Hemostatic material capable of slowly releasing blood coagulation factors as well as preparation method and application thereof

Abstract

The invention provides a preparation method of a hemostatic material capable of slowly releasing blood coagulation factors, which comprises the following steps: S1, dispersing collagen into a buffer solution with the pH value of 4-8, adding the blood coagulation factors into the buffer solution, and uniformly mixing to obtain a mixture; s2, adding salt into the mixture to carry out salting-out precipitation, then retaining the generated precipitate in a salting-out solution, crushing, and uniformly mixing; s3, adjusting the temperature of the mixture to 2-6 DEG C, adding a coupling agent, stirring, and reacting for 1-10 hours; and S4, dialyzing the reaction product, and crushing a retention solution obtained by dialysis into gel, thereby obtaining the hemostatic material. The invention further provides a hemostatic material capable of slowly releasing the blood coagulation factors and application of the hemostatic material in the aspect of hemostasis. The hemostatic material provided by the invention can be slowly and continuously released under the condition that the activity of the blood coagulation factors is reserved, so that the local stable blood coagulation environment of a wound can be maintained for a long time, and the risk of postoperative bleeding relapse is remarkably reduced.

Description

Technical Field

[0001] This application relates to the field of medical hemostatic materials technology, specifically to hemostatic materials capable of slowly releasing clotting factors, their preparation methods, and their applications. Background Technology

[0002] Bleeding is one of the most common and difficult problems to control in surgery and trauma. When the body suffers severe trauma, the body's own blood clotting is insufficient to form a rapid clotting, so it is necessary to add hemostatic materials to the wound site to control bleeding. Ideal hemostatic materials have the following performance requirements: (1) good hemostatic effect; (2) good biocompatibility; (3) easy to use, without the need for secondary debridement.

[0003] Postoperative wound bleeding is a common complication of surgery. Its harm extends beyond the superficial phenomenon of "wound seepage," posing a multi-dimensional threat to the patient's postoperative recovery and even life by disrupting physiological balance, interfering with the treatment process, and inducing a chain of complications. Existing hemostatic products focus primarily on stopping bleeding during surgery, but there are no effective materials available to inhibit secondary bleeding after surgery.

[0004] Coagulation factors are a group of proteins in blood plasma whose main function is to promote blood clotting through a cascade reaction. Currently, they are primarily used clinically via intravenous injection, a systemic administration method that cannot achieve targeted or directed delivery. Furthermore, coagulation factors degrade rapidly in the blood, failing to achieve long-lasting hemostasis. Collagen sponges are commonly used hemostatic materials in surgery. Through physical hemostasis, the sponge structure absorbs exudate and aids blood clotting by compressing and filling the wound, exhibiting good hemostatic effects and absorbability. Coupling coagulation factors to collagen can significantly improve hemostatic efficacy.

[0005] CN 111643715A discloses a hemostatic gel composed of collagen and thrombin. In use, a lyophilized powder of collagen and thrombin is mixed with a gelling catalyst and then injected into the bleeding wound. However, due to the poor solubility of collagen, it preferentially cross-links thrombin, leading to excessive cross-linking. This not only fails to achieve cross-linking between collagen and thrombin but also causes thrombin polymerization and inactivation. Consequently, it cannot achieve slow, continuous release of thrombin for long-term hemostasis.

[0006] However, it is difficult to precisely control the reaction sites during the conjugation of collagen with coagulation factors, which can lead to the shielding of the hemostatic activity of collagen or the catalytic sites of coagulation factors, reducing their hemostatic effect and recyclability. Chemical reagents, temperature, pH, and other conditions during the conjugation process can easily disrupt the natural conformations of both collagen and coagulation factors, resulting in decreased activity. In large-scale production, it is difficult to precisely control the number of coagulation factors conjugated to each collagen molecule, making it difficult to guarantee batch consistency and affecting product stability. The conjugate must simultaneously meet the requirements of hemostatic efficiency and tissue compatibility to avoid inducing local inflammation or foreign body reactions.

[0007] There is an urgent need for an absorbable hemostatic material that can achieve slow and continuous release of thrombin to achieve long-lasting hemostasis, thus solving the problems encountered in clinical practice. Summary of the Invention

[0008] This application provides a method for preparing a hemostatic material capable of slowly releasing coagulation factors, comprising:

[0009] Step S1: Disperse collagen in a buffer solution with a pH of 4-8, add clotting factors to the buffer solution, mix well, and obtain a mixture;

[0010] Step S2: Add salt to the mixture to induce salting-out precipitation, then crush the resulting precipitate in the salting-out solution and mix thoroughly.

[0011] Step S3: Adjust the temperature of the mixture to 2~6℃, add the coupling agent, stir, and react for 1~10h;

[0012] Step S4: Dialyze the reaction product, and crush the retained solution obtained from dialysis into a gel to obtain the hemostatic material.

[0013] Preferably, the buffer solution is one of the following: phosphate buffer solution, carbonate buffer solution, Tris-HCl buffer system, and borate buffer system.

[0014] Preferably, the salt is one of sodium chloride, potassium chloride, magnesium chloride, sodium sulfate, magnesium sulfate, potassium phosphate, sodium phosphate, sodium citrate, and ammonium acetate.

[0015] Preferably, the coupling agent is a dialdehyde substance, and more preferably, the coupling agent is one of glutaraldehyde, succinaldehyde, and adipaldehyde.

[0016] Preferably, a dialysis membrane with a capacity of 5000-10000 Da is used during dialysis.

[0017] Preferably, in step S4, after dialysis of the reaction product, the method further includes: adding calcium-containing inorganic salts to the retention solution obtained by dialysis, and then pulverizing the retention solution at high speed into a gel to obtain the hemostatic material;

[0018] Preferably, the calcium-containing inorganic salt is one of calcium chloride, calcium citrate, calcium lactate, or calcium acetate. Calcium-containing inorganic salts can activate thrombin through calcium ions.

[0019] Preferably, the buffer solution is a phosphate buffer solution, the calcium-containing inorganic salt is calcium chloride, and by weight, the amount of collagen is 0.2 to 2 parts, the amount of coagulation factor is 50 to 2000 IU, the amount of calcium chloride is 0.01 to 0.1 parts, and the amount of phosphate buffer solution is 90 to 110 parts.

[0020] Preferably, the amount of collagen used is 0.2 to 0.5 parts, the amount of coagulation factor is 50 to 400 IU, the amount of calcium chloride is 0.01 to 0.03 parts, and the amount of phosphate buffer solution is 98 to 100 parts.

[0021] Preferably, in step S2, before salting out precipitation, the following steps are also included:

[0022] The mixture was frozen at -15°C to -25°C for 2 to 18 hours, and then slowly thawed at 2 to 10°C for 8 to 48 hours. This freezing and thawing process allows collagen and clotting factors to self-assemble through intermolecular hydrogen bonding, thus facilitating subsequent reactions.

[0023] Preferably, the salt is sodium chloride. Step S2 further includes: adding sodium chloride to the thawed mixture to prepare a 20%-28% sodium chloride solution for salting out precipitation, and then keeping the precipitate in the salting-out solution and pulverizing it at high speed to mix it evenly.

[0024] Preferably, the coupling agent is glutaraldehyde, and step S3 includes: adjusting the temperature of the mixture to 3~5℃, adding glutaraldehyde, stirring, and reacting for 3~5 hours.

[0025] Preferably, the collagen is one or more of fresh, undried bovine type I collagen, porcine collagen, or recombinant collagen, and the coagulation factor is one or more of human coagulation factor, bovine coagulation factor, or recombinant coagulation factor.

[0026] This application also provides a hemostatic material capable of slowly releasing clotting factors, wherein the hemostatic material is prepared by the above-described preparation method.

[0027] This application also provides the application of the above-mentioned hemostatic material capable of slowly releasing clotting factors in hemostasis. Preferably, this application also provides the application of the above-mentioned hemostatic material capable of slowly releasing clotting factors in the preparation of in vivo and in vitro wound hemostatic materials. More preferably, the hemostatic material is a wound hemostatic material for tissues, organs, and irregular areas of the body surface; or a functional material that promotes the healing of burns and wounds.

[0028] This application provides a method for preparing a hemostatic material capable of slowly releasing coagulation factors, the hemostatic material itself, and its application. It employs heterogeneous coupling technology. First, salting-out denaturation alters the spatial configuration of the coagulation factor, protecting its hydrophobic catalytic active centers. Then, a coupling agent is added. At this point, the cross-linking agent only reacts with the hydrophilic portion of the coagulation factor, achieving cross-linking at the solid-liquid interface. This allows for precise control of the cross-linking sites, minimizing the degradation of the coagulation factor's activity. Furthermore, the heterogeneous coupling method, through salting-out, causes the coagulation factor to precipitate from the solution, resulting in a heterogeneous reaction. This significantly reduces the coupling reaction rate, ensuring controllable amounts of collagen-coupled coagulation factors. When the clotting factors in the hemostatic material are cross-linked with collagen, their solubility in water changes. They can only be slowly released into the water as the collagen degrades, thus achieving a slow and continuous release of the clotting factors. This allows for the slow and continuous release of the clotting factors while preserving their activity, which helps maintain a stable local coagulation environment at the wound site for a longer period of time. It has a very good long-lasting hemostatic effect and significantly reduces the risk of postoperative bleeding recurrence.

[0029] This absorbable fluid hemostatic material has several core advantages: 1. Enhanced stability: The coupled structure protects coagulation factors from enzymatic degradation or environmental factors, prolonging their effective action time on the wound surface. 2. Improved targeting and retention: Collagen can adsorb onto damaged blood vessel walls, allowing coagulation factors to precisely anchor to the wound surface and reducing systemic diffusion. 3. Accelerated coagulation process: Collagen is a natural hemostatic matrix; after coupling, it allows coagulation factors to accumulate locally on the wound surface, significantly shortening coagulation time. 4. Reduced medication risks: The local accumulation effect reduces the total amount of coagulation factors used, lowering the risk of side effects such as thrombosis and immunogenicity that may be caused by systemic administration. Attached Figure Description

[0030] Figure 1 This is a bar chart showing the detection results of coagulation factor content in the products obtained in Example 4, Comparative Example 1, and Comparative Example 2;

[0031] Figure 2 This is a curve showing the release of coagulation factors from the product obtained in Example 4 under a simulated wound bleeding environment;

[0032] Figure 3 This is a curve showing the release of coagulation factors from the product obtained in Comparative Example 1 under a simulated wound bleeding environment.

[0033] Figure 4 This is a curve showing the release of clotting factors from the product obtained in Comparative Example 2 under simulated wound bleeding conditions. Detailed Implementation

[0034] The preferred embodiments of this application will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are merely some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are within the scope of protection claimed in this application.

[0035] Example 1

[0036] 200 mg of bovine type I collagen was dispersed in 100 ml of 0.1 M phosphate buffer solution at pH 4.5, and then 400 IU of recombinant human coagulation factor II was added. The mixture was then pulverized at 15,000 rpm and mixed thoroughly. The mixture was then frozen at -20°C for 8 hours and slowly thawed at 4-8°C for 24 hours. 20 g of sodium chloride was then added to prepare a 20% sodium chloride salting-out solution. After salting-out precipitation, the precipitate was retained in the salting-out solution, pulverized at high speed and mixed thoroughly, and then cooled to 4°C. 1.0 ml of 50% glutaraldehyde aqueous solution was then slowly added and stirred for 4 hours. The mixture was then loaded into a 120 ml dialysis membrane with a molecular weight cutoff of 10,000 Da and dialyzed five times in 1000 ml of 0.1 M phosphate buffer solution at pH 7.4. 20 mg of calcium chloride was added to the dialysis residue, and the mixture was pulverized at high speed to obtain 120 ml of gel-like fluid, which is the hemostatic material capable of slowly releasing coagulation factors.

[0037] Example 2

[0038] 500 mg of bovine type I collagen was dispersed in 100 ml of 0.1 M phosphate buffer solution at pH 5.0, and then 400 IU of recombinant human coagulation factor II was added. The mixture was then pulverized at 15,000 rpm and homogenized. The mixture was then frozen at -20°C for 8 hours and slowly thawed at 4-8°C for 24 hours. 20 g of sodium chloride was then added to prepare a 20% sodium chloride salting-out solution. After salting-out precipitation, the precipitate was retained in the salting-out solution, pulverized at high speed and homogenized, and then cooled to 4°C. 1.0 ml of 50% glutaraldehyde aqueous solution was then slowly added and stirred for 4 hours. The solution was then loaded into a 120 ml dialysis membrane with a molecular weight cutoff of 10,000 Da and dialyzed five times in 1000 ml of 0.1 M phosphate buffer solution at pH 7.4. 20 mg of calcium chloride was added to the dialysis residue, and the mixture was pulverized at high speed to obtain 120 ml of gel-like fluid, which is the hemostatic material capable of slowly releasing coagulation factors.

[0039] Example 3

[0040] 200 mg of bovine type I collagen was dispersed in 100 ml of 0.1 M phosphate buffer solution at pH 4.5, and then 400 IU of recombinant human coagulation factor X was added. The mixture was then pulverized at 15,000 rpm and mixed thoroughly. The mixture was then frozen at -20°C for 8 hours and slowly thawed at 4-8°C for 24 hours. 20 g of sodium chloride was then added to prepare a 20% sodium chloride salting-out solution. After salting-out precipitation, the precipitate was retained in the salting-out solution, pulverized at high speed and mixed thoroughly, and then cooled to 4°C. 1.0 ml of 50% glutaraldehyde aqueous solution was then slowly added and stirred for 4 hours. The mixture was then loaded into a 120 ml dialysis membrane with a molecular weight cutoff of 10,000 Da and dialyzed five times in 1000 ml of 0.1 M phosphate buffer solution at pH 7.4. 20 mg of calcium chloride was added to the dialysis residue, and the mixture was pulverized at high speed to obtain 120 ml of gel-like fluid, which is the hemostatic material capable of slowly releasing coagulation factors.

[0041] Example 4

[0042] 400 mg of bovine type I collagen was dispersed in 100 ml of 0.1 M phosphate buffer solution at pH 4.5. Then, 400 IU of human coagulation factor II, 200 IU of human coagulation factor VII, 300 IU of human coagulation factor X, and 400 IU of human coagulation factor IX were added. The mixture was then rapidly pulverized at 15,000 rpm and thoroughly mixed. The solution was then frozen at -20°C for 8 hours, followed by slow thawing at 4-8°C for 24 hours. 20 g of sodium chloride was added to prepare a 20% sodium chloride salting-out solution. After salting-out precipitation, the precipitate was retained in the salting-out solution, rapidly pulverized and thoroughly mixed, and then cooled to 4°C. 1.0 ml of 50% glutaraldehyde aqueous solution was then slowly added, and the mixture was stirred for 4 hours. The solution was then packed into a 120 ml dialysis membrane with a molecular weight cutoff of 10,000 Da and placed in 1000 ml of 0.1 M phosphate buffer solution at pH 7.4. Dialyze the sample seven times in M ​​phosphate buffer solution, add 50 mg of calcium chloride to the dialysis residue, and then pulverize it at high speed to obtain 120 ml of gel-like fluid, which is the hemostatic material that can slowly release clotting factors.

[0043] Comparative Example 1

[0044] Simple mixing of collagen and coagulation factors: 400 mg of bovine type I collagen was dispersed in 100 ml of 0.1 M phosphate buffer solution at pH 7.4, and then 400 IU of human coagulation factor II, 200 IU of human coagulation factor VII, 300 IU of human coagulation factor X, and 400 IU of human coagulation factor IX were added. The mixture was then pulverized at 15,000 rpm to obtain 100 ml of turbid liquid.

[0045] Comparative Example 2

[0046] Direct cross-linking of collagen with coagulation factors: 400 mg of bovine type I collagen was dispersed in 100 ml of 0.1 M phosphate buffer solution at pH 7.4. Then, 400 IU of human coagulation factor II, 200 IU of human coagulation factor VII, 300 IU of human coagulation factor X, and 400 IU of human coagulation factor IX were added. Then, 1.0 ml of 50% glutaraldehyde aqueous solution was slowly added. After stirring and reacting for 4 h, the mixture was loaded into a 120 ml dialysis membrane with a molecular weight cutoff of 10000 Da. The membrane was then dialyzed 7 times in 1000 ml of 0.1 M phosphate buffer solution at pH 7.4. After dialysis, 50 mg of calcium chloride was added, and the mixture was pulverized at high speed to obtain 120 ml of gel-like fluid.

[0047] Coagulation factor content detection

[0048] Take 1.2 ml of the gel-like fluids obtained in Example 4, Comparative Example 1, and Comparative Example 2 respectively, add 10 mL of NH4HCO3 buffer (50 mmol / L, pH 8.0), denature at 100 °C for 10 min, cool to room temperature, add 80 μg of sequence-pure trypsin, and enzymatically digest at 37 °C for 16 h. Analyze the coagulation factor content of the digested products by HPLC-MS using the characteristic peptide method.

[0049] The chromatographic column was a Zorbax SBC18 [150 × 2.1 mm (ID), 5 μm]; mobile phase A: water (containing 0.1% formic acid), mobile phase B: 60% acetonitrile (containing 0.1% formic acid); gradient 0–120 min, 5%–100% B; injection volume 50 μL; flow rate 0.2 mL / min. Mass spectrometry conditions: ion source spray voltage 4.5 kV, capillary temperature 300 °C, scan range m / z 300–2000, both precise mass number scan and secondary mass spectrometry scan were data-dependent scans, and the collision energy was 35%.

[0050] Test results as follows Figure 1 As shown. By Figure 1 It can be seen that, compared with Comparative Example 1, although the preparation method used in this invention cross-links collagen and coagulation factors, the hemostatic material obtained has little effect on the content of each coagulation factor; while the direct cross-linking method used in Comparative Example 2 leads to over-cross-linking of coagulation factors, and the content of coagulation factors is significantly reduced.

[0051] Coagulation factor sustained release effect test

[0052] Take 2 ml of the products obtained in Comparative Examples 1 and 2 of Example 4, respectively, and inject them into 4 ml of bovine serum. Incubate aseptically. Take 100 μL samples at 0 h, 12 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, 168 h, and 192 h. Detect the coagulation factor content according to the method described in the section on coagulation factor content detection above. The detection results of the product in Example 4 are as follows: Figure 2 As shown, the detection results of the product in Comparative Example 1 are as follows: Figure 3 As shown, the detection results of the product in Comparative Example 2 are as follows: Figure 4 As shown.

[0053] Depend on Figure 2 , Figure 3 and Figure 4 It is known that the hemostatic material prepared by the method of the present invention can achieve slow and long-term release of coagulation factors, thereby maintaining a stable local coagulation environment at the wound site for a longer period of time and significantly reducing the risk of postoperative bleeding recurrence. The material obtained by the simple mixing method will release all the coagulation factors in a short time, which will lead to rapid inactivation of the coagulation factors in the human body environment and will not achieve long-term hemostasis. The material obtained by the direct cross-linking method has a significantly reduced content of coagulation factors due to over-cross-linking and cannot achieve the hemostatic effect.

[0054] Based on the various embodiments of this application described above, in the absence of explicit denial or conflict, the technical features of one embodiment may be advantageously combined with one or more other embodiments.

[0055] While specific embodiments of this application have been described in detail by way of examples, those skilled in the art should understand that the above examples are for illustrative purposes only and are not intended to limit the scope of this application. Those skilled in the art should understand that modifications can be made to the above embodiments or equivalent substitutions can be made to some technical features without departing from the scope and spirit of this application. The scope of this application is defined by the appended claims.

Claims

1. A method for preparing a hemostatic material capable of slowly releasing clotting factors, characterized in that, include: Step S1: Disperse collagen in a buffer solution with a pH of 4-8, add clotting factors to the buffer solution, mix well, and obtain a mixture; Step S2: Add salt to the mixture to induce salting-out precipitation, then crush the resulting precipitate in the salting-out solution and mix thoroughly. Step S3: Adjust the temperature of the mixture to 2~6℃, add the coupling agent, stir, and react for 1~10h; Step S4: Dialyze the reaction product, and crush the retained solution obtained from dialysis into a gel to obtain the hemostatic material.

2. The preparation method according to claim 1, characterized in that, The buffer solution is one of the following: phosphate buffer solution, carbonate buffer solution, Tris-HCl buffer system, and borate buffer system; Preferably, the salt is one of sodium chloride, potassium chloride, magnesium chloride, sodium sulfate, magnesium sulfate, potassium phosphate, sodium phosphate, sodium citrate, and ammonium acetate; Preferably, the coupling agent is a dialdehyde substance, and more preferably, the coupling agent is one of glutaraldehyde, succinaldehyde, and adipaldehyde.

3. The preparation method according to claim 2, characterized in that, In step S4, after dialysis of the reaction product, the method further includes: adding calcium-containing inorganic salts to the retention solution obtained by dialysis, and then pulverizing the retention solution at high speed into a gel to obtain the hemostatic material; Preferably, the calcium-containing inorganic salt is one of calcium chloride, calcium citrate, calcium lactate, or calcium acetate.

4. The preparation method according to claim 3, characterized in that, The buffer solution is a phosphate buffer solution, and the calcium-containing inorganic salt is calcium chloride. By weight, the amount of collagen is 0.2 to 2 parts, the amount of coagulation factor is 50 to 2000 IU, the amount of calcium chloride is 0.01 to 0.1 parts, and the amount of phosphate buffer solution is 90 to 110 parts.

5. The preparation method according to claim 4, characterized in that, The dosage of collagen is 0.2-0.5 parts, the dosage of coagulation factor is 50-400 IU, the dosage of calcium chloride is 0.01-0.03 parts, and the dosage of phosphate buffer solution is 98-100 parts.

6. The preparation method according to claim 2, characterized in that, In step S2, before salting out precipitation, the following steps are also included: The mixture was frozen at -15℃ to -25℃ for 2 to 18 hours, and then slowly thawed at 2 to 10℃ for 8 to 48 hours. Preferably, the salt is sodium chloride. Step S2 further includes: adding sodium chloride to the thawed mixture to prepare a 20%-28% sodium chloride solution for salting out precipitation, and then keeping the precipitate in the salting-out solution and pulverizing it at high speed to mix it evenly.

7. The preparation method according to claim 6, characterized in that, The coupling agent is glutaraldehyde. Step S3 includes: adjusting the temperature of the mixture to 3~5℃, adding glutaraldehyde, stirring, and reacting for 3~5 hours.

8. The preparation method according to claim 1, characterized in that, Collagen is one or more of fresh, undried bovine type I collagen, porcine skin collagen, or recombinant collagen; coagulation factors are one or more of human coagulation factors, bovine coagulation factors, or recombinant coagulation factors.

9. A hemostatic material capable of slowly releasing clotting factors, characterized in that, The hemostatic material is prepared by the preparation method according to any one of claims 1-8.

10. The application of the hemostatic material of claim 9, which is capable of slowly releasing clotting factors, in hemostasis.

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