Application of Floccuronic acid polysaccharide in the preparation of liquid hemostatic materials for treating non-compressible bleeding and secondary bleeding.

Abstract

The application discloses application of Floccuronic acid polysaccharide in preparation of a liquid hemostatic material for treating incompressible hemorrhage and secondary hemorrhage. The liquid hemostatic material is a solution containing Floccuronic acid polysaccharide, the Floccuronic acid polysaccharide solution can accelerate blood cell aggregation and enhance fibrin clot structure, thereby accelerating the blood coagulation process and forming a dense clot, which shows excellent hemostatic effect in an animal liver model and a rat arterial rebleeding model, and has wide application prospects in treatment of incompressible hemorrhage and secondary hemorrhage as the liquid hemostatic material.

Description

Technical Field

[0001] This invention belongs to the field of hemostatic materials technology, and relates to the application of a floccuronic acid polysaccharide in the preparation of a liquid hemostatic material for treating non-compressible bleeding and secondary bleeding. Background Technology

[0002] While the body's natural blood clotting process is effective in preventing excessive bleeding and treating minor wounds, it often falls short in situations requiring immediate and efficient hemostasis. Because the nature and severity of bleeding vary, customized hemostatic materials suitable for different situations and user-friendly operation are necessary.

[0003] The mechanisms of action of existing hemostatic materials mainly include physical barriers, blood flow restriction, primary hemostasis related to platelets, secondary hemostasis related to coagulation factors, and adhesiolytic agents that enhance clot formation. The most common hemostasis methods utilize physical barriers and blood flow restriction; for example, minor bleeding can be managed with pressure gauze, while limb bleeding can be controlled with a tourniquet. Accessible wounds where tourniquets cannot be used may be effectively treated with hemostatic dressings such as QuikClot Combat Gauze, which can activate factor XII, accelerate the coagulation cascade, and has been reported to have a hemostatic success rate of 85.2%. Besides the aforementioned local applications, there are also cases of internal bleeding, postoperative bleeding, and intraoperative bleeding that can lead to serious complications or even death. These difficult-to-control hemostatic situations are usually treated with systemic hemostatic agents such as fibrin products, injectable hemostatic gels, or injectable hemostatic bodies.

[0004] Given the antigenicity and complex preparation processes of systemic hemostatic agents, and the difficulty of addressing situations where bleeding sites are inaccessible or where direct pressure hemostasis is not feasible, there is a need to develop new topical medications that possess both high hemostatic efficacy and suitability for restricted or difficult-to-access sites. Some literature mentions that liquid hemostatic materials can be directly injected during surgery or applied intraoperatively to organs, tissues, or cavities, suitable for restricted or difficult-to-access sites. Furthermore, some liquid hemostatic materials also show promise for intravenous application.

[0005] Chinese patent CN113755407B and literature (Chen S, Cheng R, Xu X, et al. The structure and flocculation characteristics of a novel exopolysaccharide from a Paenibacillus isolate. Carbohydr Polym. 2022 Sep 1; 291:119561.) both report a microbial polysaccharide, floccuronic acid (FA), secreted by Paenibacillus sp. M21629. This FA has abundant carboxyl groups, a high molecular weight (number average molecular weight Mn = 8.66 × 10⁷ Da, weight average molecular weight Mw = 2.38 × 10⁸ Da, size average molecular weight Mz = 3.44 × 10⁸ Da), and exhibits excellent flocculation ability for kaolin and coal powder through a bridging mechanism. Currently, there are no literature reports on the use of FA polysaccharide as a hemostatic material. Summary of the Invention

[0006] The purpose of this invention is to provide the application of FA polysaccharide in the preparation of liquid hemostatic materials for treating non-compressible bleeding and secondary bleeding. When used as a liquid hemostatic material, FA polysaccharide has the advantages of good hemostatic effect, good biocompatibility, and low price.

[0007] In this invention, uncompressible bleeding refers to bleeding in which a large amount of blood flows freely from a ruptured blood vessel when it cannot be effectively controlled by external pressure or bandaging. This bleeding can lead to serious life-threatening situations and requires emergency treatment. Uncompressible bleeding typically occurs in the following situations: rupture of large blood vessels, such as aortic rupture, hepatic or splenic artery rupture, etc.; severe trauma, such as severe stab wounds, gunshot wounds, internal bleeding caused by impact or compression, etc.; surgical complications, such as vascular rupture or postoperative bleeding during or after surgery. Secondary bleeding refers to the recurrence of bleeding from a wound that has previously stopped. This bleeding can occur after surgery, during wound healing, or during recovery from other surgical procedures. Secondary bleeding is usually unpredictable, but it can lead to complications such as blood loss, infection, and delayed wound healing. The causes of secondary bleeding may include the following: Infection: Infection may affect the wound healing process and cause blood vessels to rupture, thus causing secondary bleeding; Abnormal blood clotting function: Certain diseases, medications, or genetic factors may cause abnormal blood clotting function, increasing the risk of secondary bleeding; Wound tension: If the wound is subjected to severe tension, such as strenuous exercise, coughing, or pulling, it may cause the already formed blood clot to rupture, triggering secondary bleeding; Insufficient blood supply: If the blood supply around the wound is insufficient, or if the formation of new blood vessels is abnormal, it may also lead to secondary bleeding.

[0008] The structural formula of the FA polysaccharide described in this invention is as follows:

[0009]

[0010] Where n = 10 4 -10 6 .

[0011] The liquid hemostatic material of the present invention is a solution containing FA polysaccharide.

[0012] Furthermore, the FA polysaccharide in the liquid hemostatic material is sterile, high-purity FA polysaccharide.

[0013] Specifically, the sterile, high-purity FA polysaccharide is prepared through the following steps:

[0014] (1) Prepare a 0.5 wt% solution of crude FA polysaccharide, add 0.1 wt% NaOH, react at 105℃ for 10 minutes to remove protein, filter the polysaccharide solution after reaction through a 1 μm fiber membrane, adjust the pH of the filtrate to neutral, add alcohol to precipitate, and obtain high-purity FA polysaccharide.

[0015] (2) Dissolve high-purity FA polysaccharide in deionized water and stir at room temperature and stirring speed of 400-800 rpm for 1-2 hours to obtain high-purity FA polysaccharide solution.

[0016] (3) The high-purity FA polysaccharide solution was sterilized at 121℃ for 20 min to obtain a sterile high-purity FA polysaccharide solution.

[0017] Furthermore, in the solution containing FA polysaccharide, the concentration of FA polysaccharide is 0.1wt%-10wt%.

[0018] Furthermore, the liquid hemostatic material also contains excipients such as antibacterial agents.

[0019] Furthermore, the dosage form of the liquid hemostatic material is a topical ointment, injection, or spray.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] (1) This invention is the first to discover that FA polysaccharide can flocculate red blood cells, making them denser, and can affect the formation of fibrin and change its structure, making the fibrin clot rheological strength stronger, thus forming a denser blood clot, promoting the blood coagulation process, and thus achieving a hemostatic effect. Using a solution containing FA polysaccharide as a liquid hemostatic material, no pressure is required during the hemostatic process, making it suitable for treating non-compressible bleeding and secondary bleeding.

[0022] (2) When solutions containing FA polysaccharides are used as liquid hemostatic materials, they have good permeability and can penetrate deep into the wound to quickly come into contact with blood. This permeability can quickly form clots or gel-like substances, promote blood coagulation and seal the bleeding site, and achieve hemostasis in a short time. It is suitable for emergency treatment and surgery and other scenarios that require rapid hemostasis. Attached Figure Description

[0023] Figure 1 The images show the results of the fresh whole blood clot test with FA polysaccharide solution. (a) shows the actual images of the fresh blood clots after 10 minutes in the saline group and the 0.1% FA polysaccharide group, and (b) shows the actual images of the fresh blood clots tilted in the saline group and the 0.1% FA polysaccharide group.

[0024] Figure 2 The image shows the effect of FA solution in treating non-compressible bleeding. Figure 2 (a) Photographs of the saline group and the 10% FA group in a rat liver biopsy model. Figure 2 (b) is a statistical graph of blood loss measured on each filter paper in the rat liver puncture model.

[0025] Figure 3 The image shows the effect of FA solution in treating secondary bleeding. Figure 3 (a) Photographs of wounds treated with saline, 5% FA, and 10% FA, taken at 10 minutes and 15 minutes, respectively. Figure 3(b) is a statistical chart of blood loss in each group during the rat secondary hemorrhage model test.

[0026] Figure 4 The diagram shows the biocompatibility of FA polysaccharide. Figure 4 (a) Routine blood test results in mice one week after subcutaneous implantation of FA polysaccharide. Figure 4 (b) H&E stained sections of mouse subcutaneous tissue treated with physiological saline and FA. Detailed Implementation

[0027] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. Unless otherwise specified, all technical and scientific terms used in this invention have the meanings commonly understood by one of ordinary skill in the art. Unless otherwise specified, the reagents or materials used in the following embodiments can be commercially available or synthesized by referring to existing methods.

[0028] Example 1

[0029] Preparation of sterile FA polysaccharide solution:

[0030] (1) Dissolve crude FA polysaccharide powder in pure water to obtain a FA polysaccharide solution with a concentration of 0.5wt%, then add 0.1wt% NaOH, stir and mix well, react at 105℃ for 10 minutes, then filter the polysaccharide solution after reaction through a 1μm fiber membrane, adjust the pH of the filtrate to neutral, add alcohol, and precipitate to obtain high-purity FA polysaccharide.

[0031] (2) Dissolve high-purity FA polysaccharide in deionized water and stir at room temperature and stirring speed of 400-800 rpm for 1-2 hours to obtain high-purity FA polysaccharide solutions with concentrations of 0.1wt%, 4wt%, 5wt%, and 10wt%, respectively.

[0032] (3) High-purity FA polysaccharide solutions of different concentrations were sterilized at 121℃ for 20 min to obtain sterile FA polysaccharide solutions of 0.1wt%, 4wt%, 5wt%, and 10wt%, respectively.

[0033] Example 2

[0034] Fresh whole blood clot test of FA polysaccharide solution:

[0035] To determine the clot time of the FA polysaccharide solution, 900 μL of fresh blood from mice was immediately added to a 1.5 cm diameter glass bottle containing 100 μL of 0.1 wt% FA polysaccharide solution or physiological saline. The bottle was shaken for 15 seconds to mix the fresh blood with the FA polysaccharide solution or physiological saline. After 10 minutes of clot formation, the bottle was adhered to a white board and observed at an angle. Finally, the clot was placed on filter paper to absorb the flowing blood for weight measurement, providing a preliminary assessment of the hemostatic ability of the FA polysaccharide solution.

[0036] like Figure 1 As shown, the blood clots in the FA group (0.417±0.108g) were larger and slightly more adhesive than those in the saline group (0.343±0.075g), which preliminarily proves that the FA polysaccharide solution has coagulation ability.

[0037] Example 3

[0038] Experiments on the use of FA polysaccharide solution as a liquid hemostatic material for the treatment of non-compressible bleeding:

[0039] Uncompressible bleeding was simulated using a rat liver circumcision model. Rats were first anesthetized in an induction chamber inhaling 4% isoflurane and 100% oxygen, followed by an intraperitoneal injection of 5% chloral ester. During the surgery, the rats were placed on a heating pad, and a plastic sheet was used to isolate the liver from bodily fluids. A 4 mm diameter biopsy punch was then used to make a 4 mm diameter incision in the liver. After allowing free bleeding for 5 seconds, excess blood was wiped away with gauze. Then, 100 μL of physiological saline, 4 wt% FA polysaccharide solution, and 10 wt% FA polysaccharide solution were applied to the injury site using pipettes. Pre-weighed filter paper was placed near the wound and changed every minute to measure blood loss.

[0040] like Figure 2 As shown in Figure (a), compared to the saline group, the 10w%FA group showed a significant reduction in blood loss within just 2 minutes, demonstrating a remarkable hemostatic effect. This visually demonstrates the effectiveness of FA polysaccharide solution as a liquid hemostatic material in treating non-compressible bleeding.

[0041] like Figure 2 As shown in Figure (b), by measuring the cumulative blood loss on each filter paper, it was found that all polysaccharide groups at different concentrations had hemostatic effects compared to the saline group, with high-concentration FA polysaccharide showing better hemostatic effects. These results indicate that FA polysaccharide solution, as an excellent hemostatic agent, has potential application prospects in the treatment of non-compressible bleeding.

[0042] Example 4

[0043] Experiments on the use of FA polysaccharide solution as a liquid hemostatic material for the treatment of secondary bleeding:

[0044] Secondary hemorrhage was simulated using a rat femoral artery rebleeding model. Rats were anesthetized in an induction chamber supplied with 4% isoflurane and 100% oxygen, followed by an intraperitoneal injection of 5% chloral ester. During the procedure, the rats were placed on a heating pad, and a catheter was inserted through a 24G intravenous catheter. Ophthalmic scissors were used to dissect the skin and soft tissue to expose the femoral artery. Arterial bleeding was then induced by incising the femoral artery with a scalpel, and 500 μL of normal saline, 5 wt% FA, or 10 wt% FA polysaccharide solution were immediately added to mix with the forming blood clot. Pre-weighed gauze was placed on the wound to record blood loss. After hemostasis, normal saline was infused at a rate of 800 μL / min starting at 10 minutes. Blood loss due to secondary hemorrhage during the reperfusion phase was recorded on pre-weighed gauze and changed every 5 minutes.

[0045] like Figure 3 As shown, the blood loss in the FA group was slightly less than that in the saline group within the first 10 minutes of manual pressure hemostasis, suggesting that the gradual aggregation of blood cells may have limited effectiveness in situations requiring rapid bleeding control. However, after saline infusion, the secondary bleeding situation in the FA group was significantly improved, and the amount of bleeding decreased with increasing polysaccharide concentration. These results strongly demonstrate that FA polysaccharide solution enhances the stability of blood clots and holds promise for playing a significant role in postoperative secondary bleeding and similar clinical situations.

[0046] Example 5

[0047] Safety evaluation of FA polysaccharides:

[0048] To verify the biocompatibility of FA polysaccharide, FA polysaccharide and physiological saline were implanted subcutaneously into mice, respectively. One week later, blood routine indicators of each group of mice were measured, including: basophil count (BASO), eosinophil count (EO), monocyte count (MONO), lymphocyte count (LYMPH), and neutrophil count (NEUT). Subcutaneous tissue from each group of mice was also collected for H&E staining to observe whether there was an inflammatory response.

[0049] like Figure 4 As shown in Figure (a), the implanted FA polysaccharide had almost no effect on the various cell counts in the blood being tested.

[0050] like Figure 4 As shown in Figure (b), H&E-stained sections of mouse subcutaneous tissue showed no signs of inflammation. These results collectively demonstrate that FA polysaccharide is a safe and biocompatible material, consistent with the biocompatibility and biodegradability exhibited by most microbial polysaccharides.

Claims

1. The application of floccuronic acid polysaccharide in the preparation of liquid hemostatic materials for treating non-compressible bleeding and secondary bleeding, wherein the structural formula of the floccuronic acid polysaccharide is: where n = 10 4 -10 6 .

2. The application according to claim 1, characterized in that, The liquid hemostatic material is a solution containing Floccuronicacid polysaccharide.

3. The application according to claim 2, characterized in that, The floccuronic acid polysaccharide mentioned above is a sterile, high-purity floccuronic acid polysaccharide, prepared through the following steps: (1) Prepare a 0.5 wt% solution of crude Floccuronic acid polysaccharide, add 0.1 wt% NaOH, react at 105℃ for 10 minutes to remove protein, filter the polysaccharide solution after reaction through a 1 μm fiber membrane, adjust the pH of the filtrate to neutral, add alcohol to precipitate, and obtain high-purity Floccuronic acid polysaccharide. (2) Dissolve high-purity Floccuronic acid polysaccharide in deionized water and stir at room temperature and stirring speed of 400-800 rpm for 1-2 hours to obtain high-purity Floccuronic acid polysaccharide solution. (3) The high-purity Floccuronic acid polysaccharide solution was sterilized at 121℃ for 20 min to obtain a sterile high-purity Floccuronic acid polysaccharide solution.

4. The application according to claim 1, characterized in that, The concentration of Floccuronic acid polysaccharide in the solution is 0.1wt%-10wt%.

5. The application according to claim 1, characterized in that, The liquid hemostatic material also contains an antibacterial agent.

6. The application according to claim 1, characterized in that, The liquid hemostatic material is available in the form of a topical ointment, injection, or spray.

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