A covalently cross-linked chitosan hemostatic material and a preparation method thereof

Abstract

The invention discloses a chitosan hemostatic material formed by covalent crosslinking and a preparation method thereof, and relates to the field of medical technology. The chitosan hemostatic material formed by covalent crosslinking is obtained by chemically crosslinking chitosan through covalent bonds using a new crosslinking agent; the crosslinking agent used is 1,2:4,5-dianhydroxylitol. The preparation method of the chitosan hemostatic material formed by covalent crosslinking, the steps are as follows: 1) chitosan is made into an aqueous solution; 2) the crosslinking agent is added to the chitosan aqueous solution, stirred and mixed, and freeze-dried. The present invention adopts 1,2:4,5-dianhydroxylitol to crosslink chitosan under acidic conditions. Wherein, the ethylene oxide group in 1,2:4,5-dianhydroxylitol undergoes a ring-opening reaction with the hydroxyl group on the chitosan under acidic conditions, and a crosslinked product is formed by generating an ether bond. The crosslinking agent used in the method of the present invention is stable in nature, biodegradable, safe and non-toxic.

Description

Technical Field

[0001] The invention relates to the field of medical technology, and more particularly to a covalently cross-linked chitosan hemostatic material and a preparation method thereof. Background Art

[0002] Bleeding of human tissue often occurs in various situations, such as surgery, arterial puncture (arterial interventional diagnosis and interventional treatment), daily life trauma and war injuries. Large amounts of blood loss in the human body can lead to serious consequences and even threaten life. Uncontrolled blood loss from trauma is the top two causes of death in civilians and battlefields in daily life. Therefore, the research and development of hemostatic materials has always been of great significance.

[0003] So far, in the treatment of surgical bleeding, the hemostatic materials used clinically can be divided into three categories according to their different mechanisms of action: aggregation agents, coagulants, and adhesives.

[0004] and coagulation factors; coagulants can quickly start the endogenous coagulation pathway; adhesives adhere to blood cells and surrounding tissues through cross-linking, and locally aggregate into blocks to fill the wound. In addition, natural plant hemostatics have also received attention from researchers at home and abroad. Chitosan, also known as polyglucosamine (1-4)-2-amino-β-D glucose, is a degradable organic matter extracted from the shells of shrimp and shellfish, which has good wound healing and hemostatic effects. The hemostatic effect of chitosan sponge is believed to be that the capillary action of the sponge quickly absorbs serum, increases the concentration of red blood cells and platelets on its surface, causes them to aggregate on the surface of the sponge and seal the wound, stops bleeding before blood clots form, and reduces blood loss. At the same time, the positive charge on the surface of the chitosan sponge can activate the coagulation mechanism involving red blood cells, thereby accelerating the coagulation of blood containing anticoagulants such as heparinized blood. . Studies have shown that chitosan is metabolized by the lysosomal pathway in the body, and the product is glucosamine, which has good biological safety and does not affect the recanalization of deep blood vessels.

[0005] At the same time, there are also domestic and foreign patents that disclose the research results of chitosan hemostatic agents. For example, U.S. Patent US4532134 involves the optimization of conditions such as the degree of deacetylation, molecular weight, solvent used for dissolution, concentration of solution after dissolution, and pH of the chitosan material, and prepares chitosan hemostatic agents. Animal experiments have proved that chitosan hemostatic agents not only have good hemostatic effects, but also have the effects of inhibiting fibrosis and promoting tissue regeneration.

[0006] Patents for preparing hemostatic sponges using chitosan as a raw material include Chinese invention patents 200810198164.4, 201010291789.2, 200680030822.8, 200710115108.5, 201110382475.8, 201210545888.8, and 201210033793.8. Among them, Chinese invention patent 201010291789.2 discloses a novel chitosan extraction method, and a chitosan hemostatic sponge is prepared by the new extraction process, and a porous and easily absorbable hemostatic material is obtained after freeze-drying, and animal experiments have proved that this hemostatic material has excellent hemostatic properties. Studies have found that the mechanical strength of chitosan sponges prepared directly using chitosan as raw material is poor, so some patents for related improved methods have appeared one after another. For example, Chinese invention patent 200810198164.4 and Chinese invention patent applications 200710115108.5, 200210009638.9, and 200910248822.0 disclose related technologies for preparing hemostatic sponges by mixing other natural polysaccharide materials with chitosan. However, the relevant preparation process is complicated and the wet stability of the obtained hemostatic agent is poor (dissolves when encountering blood or becomes gelatin). For example, the porous hemostatic sponge prepared with chitosan and gelatin as the main components, which is the most studied at present, is easily dissolved in wounds with heavy bleeding and cannot play a coagulation role (Wu Bin, Sun Yun, etc. A method for preparing hemostatic sponges for auxiliary materials [P]. Chinese invention patent application 201210545888.8, 2012-12-14).

[0007] In order to solve the above problems, the technology of preparing hemostatic agents by cross-linking chitosan with cross-linking agents has been developed. For example, patent application 201310463753.1 discloses the technology of adding glycerol to chitosan solution, cross-linking and curing with formaldehyde, freezing and forming, washing off the formaldehyde, slicing and encapsulating. It is said that the chitosan prepared by this method has good tissue compatibility with the human body, is non-toxic, promotes skin regeneration, accelerates healing, and reduces scars; patent application 03112762.2 also provides a method for preparing sponges by cross-linking chitosan and other polysaccharides with glutaraldehyde or formaldehyde. However, the cross-linking agents used in these patents are toxic themselves.

[0008] Patent CN104474576A uses 1,4-butanediol diglycidyl ether (BDDE) as a crosslinking agent for chitosan, but its raw material 1,4-butanediol is a precursor of γ-hydroxybutyric acid (GHB, a central nervous system poison), which is oxidized in vivo to produce GHB. Patent CN104474575A uses polyethylene glycol diglycidyl ether (GDGE) as a crosslinking agent for chitosan, and the raw material polyethylene glycol or ethylene glycol is also a chemical raw material, not a pure natural non-toxic ingredient.

[0009] In summary, the currently disclosed chitosan hemostatic material preparation technology cannot fully meet clinical needs due to various reasons. Summary of the invention

[0010] In view of this, the present invention provides a novel covalently cross-linked chitosan hemostatic material and a preparation method thereof.

[0011] In order to achieve the above object, the present invention adopts the following technical solution:

[0012] A covalently cross-linked chitosan hemostatic material is obtained by chemically cross-linking chitosan with a cross-linking agent through covalent bonds.

[0013] Preferably, the cross-linking agent is 1,2:4,5-dianhydro-xylitol (Xylitol, 1,2:4,5-dianhydro-) (CAS Registry Number 63976-13-6).

[0014] Its significant advantage is that its water solubility is higher than that of 1,4-butanediol diglycidyl ether. More importantly, in the cross-linked product, its metabolite is xylitol, which is a natural and non-toxic ingredient, so its safety is far superior to the metabolite of 1,4-butanediol diglycidyl ether.

[0015] Preferably, the molar ratio of the 1,2:4,5-dianhydroxylitol to the monosaccharide units in the chitosan molecule is 0.001:1 to 1:1.

[0016] The choice of this ratio is critical because it directly affects the degree of cross-linking and performance of the material. A ratio below 0.001:1 results in too little cross-linking, making the material easily soluble in the blood, while a ratio above 1:1 can result in excessive cross-linking, making the material too hard and unable to adhere tightly to the wound.

[0017] Preferably, the molar ratio of the 1,2:4,5-dianhydroxylitol to the monosaccharide units in the chitosan molecule is 0.05:1 or 0.10:1 or 0.20:1.

[0018] This specific ratio selection can significantly shorten the hemostasis time, thereby achieving the effect of rapid hemostasis. By precisely controlling this molar ratio, it can be ensured that the material can quickly form an effective hemostatic barrier when it contacts the wound, reduce blood loss, and buy precious treatment time for the patient.

[0019] Another object of the present invention is to provide a method for preparing a covalently cross-linked chitosan hemostatic material, the synthesis route of which is as follows:

[0020]

[0021] The specific steps include:

[0022] 1) preparing chitosan into a 1-5% (W / V) chitosan solution;

[0023] 2) adding the cross-linking agent into the chitosan solution, stirring and mixing the mixture thoroughly, placing the mixture in a mold and letting it stand for a period of time, then pouring the mixture into a freeze drying container and freeze drying the mixture to obtain the hemostatic material.

[0024] Preferably, the concentration of the chitosan solution in step 1) is 2% or 3% (W / V).

[0025] Preferably, the chitosan solution is a chitosan aqueous solution prepared by using water, an aqueous solution containing an organic solvent, or an acid aqueous solution.

[0026] Preferably, the aqueous solution containing an organic solvent is an aqueous solution of dimethyl sulfoxide with a concentration of 0-25% (W / V).

[0027] Preferably, the acid aqueous solution includes an organic acid aqueous solution or an inorganic acid aqueous solution; the concentration of the organic acid aqueous solution is 0.5-10% (W / V); the concentration of the inorganic acid aqueous solution is 0.5-3% (W / V).

[0028] Preferably, the organic acid includes any one of formic acid, acetic acid, propionic acid, glycolic acid and methanesulfonic acid; the inorganic acid includes any one of hydrochloric acid, sulfuric acid, phosphoric acid and sulfurous acid.

[0029] Preferably, the aqueous acid solution is an aqueous acetic acid solution.

[0030] Preferably, the standing time is 1-2 hours.

[0031] It can be seen from the above technical solution that compared with the prior art, the present invention has the following beneficial effects:

[0032] The present invention uses 1,2:4,5-dianhydroxylitol as a crosslinking agent to crosslink chitosan under acidic conditions. The ethylene oxide group of 1,2:4,5-dianhydroxylitol undergoes a ring-opening reaction with the hydroxyl group on chitosan under acidic conditions to form a crosslinked product by generating an ether bond. The crosslinking agent used in the method of the present invention is safe, non-toxic and biodegradable. The degradation products, xylitol and 2-aminoglucose, are both very safe molecules. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0034] Figure 1 This is a photo of the chitosan sponge prepared in Example 1 of the present invention. DETAILED DESCRIPTION

[0035] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] Example 1

[0037] This embodiment provides a method for preparing a novel covalently cross-linked chitosan hemostatic material, as follows:

[0038] Chitosan (viscosity 100 mPa·s, deacetylation degree 90%) (0.64 g, molar weight of monosaccharide unit 42.4 mmol) was weighed and dissolved in 30 ml of 2% acetic acid aqueous solution to obtain a 2% chitosan solution (w / v), which was shaken on a shaker at 40°C for 24 h to completely dissolve. 1,2:4,5-dianhydroxylitol (0.05 eq) was added, mechanically stirred and mixed, and then poured into a mold with a diameter of 6 cm and allowed to stand for 1-2 h, and freeze-dried to obtain a sponge-like product with a thickness of 5 mm.

[0039] Example 2

[0040] This embodiment provides a method for preparing a novel covalently cross-linked chitosan hemostatic material, which is completely the same as the steps in Example 1, except that the amount of 1,2:4,5-dianhydroxylitol added is 0.10 eq.

[0041] Example 3

[0042] This example provides a method for preparing a novel covalently cross-linked chitosan hemostatic material, which is completely the same as the steps in Example 1, except that the amount of 1,2:4,5-dianhydroxylitol added is 0.20 eq.

[0043] Solubility test

[0044] In order to prove that the prepared chitosan sponge is formed by cross-linking, the solubility test of the chitosan sponge products without and with the addition of cross-linking agent was carried out.

[0045] 1. 2% (V / V) acetic acid aqueous solution

[0046] The sponge products with the least amount of crosslinking agent added, i.e., Example 1 and the sponge without crosslinking agent, were selected, 60 mg each, placed in 30 ml centrifuge tubes, and 20 ml of 2% acetic acid aqueous solution was added to each. The sponges were placed in an oscillator (rotation speed of 300 rpm) and shaken for 30 minutes. The results showed that the sponge without crosslinking agent was completely dissolved in the 2% acetic acid aqueous solution, while Example 1 only swelled but did not dissolve.

[0047] 2. Aqueous solution

[0048] The sponge products with the least amount of crosslinking agent added, i.e., the sponge formed in Example 1 and the sponge formed without the crosslinking agent, each about 1×1 cm, were selected and placed in a disposable culture dish, and 10 ml of aqueous solution was added to each. After standing for 10 minutes, the results showed that the sponge without the crosslinking agent was in a gel state, while the sponge formed in Example 1 became a swollen sponge.

[0049] Water absorption test

[0050] The water absorption mass of the hemostatic sponge product of the present invention is 25 to 40 times that of the sponge itself. The experimental method refers to the experimental method in (Fu Xiaobing, Wu Zhigu, Modern Trauma Accessories Theory and Practice, Beijing: Chemical Industry Press, 2007: 651) to test the water absorption of the hemostatic sponge product in the specific implementation scheme. Since the phosphate buffer is closer to human body fluids than water, the distilled water used in the experiment in the reference was replaced with a phosphate buffer pH 7.4 in the test.

[0051] Coagulation test

[0052] 1. Cut off the tail of the rat to stop bleeding

[0053] Taking rat tail amputation model as experimental object, the rat tail is cut off in the middle, and the hemostatic effect of gauze, chitosan, and chitosan with different cross-linking degrees is compared. The average results of three experiments are shown in Table 1. In the experimental group, the hemostatic time mean of chitosan with three different cross-linking degrees is 68s, 67s, and 70s, respectively. The chitosan control group hemostatic time mean is 95s. The gauze control group still slowly oozes blood after 180s. In the control group (patent CN104474576A and CN104474575A) with BDDE and GDGE as cross-linking agent (0.05eq), the clotting time is about 80 seconds. In the experimental group, the hemostatic sponges with cross-linking ratios of 0.05, 0.10, and 0.20, there is no significant difference in hemostatic time, and compared with the control group, the hemostatic time is significantly reduced.

[0054] Table 1. The time for hemostasis of tail amputation in rats

[0055]

[0056] 2. Coagulation test

[0057] The coagulation rate of the porous sponge-like hemostatic dressing prepared by the examples 1 to 3 of the present invention and the comparative chitosan was compared. The test method was as follows: the porous sponge-like hemostatic dressing prepared by the examples 1 to 3 and the comparative preparation was sampled, the samples were cut into 1 cm × 1 cm squares, placed in a 50 mL glass beaker, and kept warm at 37 ° C for 1 min in a constant temperature incubator; then 0.25 mL of blood sample was dripped into the sample until the blood was completely absorbed; after being placed in the constant temperature incubator for 1 min, 20 mL of pure water solution was slowly added to the beaker, and then the beaker was placed in a constant temperature incubator at 37 ° C for 10 min, and then 10 mL of solution was taken and centrifuged at 800 rpm / min for 5 min to obtain the supernatant. The obtained supernatant was detected by ultraviolet spectrophotometer at a wavelength of 540 nm, and the absorption value was recorded. As a reference, 0.25 mL of blood was directly dissolved in 20 mL of pure water as a 100% control.

[0058] Calculate according to the following formula: V = (1-X) × 100%;

[0059] Where: V—product coagulation rate;

[0060] X is the ratio of the absorption value of the test sample to the absorption value of the control sample. The test was repeated three times. The average test results are shown in Table 2.

[0061] Table 2 Coagulation performance data of Examples 1 to 3 and comparative chitosan

[0062]

[0063] The above experimental results show that the novel cross-linked chitosan hemostatic sponge of the present invention has excellent hemostatic performance. It is superior to chitosan in terms of coagulation time and coagulation rate, and the coagulation rate is equivalent to that of the BDDE and GDGE control groups, but the coagulation time is significantly shortened. The human hemostatic mechanism mainly relies on platelet aggregation to activate coagulation factors to form blood clots. At present, the coagulation mechanism known to start coagulation takes at least 90 seconds (Luo Xiaoxun. Chitosan hemostatic agent [P] Chinese invention patent application, 200710028001.7, 2007-5-14). The hemostatic mechanism of chitosan is mainly tissue adhesion, and it has no intrinsic coagulation activity. As the only cationic alkaline polysaccharide in nature, chitosan can also cause red blood cell aggregation by reacting with cell surface anions, and finally adhere to tissue to seal wounds. In addition to the hemostatic effect, chitosan has a similar molecular structure to the cell mucopolysaccharide matrix, which can provide a favorable microenvironment for tissue regeneration and has antibacterial, moisturizing, analgesic and other effects. The xylitol cross-linked chitosan of the present invention will have great application potential in medical treatment and first aid.

[0064] In this specification, each embodiment is described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the embodiments can be referred to each other. For the device disclosed in the embodiment, since it corresponds to the method disclosed in the embodiment, the description is relatively simple, and the relevant parts can be referred to the method part.

[0065] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A covalently cross-linked chitosan hemostatic material, characterized in that: It is obtained by chemically cross-linking chitosan through covalent bonds using a cross-linking agent; The cross-linking agent is 1,2:4,5-dianhydroxylitol; The molar ratio of the 1,2:4,5-dianhydroxylitol to the monosaccharide units in the chitosan molecule is 0.05:1 or 0.10:1 or 0.20:

1.

2. The method for preparing a covalently cross-linked chitosan hemostatic material according to claim 1, characterized in that: The synthetic route is as follows: The specific steps include: 1) preparing chitosan into a 1-5% (W / V) chitosan solution; 2) adding a cross-linking agent to the chitosan solution, stirring and mixing the mixture, placing the mixture in a mold and letting it stand for a period of time, and then placing the mixture in a freeze drying container and freeze drying the mixture to obtain the hemostatic material.

3. The method for preparing a covalently cross-linked chitosan hemostatic material according to claim 2, characterized in that: The chitosan solution is a chitosan aqueous solution prepared by using water, an aqueous solution containing an organic solvent, or an acid aqueous solution.

4. The method for preparing a covalently cross-linked chitosan hemostatic material according to claim 3, characterized in that: The aqueous solution containing an organic solvent is a dimethyl sulfoxide aqueous solution with a concentration of 0-25% (W / V).

5. The method for preparing a covalently cross-linked chitosan hemostatic material according to claim 3, characterized in that: The acid aqueous solution includes an organic acid aqueous solution or an inorganic acid aqueous solution; the concentration of the organic acid aqueous solution is 0.5-10% (W / V); the concentration of the inorganic acid aqueous solution is 0.5-3% (W / V).

6. The method for preparing a covalently cross-linked chitosan hemostatic material according to claim 5, characterized in that: The organic acid includes any one of formic acid, acetic acid, propionic acid, glycolic acid and methanesulfonic acid; The inorganic acid includes any one of hydrochloric acid, sulfuric acid, phosphoric acid and sulfurous acid.

7. The method for preparing a covalently cross-linked chitosan hemostatic material according to claim 2, characterized in that: The standing time is 1-2h.

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