentation broth for alcohol precipitation, centrifuge (6000 g, 5 min) to collect the precipitate, and dry it at 40 °C to obtain the crude Riclin polysaccharide. Redissolve the crude Riclin polysaccharide in deionized water to prepare an aqueous solution with a concentration of 10 mg / mL, add NaOH aqueous solution to adjust the pH to 9.0, sterilize it under high pressure (30 kPa) at 105 °C for 20 min, then centrifuge (10000 g, 10 min) to obtain the supernatant, add 3 volumes of pre-cooled 95% (v / v) ethanol aqueous solution for alcohol precipitation, centrifuge (6000 g, 10 min) to collect the precipitate, and obtain Riclin polysaccharide without succinyl groups (average molecular weight is 2.5×10 6 da), abbreviated as Riclin ①;

[0055] (2) Dissolve the above Riclin polysaccharide without succinyl groups in deionized water to prepare a solution with a concentration of 2% (w / v). Add 5 wt% sodium periodate solution according to a ratio of 1:0.33 (mass ratio of Riclin polysaccharide without succinyl groups to NaIO4), adjust the pH to 8, stir and react in the dark at room temperature for 3 h, add excessive ethylene glycol to terminate the reaction, then dialyze the reaction solution in deionized water using a dialysis bag (Mw 1000 Da) for 24 h, change the water every 4 h, then pour it into the corresponding mold, alternately freeze in a -20 °C refrigerator / thaw at 20 °C outside, freeze-thaw 3 times every 12 h, obtain a homogeneous structure and then put it into a freeze dryer for freeze-drying. First cool down to -70 °C, then reduce the pressure to 0 kPa, freeze-dry at this temperature and pressure for 48 h, then wash with ethanol for 10 h and dry at 10 °C for 6 h to obtain the polysaccharide-based hemostatic sponge, abbreviated as AR.

[0056] Comparative example

[0057] This comparative example also provides a polysaccharide-based hemostatic sponge, and its preparation method includes:

[0058] (1) The crude Riclin polysaccharide was prepared by the method described in the reference example; the crude Riclin polysaccharide was redissolved in deionized water to prepare a polysaccharide solution with a concentration of 10 mg / mL, and then a chloroform-n-butanol (4:1) mixture was prepared. The polysaccharide solution and the mixture were mixed at a ratio of 1:4 (v:v). After vigorous shaking for about 30 min, the mixture was allowed to stand for layer separation. The denatured protein appeared in the middle layer, the upper layer was the aqueous phase, and the lower layer was the organic phase. The upper aqueous phase was aspirated with a pipette, and the protein at the bottom layer was removed by centrifugation (8000 g, 15 min). The supernatant was remixed with the mixture at a ratio of 1:4 (v:v), and the above operations were repeated several times to continuously extract the protein with the mixture until no visible protein layer was observed; the supernatant was collected, and 3 volumes of pre-cooled 95% ethanol were added for precipitation. After centrifugation (8000 g, 10 min), the precipitate was collected and dried at 40 °C to obtain the purified Riclin polysaccharide without removing the succinyl group, abbreviated as Riclin②;

[0059] (2) Referring to the method described in the reference example, the polysaccharide-based hemostatic sponge was prepared by using the above-mentioned Riclin polysaccharide without removing the succinyl group instead of the Riclin polysaccharide with the succinyl group removed, abbreviated as OR.

[0060] The polysaccharide-based hemostatic sponge prepared in the example and the Riclin polysaccharide with the succinyl group removed were detected by nuclear magnetic resonance hydrogen spectrum, and the results are as Figure 1 shown, Figure 1 which is the nuclear magnetic resonance hydrogen spectrum ( 1 H-NMR) diagram of the polysaccharide-based hemostatic sponge described in the example and the Riclin polysaccharide with the succinyl group removed. Referring to Figure 1 it can be seen that the polysaccharide-based hemostatic sponge showed peaks corresponding to the hemiacetal hydrogen at a chemical shift of 5 - 6.5 ppm and peaks corresponding to the free aldehyde hydrogen at a chemical shift of 9 - 10 ppm, indicating that the polysaccharide-based hemostatic sponge with the structure of polycondensation cross-linking of aldehyde group-containing Riclin polysaccharide as the skeleton was successfully synthesized in this example; the degree of aldehyde group modification of the polysaccharide-based hemostatic sponge described in this example was tested, and the actual oxidation rate and aldehyde group content reached 73.44% and 72 mg / g. Therefore, by adjusting the mass ratio of the Riclin polysaccharide with the succinyl group removed to NaIO4 in the reaction, the oxidation rate can be changed, and the actual oxidation rate is between 0.1 - 75.0%.

[0061] The polysaccharide-based hemostatic sponge prepared in this example was sputter-coated with gold and then scanned under a scanning electron microscope. The results are as Figure 2 shown, Figure 2 which is the scanning electron microscope image of the surface pore structure of the polysaccharide-based hemostatic sponge described in the example. Referring to Figure 2It can be seen that the polysaccharide-based hemostatic sponge described in this embodiment has a mutually connected pore structure and is arranged in a honeycomb-like manner. The pores provided by this structure are beneficial for the rapid absorption of liquids and the adhesion and binding of cells.

[0062] Mechanical compression performance test of the polysaccharide-based hemostatic sponge described in the embodiment

[0063] Place the polysaccharide-based hemostatic sponges (cylindrical) prepared in the examples and comparative examples on a universal mechanical testing machine, add about 0.7 mL of water with a dropper or syringe to fill them, and then perform compression; in the compression test, compress the cylindrical water sponge at a speed of 5 mm / min by 80%, and then recover it to 0% of the original length at the same speed for 3 cycles, and record the stress-strain curve; during each compression, the water in the sponge will be squeezed out, and during recovery, part of the water will be adsorbed back by the sponge to obtain its stress-strain curve and data graph, and the process is recorded by a camera; the test results are as Figure 3 shown, Figure 3 For the 80% recovery diagram and compression curve of the polysaccharide-based hemostatic sponges described in the examples and comparative examples. Refer to Figure 3 It can be seen that the polysaccharide-based hemostatic sponge described in the embodiment has good compression performance and shape recovery performance; specifically, Figure 3 a shows that the polysaccharide-based hemostatic sponge described in the embodiment does not break even when the strain reaches 80%, and almost perfectly recovers the liquid exuded during the compression process, Figure 3 b shows that the polysaccharide-based hemostatic sponge described in the embodiment reaches a maximum bearing pressure of 19.6 kPa at a compression ratio of 80%, and the liquid-containing state has a strong resistance to compression, Figure 3 c shows that the polysaccharide-based hemostatic sponge described in the comparative example is compressed 3 times in a cycle, and the maximum bearing pressure values after each compression strain reaches 80% are different, while Figure 3 b shows that the polysaccharide-based hemostatic sponge described in the embodiment is compressed 33 times in a cycle, and the maximum bearing pressure values after each compression strain reaches 80% are basically the same, indicating that the polysaccharide-based hemostatic sponge described in the embodiment has better mechanical compression performance than the polysaccharide-based hemostatic sponge described in the comparative example.

[0064] Antibacterial performance test of the polysaccharide-based hemostatic sponge described in the embodiment

[0065] Using the inhibition zone test method, with an initial concentration of 1×10 7 CFU·mL -1The Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus suspensions were respectively spread on Luria-Bertani (LB) agar medium in a petri dish; the polysaccharide-based hemostatic sponges (cylindrical, 20 mg, 1 cm in diameter) prepared in the examples and comparative examples, the Riclin polysaccharide with succinyl groups removed prepared in the examples, and the Riclin polysaccharide without succinyl groups removed prepared in the comparative examples were respectively placed on the LB agar medium containing Escherichia coli and Staphylococcus aureus. After incubation at 37 °C for 24 h, the formation of inhibition zones was observed;

[0066] Using the dilution plating method and LB liquid medium, the polysaccharide-based hemostatic sponges (cylindrical, 20 mg, 1 cm in diameter) prepared in the examples and comparative examples, the Riclin polysaccharide with succinyl groups removed prepared in the examples, and the Riclin polysaccharide without succinyl groups removed prepared in the comparative examples were mixed with the bacterial suspensions (5 mg / mL) of Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus with a concentration of 1×10 6 CFU·mL -1 and incubated at 37 °C for 12 h. 100 μL of the mixed bacterial suspension was taken, diluted 1000 times, and 100 μL of the diluted bacterial solution was spread on Luria-Bertani (LB) agar medium in a petri dish. After incubation at 37 °C for 24 h, the colony growth was observed.

[0067] The test results are as Figure 4 shown, Figure 4 are the inhibition zone and dilution plating culture diagrams after the polysaccharide-based hemostatic sponges, the Riclin polysaccharide with succinyl groups removed, and the Riclin polysaccharide without succinyl groups removed described in the examples and comparative examples acted on bacteria. Referring to Figure 4 it can be seen that in the inhibition zone experiment, obvious inhibition zones were observed in the AR group compared with the blank group, Riclin group, and OR group; in the dilution plating experiment, the number of colonies produced after co-culture in the AR group was significantly lower than that in the blank group, Riclin group, and OR group; thus, it is shown that compared with the polysaccharide-based hemostatic sponges described in the comparative examples, the polysaccharide-based hemostatic sponges described in the examples have obvious inhibitory effects on both Escherichia coli and Staphylococcus aureus and have better antibacterial effects in practical applications.

[0068] Hemostatic ability test of the polysaccharide-based hemostatic sponge described in the example

[0069] (1) Hemostatic ability test for animal liver perforation: Male healthy piglets (10 - 15 kg) were anesthetized with sodium pentobarbital (3% concentration, 1 mL·kg -1)Anesthetize pigs by intraperitoneal injection. After complete anesthesia, make an incision near the xiphoid process in the upper abdomen to expose the largest lobe of the liver on filter paper. During the production of some animals, the blood vessels are clamped with hemostatic forceps. At the same time, place a gauze under the filter paper with the liver to record the amount of blood flowing out of the filter paper. Place a plastic film at the bottom to prevent peritoneal fluid from exuding, removing the interference of non-liver bleeding. Use a punch (20 mm in diameter) to make a hole, and take out the visceral mass at the perforation until bleeding occurs. To seal the area above this hole, immediately press with A (about 0.3 g) for 5 seconds. Judge the hemostasis time by the continuous oozing of blood on the material stopping, and the blood on the filter paper no longer flowing and dripping, and record the hemostasis time. After bleeding for 5 - 10 minutes, remove the material from the wound, record the blood loss, and the hemostasis process is recorded by a camera. Except for the untreated control group, the rest are treated with medical gauze (about 1 g).

[0070] (2) Test on the hemostatic ability of animal femoral artery transection: Anesthetize male healthy piglets (10 - 15 kg) by intraperitoneal injection of sodium pentobarbital (3% concentration, 1 mL·kg -1 )Anesthetize pigs by intraperitoneal injection. After complete anesthesia, remove the hair before surgery. Incise the overlying muscle layer in the inguinal region to expose the femoral artery, cut the femoral artery to cause arterial bleeding. After 10 s of natural bleeding, wipe the blood overflowing in the first 10 s with gauze, manually press AR (about 1 g) on the wound. After successful hemostasis, end the compression. This compression period is the hemostasis time of the material. Remove the hand pressure and then observe, record the time and observe the wound condition. After bleeding for 5 - 10 minutes, remove the material from the wound, observe the condition of the femoral artery rupture, and record the blood loss at the same time. The hemostasis process is recorded by a camera, and the rest are treated with medical hemostatic gauze (about 8 g).

[0071] The test results are as Figure 5 shown, Figure 5 The application diagrams of the polysaccharide-based hemostatic sponge and gauze described in the examples and comparative examples in the porcine liver perforation bleeding model and the porcine femoral artery transection bleeding model. Referring to Figure 5 e - f, it can be seen that in the porcine liver perforation bleeding model, compared with the polysaccharide-based hemostatic sponge (average blood loss of 11.64 g) and gauze (average blood loss of 23.13 g) described in the comparative example, the polysaccharide-based hemostatic sponge described in the example has a liver perforation blood loss (average blood loss of 5.32 g) at a relatively low dosage, which is only 23% of the commercial medical gauze and only 45% of the polysaccharide-based hemostatic sponge described in the comparative example. It can be seen that the hemostatic effect of the polysaccharide-based hemostatic sponge described in the example in the large animal bleeding model is much higher than that of the medical gauze and the polysaccharide-based hemostatic sponge described in the comparative example; and the blood loss of the polysaccharide-based hemostatic sponge described in the example remains at 2 - 8 g after multiple tests, and the non-compressive hemostasis time is reduced to within 150 s; Referring to Figure 5As can be seen from g-h, in the porcine femoral artery transection and bleeding model, compared with the polysaccharide-based hemostatic sponge (average blood loss of 8.63 g) and gauze (average blood loss of 34.42 g) described in the comparative example, the polysaccharide-based hemostatic sponge (average blood loss of 4.13 g) described in the example has a blood loss of only 12% of the commercial medical gauze and only 48% of the polysaccharide-based hemostatic sponge described in the comparative example under relatively low dosage. It can also be seen that the hemostatic effect of the polysaccharide-based hemostatic sponge described in the example in the large animal bleeding model is much higher than that of the medical gauze and the polysaccharide-based hemostatic sponge described in the comparative example; and the blood loss of the polysaccharide-based hemostatic sponge described in the example in multiple tests is maintained at 1-8 g, and the compression hemostasis time is reduced to within 100 s and maintained below 50 s.

[0072] Blood compatibility test of the polysaccharide-based hemostatic sponge described in the example

[0073] Take fresh volunteer blood (ACD), extract RBC according to the above method, dilute it to 2% with PBS, and then mix 10 mg, 20 mg, and 30 mg of the polysaccharide-based hemostatic sponge prepared in the example and Riclin polysaccharide without succinyl groups with 4 mL of the red blood cell suspension and incubate at 37 °C for 1 h; then take 1 mL of the mixed solution and centrifuge it in an ep tube (1200 g, 10 min), observe the hemolysis situation of each group, take 100 uL of the supernatant and measure the OD value of each material at 540 nm using a microplate reader (Thermo Fisher) (OD experiment ), use the addition of PBS as a negative control (OD negative ), and the addition of 2% Triton X-100 as a positive control; the hemolysis rate is calculated according to the following formula:

[0074]

[0075] According to the hemolysis rates at different concentrations, draw a hemolysis schematic diagram, and the results are as Figure 6 shown, Figure 6 is the blood compatibility test diagram of the polysaccharide-based hemostatic sponge described in the example and Riclin polysaccharide without succinyl groups. Referring to Figure 6 it can be seen that the polysaccharide-based hemostatic sponge described in the example has good blood compatibility, with a hemolysis rate lower than 1%, and hardly has a harmful effect on red blood cells; the polysaccharide-based hemostatic sponge described in the example basically does not change the blood compatibility compared with Riclin polysaccharide and still maintains good blood compatibility.

[0076] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, makes equivalent substitutions or changes, and all should be covered within the protection scope of the present invention.

Claims

1. A polysaccharide-based hemostatic sponge with both antibacterial and highly efficient hemostatic properties, characterized in that, It is obtained by removing the succinyl group from Riclin polysaccharide and then oxidizing it to form a hydrogel, and then freeze-drying it to form a porous structure. The Riclin polysaccharide is obtained by purifying the crude Riclin polysaccharide by the alkali purification method to remove the succinyl group. The crude Riclin polysaccharide is fermented by Agrobacterium sp. ZCC3656 with the preservation number of CCTCC NO: M2018797. The specific process of "purifying the crude Riclin polysaccharide by the alkali purification method to remove the succinyl group" includes: dissolving the crude Riclin polysaccharide in water, adding an inorganic base for hydrolysis reaction, after centrifugal separation, subjecting the obtained supernatant to alcohol precipitation, and the obtained precipitate is the Riclin polysaccharide with the succinyl group removed. The temperature of the hydrolysis reaction is 100 - 110 °C, the time is 15 - 30 min, the pH is 8.0 - 9.5, and the pressure is 5 - 45 kPa.

2. The polysaccharide-based hemostatic sponge with both antibacterial and highly efficient hemostatic properties according to claim 1, characterized in that, The protein content of the Riclin polysaccharide with the succinyl group removed is 1 - 5 wt%.

3. The polysaccharide-based hemostatic sponge with both antibacterial and highly efficient hemostatic properties according to claim 1 or 2, characterized in that The structural formula of the Riclin polysaccharide with the succinyl group removed is as follows: Wherein, n is an integer greater than 0.

4. The polysaccharide-based hemostatic sponge with both antibacterial and highly efficient hemostatic properties according to claim 1 or 2, characterized in that, The specific process of "oxidizing to form a hydrogel" includes: dissolving the Riclin polysaccharide with the succinyl group removed in water, adding an oxidant for oxidation reaction to obtain an aldehyde group-containing polysaccharide, and after cross-linking reaction of the aldehyde group-containing polysaccharide in water, a hydrogel is formed.

5. The polysaccharide-based hemostatic sponge with both antibacterial and highly efficient hemostatic properties according to claim 4, wherein The oxidant is at least one of sodium periodate, sodium hypochlorite or hydrogen peroxide. The mass ratio of the oxidant to the Riclin polysaccharide with the succinyl group removed is 0.175 - 1.25:

1. The temperature of the oxidation reaction is 15 - 25 °C, the time is 2 - 5 h, and the pH is 7 - 9.

6. The polysaccharide-based hemostatic sponge with both antibacterial and highly efficient hemostatic properties according to claim 1 or 2, characterized in that, The specific process of "freeze-drying to form a porous structure" includes: first subjecting the hydrogel to freeze-thaw to form an aqueous solution with uniform dispersion of substances, and then performing vacuum freeze-drying to form a porous structure, thus obtaining the polysaccharide-based hemostatic sponge.

7. The polysaccharide-based hemostatic sponge with both antibacterial and highly efficient hemostatic properties according to claim 6, wherein The number of freeze-thaw cycles is 1 - 4 times, the freezing temperature is -10 °C to -20 °C, and the melting temperature is 4 - 25 °C. The temperature of the vacuum freeze-drying is -60 °C to -80 °C, the pressure is 0 - 10 kPa, and the time is 24 - 60 h.

8. The polysaccharide-based hemostatic sponge with both antibacterial and highly efficient hemostatic properties according to claim 1 or 2, characterized in that, Before "freeze-drying to form a porous structure", the hydrogel is also dialyzed.

9. The polysaccharide-based hemostatic sponge with both antibacterial and highly efficient hemostatic properties according to claim 8, characterized in that, The cut-off molecular weight Mw of the dialysis bag used for dialysis is 500 - 14000 da, the replacement frequency of the dialysis water is 3 - 8 h / time, and the dialysis time is 12 - 24 h.

10. A method for preparing the polysaccharide-based hemostatic sponge with both antibacterial and highly efficient hemostatic properties according to any one of claims 1-9, characterized in that, Including: After removing the succinyl group from Riclin polysaccharide, performing an oxidation reaction with an oxidant to obtain an aldehyde group-containing polysaccharide; after cross-linking reaction of the aldehyde group-containing polysaccharide in water, obtaining a hydrogel; after freeze-drying the hydrogel, the polysaccharide-based hemostatic sponge is obtained.

11. Use of the polysaccharide-based hemostatic sponge according to any one of claims 1 - 9 in the preparation of tissue trauma hemostasis, tissue trauma repair or tissue trauma antibacterial products.

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