阅读说明：本技术 一种止血凝胶及其制备方法和应用 (Hemostatic gel and preparation method and application thereof ) 是由 喻青松 甘志华 牛昆 于 2020-06-09 设计创作，主要内容包括：本发明公开一种止血组合物及其制备方法和应用。所述止血组合物为凝胶的形式,包含壳聚糖和褐藻多糖硫酸酯,二者的重量比为1:(0.5-10)。本发明提供的止血凝胶组合物有较好的流动性和可塑性的糊状物质,在腋窝、腹股沟等不可压迫型出血模型中,可以较好的填充空隙并密闭创面。可以应用在多种复杂的不规则创口,且可以很好的封闭创面并快速止血。适用于单兵作战中枪械贯穿伤等常规止血敷料无法有效填塞止血的深部大出血现场急救止血,可有效防止大出血导致的死亡,为下一步的送医和治疗争取了宝贵的抢救时间。(The invention discloses a hemostatic composition, a preparation method and application thereof. The hemostatic composition is in the form of gel, and comprises chitosan and fucoidan sulfate in a weight ratio of 1 (0.5-10). The hemostatic gel composition provided by the invention has good fluidity and plasticity of pasty substances, and can better fill gaps and seal wound surfaces in incompressible bleeding models such as armpits and groin. Can be applied to various complicated irregular wounds, and can well seal the wound surface and stop bleeding quickly. The hemostatic dressing is suitable for on-site emergency hemostasis of deep major hemorrhage, wherein the conventional hemostatic dressings such as firearms puncture wound and the like cannot effectively fill hemostasis in single-soldier operation, can effectively prevent death caused by major hemorrhage, and strives for precious rescue time for next medical delivery and treatment.)

1. A hemostatic composition, comprising a gel comprising chitosan and fucoidan.

2. The hemostatic composition according to claim 1, wherein the weight ratio of the chitosan to the fucoidan sulfate is 1 (0.5-10), preferably 1 (1-6), 1 (1-5), 1 (2-5) or 1 (2-4).

3. Hemostatic composition according to claim 1 or 2, wherein the gel further comprises water, preferably one of ultrapure water and redistilled water.

4. The hemostatic composition of any one of claims 1-3, wherein the fucoidan comprises fucose and sulfate groups, wherein the fucose is present in an amount of 15% to 35% by weight; preferably, the weight percentage content of the sulfate radical is 15% -35%;

preferably, the purity of the fucoidan is more than or equal to 95 percent;

preferably, the deacetylation degree of the chitosan is more than or equal to 95 percent;

preferably, the viscosity of the chitosan is 100-500mpa · s;

preferably, the chitosan and brown algae polysaccharide sulfate are powder.

5. Hemostatic composition according to any one of claims 1 to 4, wherein the complex viscosity of the gel is 1500-;

preferably, the composition also optionally comprises at least one loading substance, wherein the loading substance utilizes gel as a loading platform; preferably, the cargo is one, two or more of a hemostatic drug, an antibacterial drug and a healing promoting drug;

preferably, the composition is coated onto a substrate (e.g., a medical textile) or the composition is enclosed in a suitable container.

6. A process for the preparation of a composition according to any one of claims 1 to 5, characterized in that it comprises the following steps: mixing chitosan, fucoidan and water, and standing to form gel.

7. The method of claim 6, wherein the composition is prepared by a method comprising the steps of:

(1) respectively preparing chitosan aqueous solution and fucoidan aqueous solution;

preferably, the concentration of the chitosan aqueous solution is 0.1-0.5 g/mL;

preferably, the concentration of the fucoidan sulfate aqueous solution is 0.5-1.5 g/mL;

(2) pouring the chitosan aqueous solution into the fucoidan aqueous solution, uniformly mixing, preferably stirring, and standing to obtain gel;

preferably, the standing time is 5-40 h.

8. The method of claim 6 or 7, further comprising the steps of: uniformly coating the gel on a substrate;

preferably, the preparation method further optionally comprises the steps of: filling the gel into a suitable container;

preferably, the preparation method further optionally comprises the steps of: a step of sterilizing the gel and/or the substrate coated with the gel.

9. Use of a composition according to any one of claims 1 to 5 in the preparation of a haemostatic formulation.

10. A haemostatic formulation comprising a composition according to any of claims 1-5; preferably, the hemostatic preparation is a patch, an ointment or an injection;

preferably, the hemostatic preparation is for hemostasis at non-compressible wounds and/or irregular wounds.

Technical Field

The invention belongs to the field of medicines, and particularly relates to a hemostatic gel, and a preparation method and application thereof.

Background

Major hemorrhage is a major cause of battlefield death. Especially in modern war, the main needs for direct intervention of ground fighters are urban war (i.e. roadway war) and special battle in later period, and under these two conditions, the battlefield is generally far away from the base, and fighters are relatively dispersed, which is not beneficial to emergency treatment of the battlefield. Therefore, the development of the high-efficiency emergency hemostatic material suitable for the battlefield used by the individual soldier is of great significance to battlefield rescue in the modern war background.

At present, with the importance of army in various countries on body armor, the intracavity bleeding situation is reduced, and the limb bleeding is still the most common situation in the battlefield. According to the results of the military survey, the number of deaths caused by blood loss of limbs accounts for more than half of the number of deaths that can be prevented, and especially, the bleeding of the neck, armpit, groin and other parts where tourniquets cannot be applied is the main factor. In response to this problem, an important principle of the military tactical injury care course is that the survival rate of the injured person can be significantly increased by using a simple and effective hemorrhage control method such as hemostatic dressing, hemostat or a combination of the two before hospital.

It is well known that the coagulation and sealing ability of human blood itself is not sufficient to control severe bleeding symptoms, and that this ability is often significantly reduced after trauma and extensive blood loss. Therefore, the use of hemostatic dressings or devices, as well as drugs to enhance blood clotting, is critical to prevent severe bleeding and to prevent death of injured soldiers. Prior to the iraq war, the united states forces primarily treated the battlefield for blood loss using field bandages. With the progress of the iraq war and the afghanistan war, the army accelerates the development of a novel battlefield hemostatic material except gauze. These new hemostatic devices can be generally classified into two broad categories, hemostatic dressings and hemostatic granules/powders. Differentiated from the material source, the hemostatic materials mainly include inorganic hemostatic materials (zeolite, kaolin, etc., which rapidly adsorb water in plasma by a high specific surface area or stimulate the release of blood coagulation factors to accelerate blood coagulation), plant-based hemostatic materials (oxidized cellulose, oxidized regenerated cellulose, etc.), gelatin-based hemostatic materials (which initiate blood coagulation by rapidly absorbing water in plasma), collagen-based hemostatic materials, fibrin-based hemostatic materials (rich in high concentration of fibrin)Pro-and coagulation factors), coagulation factor-based hemostatic materials, and chitin-based hemostatic materials. FIG. 1 shows some of the rapid hemostatic materials that are currently common abroad, for example

And the like.

Although the application of the topical hemostatic dressing is promoted, a plurality of problems are faced in the practical application process. Firstly, the hemostatic dressing needs to directly contact with a bleeding site, so that the hemostatic dressing is generally only suitable for a large open wound, and is difficult to effectively stop bleeding for a wound with a deep bleeding part and a small opening; secondly, since the hemostatic material generally needs to be pressed by external force to assist hemostasis after contacting with a bleeding part, it is generally difficult to treat a lot of blood loss at various parts due to explosion and the like. For this reason, CoTCCC was proposed in 2016 to be used in cases of massive blood loss in the groin and underarm areas, a name of which isThe novel hemostatic material of (1). The main component of the hemostatic dressing is cellulose sponge coated by chitosan, which is different from the traditional hemostatic dressing in that the hemostatic dressing needs external force to compress,after absorbing the water in the blood, the blood rapidly expands to generate the pressure from inside to outside, so the blood can be effectively stopped without external force. The hemostasis time can be shortened to 15 seconds, which is important for emergency hemostasis in wartime. But do notThe hemostatic dressing has requirements on injured parts and wound sizes, for example, the hemostatic dressing mainly aims at wounds which are about 4.5cm and have cavity structures and cannot provide enough compressive force for other open wounds, such as armpits, groins and the like.

Based on the current situation in the field of hemostasis, an injectable gel dressing with good fluidity and good irregular wound surface sealing effect is urgently needed for non-compressible wound bleeding so as to realize rapid hemostasis under various complex hemostasis conditions.

Disclosure of Invention

The invention provides a hemostatic composition, which comprises a gel, wherein the gel comprises chitosan and fucoidan.

According to an embodiment of the invention, the weight ratio of chitosan to fucoidan is 1 (0.5-10), such as 1 (1-6), 1 (1-5), 1 (2-4), illustratively 1:1, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1: 6.

According to an embodiment of the invention, the gel further comprises water. Preferably, the gel is formed from raw materials comprising chitosan, fucoidan, and water. Wherein the water can be one of ultrapure water and redistilled water, and is preferably ultrapure water.

According to an embodiment of the present invention, the Fucoidan (FPS) is an extract of seaweed, a unique class of sulfate-bound water-soluble Fucoidan, which is present in various seaweeds, such as brown algae, kelp, Ascophyllum nodosum, Fucus vesiculosus, and the like, and is more common in brown algae. Fucoidin sulfate mainly comprises fucose and sulfate, wherein the weight percentage of the fucose is 15-35 percent, and the weight percentage of the sulfate is 15-35 percent.

According to an embodiment of the present invention, the fucoidan sulfate has a purity of 95% or more, such as 97% or more, and illustratively, has a purity of 96%, 98%, 99%, or 100%.

According to an embodiment of the present invention, the chitosan is a natural marine basic polysaccharide, which is a high molecular substance obtained by deacetylating chitin extracted from shells of crustaceans such as shrimps, crabs, and the like. Wherein the chitosan has a degree of deacetylation of at least 95%, such as at least 97%, illustratively 95%, 96%, 97%, 98%.

According to embodiments of the invention, the chitosan has a viscosity of 100-500 mPa.s, such as 100-200 mPa.s.

According to the embodiment of the invention, the chitosan and the brown algae polysaccharide sulfate are powder. For example, the particle size may be 50 to 200 mesh, for example 80 to 160 mesh.

According to an embodiment of the invention, the complex viscosity of the gel is 1500-2000 Pa.s, such as 1600-1900 Pa.s or 1700-1800 Pa.s. The gel has good fluidity and plasticity, and can be coated on various irregular surfaces, for example, the gel can be directly extruded and filled at a bleeding part with ravines, and can be molded in a closed manner.

According to an embodiment of the present invention, the gel has good biocompatibility and is harmless to the body.

According to an embodiment of the invention, the composition optionally further comprises at least one loading substance, said loading substance utilizing a gel as a loading platform. For example, the cargo is one, two or more of a hemostatic drug, an antibacterial drug, and a healing-promoting drug. Wherein the hemostatic agent, antibacterial agent and healing promoting agent may be selected from those known in the art, for example the hemostatic agent may be selected from thrombin, prothrombin complex, prothrombin, vitamin K₁And ethylenediamine diaceturate, preferably thrombin; for example, the antibacterial agent may be at least one of antibiotics, sulfonamides, imidazoles, nitroimidazoles, quinolones, and the like; the healing promoting drug may be at least one selected from vitamin E, epidermal growth factor, and the like.

According to an embodiment of the invention, the hemostatic composition is in the form of a gel, thus also referred to as "hemostatic gel composition", "hemostatic composition gel" or "hemostatic gel".

According to embodiments of the present invention, the composition may be coated on a substrate (e.g., a medical textile), and in use, a wound may be covered with the medical textile, and may be shaped by compression, suitable for use with a variety of wounds.

According to embodiments of the present invention, the composition may also be filled into suitable containers, such as syringes, medical plastic flexible bags, medical hoses, and the like, preferably syringes and hoses.

The invention also provides a preparation method of the composition, which comprises the following steps: mixing chitosan, fucoidan and water, and standing to form gel. Preferably, the chitosan and fucoidan sulfate have the weight ratio as described above.

According to an embodiment of the invention, the composition is prepared by a process comprising the steps of:

(1) respectively preparing chitosan aqueous solution and fucoidan aqueous solution;

preferably, the concentration of the chitosan aqueous solution is 0.1-0.5 g/mL; for example, 0.2 to 0.4 g/mL; illustratively, the concentration is 0.2g/mL, 0.3g/mL, 0.4 g/mL;

preferably, the concentration of the fucoidan sulfate aqueous solution is 0.5-1.5 g/mL; for example, 0.7 to 1.2 g/mL; illustratively, the concentration is 0.9g/mL, 1.0g/mL, 1.1 g/mL;

(2) pouring the chitosan aqueous solution into the fucoidan aqueous solution, uniformly mixing, and standing to obtain gel;

preferably, the chitosan aqueous solution is slowly poured into the fucoidan sulfate aqueous solution, and is stirred. Preferably, the mixing is also carried out under stirring conditions, for example stirring for 1 to 5h, preferably for 1.5 to 4h, exemplary stirring for 2 h. For example, the rotation speed of the stirring is 300-1000r/min, preferably 400-600r/min, and exemplary 500 r/min.

Preferably, the time of standing may be from 5 to 40h, such as from 8 to 24h, exemplary 10h, 12h, 15 h.

Preferably, the fucoidan is prepared by mixing the fucoidan and the chitosan in a weight ratio of 4: 1. Preferably, the aqueous solution of chitosan is 0.25g/mL, and the aqueous solution of fucoidan sulfate is 1 g/mL. Preferably, the volume ratio of the aqueous chitosan solution to the aqueous fucoidan sulfate solution is 1: 1.

According to an embodiment of the invention, the preparation process further optionally comprises the steps of: the gel is uniformly applied to a substrate, for example, the gel is uniformly applied to the medical textile.

According to an embodiment of the invention, the preparation process further optionally comprises the steps of: the gel is filled into a suitable container, such as a syringe, a soft medical plastic bag, a flexible medical tube, etc., preferably a syringe.

According to an embodiment of the invention, the preparation process further optionally comprises the steps of: a step of sterilizing the gel and/or the substrate coated with the gel.

The invention also provides application of the composition in preparing a hemostatic preparation.

The invention also provides a hemostatic preparation containing the composition, and the hemostatic preparation can be a patch, an ointment, an injection and the like.

According to the embodiment of the invention, the hemostatic preparation is used for hemostasis at non-compressible wound surfaces and/or irregular wound surfaces, such as penetrating injuries and the like caused by firearms (in single-soldier combat) such as bullets, sharp instruments and the like, and particularly for heavy bleeding at groin, armpit and other parts which cannot be suitable for traditional hemostatic products, the wound can be quickly closed, and the heavy bleeding can be prevented.

In the composition disclosed by the invention, the gel is formed by mixing chitosan, fucoidan and water. Chitosan is positively charged with amino groups in its structure under acidic conditions, and therefore, it easily binds to negatively charged blood components such as erythrocytes, leukocytes, platelets, etc., and forms a cell plug or a thromboembolus at a wound to inhibit bleeding. The fucoidan sulfate is easy to gel after being dissolved in water, and is mixed with chitosan which is rich in amino groups and has positive charges and can generate crosslinking with sulfate groups (negative charges) in the fucoidan sulfate to form gel by taking the fucoidan sulfate as a matrix. The schematic diagram of the gel network is shown in FIG. 2, wherein the positively charged one represents chitosan and the negatively charged one represents fucoidan.

The invention has the beneficial effects that:

1. the gel composition formed by chitosan and fucoidan belongs to a novel hemostatic dressing, and has better fluidity and plasticity by adjusting the proportion of the chitosan and the fucoidan, so that the balance between the adhesive capacity and the gel strength is achieved. Therefore, the gel composition can be coated on the surfaces of various complicated irregular wounds and has good sealing effect and hemostatic effect.

2. The gel composition provided by the invention has good biocompatibility and is harmless to the organism after being acted on the wound.

3. The preparation method of the gel composition provided by the invention has the advantages of mild conditions, simplicity in operation, higher yield, capability of production in enlarged quantity and capability of being effectively applied to industrial production.

4. The gel composition provided by the invention can be effectively applied to the field of hemostasis, and is particularly applied to non-compressible hemorrhage, irregular wound sealing and rapid hemostasis.

5. The gel preparation provided by the invention can better fill wound gaps and seal wound surfaces in incompressible bleeding models such as armpits, groin and the like, prevent blood from flowing out and play a role in stopping bleeding quickly. In addition, in the model of visceral hemorrhage, the tasks of quick sealing and hemostasis can be completed. The hemostatic dressing is suitable for on-site emergency hemostasis of deep major hemorrhage, wherein the conventional hemostatic dressings such as firearms puncture wound and the like cannot effectively fill hemostasis in single-soldier operation, can effectively prevent death caused by major hemorrhage, and strives for precious rescue time for next medical delivery and treatment.

Drawings

Fig. 1 is a picture of a hemostatic dressing and hemostatic powder product developed by the united states forces during the iraq and afghanistan wars. Wherein A is QuickCombatB isGauze, C isD isE is

F is

G is

FIG. 2 is a schematic diagram of the structure of the gel network formed by chitosan and fucoidan.

FIG. 3 is a flow characterization chart of hemostatic gels tested in example 5 of the present invention.

FIG. 4 is a photograph showing the hemostatic processes of hemostatic gels obtained by mixing chitosan and fucoidan sulfate according to the weight ratio of 1:1 (example 1) and 1:2 (example 2) in example 7 of the present invention.

FIG. 5 is a photograph showing the hemostatic processes of hemostatic gels obtained by mixing chitosan and fucoidan sulfate at a weight ratio of 1:4 (example 3) and 1:6 (example 4), respectively, tested in example 7 of the present invention.

FIG. 6 is a graph of the amount of bleeding from each hemostatic gel tested in example 7 of the invention in a mouse femoral artery hemostatic model.

FIG. 7 is a photograph of the wound surface and femoral artery vessel morphology 3 days after hemostasis of the femoral artery in mice tested in example 8 of the present invention.

Fig. 8 is a photograph of hemostasis in a mouse liver bleeding model for each of the hemostatic gels tested in example 9 of the present invention.

FIG. 9 is a graph of the amount of bleeding in a mouse model of liver bleeding for each hemostatic gel tested in example 9 of the present invention.

Fig. 10 is a photograph of the hemostatic process of each hemostatic gel tested in example 10 of the present invention in a rabbit liver bleeding model.

FIG. 11 is a graph of bleeding volume of each hemostatic gel tested in example 10 of the present invention in a rabbit liver bleeding model.

FIG. 12 is a photograph of the surface morphology of a liver wound after hemostasis for each hemostatic gel tested in example 10 of the present invention.

Fig. 13 is a photograph showing a method of constructing a model of rabbit femoral artery hemorrhage according to example 11 of the present invention.

Fig. 14 is a photograph showing the hemostatic process of each of the hemostatic materials according to example 11 of the present invention in a rabbit femoral artery hemorrhage model.

Fig. 15 is a graph of the amount of bleeding in a rabbit femoral artery bleeding model for each of the hemostatic materials tested in example 11 of the present invention.

Figure 16 is a photograph of the surface morphology of a femoral wound after hemostasis with the hemostatic gel tested in example 11 of the present invention.

FIG. 17 is a graph showing the results of the cytotoxicity of the hemostatic gel tested in example 12 of the present invention in MCF-7 cells.

FIG. 18 is a graph showing the results of cytotoxicity of hemostatic gels tested in example 12 of the present invention in 3T3 cells.

FIG. 19 is a photograph of the hemostasis of the thrombin loaded hemostatic gel tested in example 13 of the invention in a mouse femoral artery hemostatic model.

Detailed Description

The technical solution of the present invention will be further described in detail with reference to specific embodiments. It is to be understood that the following examples are only illustrative and explanatory of the present invention and should not be construed as limiting the scope of the present invention. All the technologies implemented based on the above-mentioned contents of the present invention are covered in the protection scope of the present invention.

Unless otherwise indicated, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods.