Nanometer multilayer composite hemostatic material and preparation method and application thereof
By alternating layers of collagen fibers and coagulation factor molecules in a nano-multilayer composite structure, the problem of poor postoperative wound bleeding inhibition effect of existing hemostatic materials is solved, achieving slow release of coagulation factors and long-lasting hemostatic effect.
Patent Information
- Application Number
- CN202610166550.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-02-05
- Publication Date
- 2026-05-01
AI Technical Summary
Existing hemostatic materials are not effective in inhibiting postoperative wound bleeding, and systemic administration of coagulation factors cannot achieve targeted and directional delivery, leading to thrombin polymerization and inactivation, thus failing to achieve long-term hemostasis.
By employing a nano-multilayer composite structure, alternating layers of collagen fibers and coagulation factor molecules are arranged. Through a sandwich-like layering preparation technique, the collagen fiber layers encapsulate the coagulation factors, enabling their slow release.
It achieves long-term, slow release of clotting factors, significantly reduces the risk of postoperative bleeding recurrence, and maintains a stable local clotting environment at the wound site.
Smart Images
Figure CN121944200A_ABST
Abstract
Description
Nanoscale multilayer composite hemostatic materials, their preparation methods and applications Technical Field
[0001] This application relates to the field of medical hemostatic materials technology, specifically to nano-multilayer composite hemostatic materials, their preparation methods, and 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 and do not require 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 helps blood coagulate by compressing and filling the wound, exhibiting good hemostatic effects and absorbability.
[0005] CN111643715A discloses a hemostatic gel composed of collagen and thrombin. In use, the collagen and thrombin are mixed with a lyophilized powder and a gelling catalyst, then injected into the bleeding wound. However, the gelling catalysts used, such as formaldehyde and glutaraldehyde, are cytotoxic and can affect wound healing. Furthermore, 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, preventing the slow, sustained release of thrombin and achieving long-term hemostasis. Summary of the Invention
[0006] To at least partially solve the above problems, according to a first aspect of this application, this application provides a nano-multilayer composite hemostatic material capable of slowly releasing coagulation factors, the nano-multilayer composite hemostatic material comprising multiple layers of oriented collagen fibers and multiple layers of oriented coagulation factor molecules, wherein the collagen fiber layers and the coagulation factor molecule layers are arranged alternately.
[0007] Preferably, the thickness of the collagen fiber layer is 3-20 μm, and the thickness of the coagulation factor molecular layer is 1-15 μm; preferably, the thickness of the collagen fiber layer is 5-10 μm, and the thickness of the coagulation factor molecular layer is 2-10 μm.
[0008] Preferably, the thickness of the nano-multilayer composite hemostatic material is 80-250 μm, and more preferably, the thickness of the nano-multilayer composite hemostatic material is 100-200 μm.
[0009] Preferably, the collagen fiber layer is prepared solely from animal-derived collagen or recombinant collagen, or prepared jointly from animal-derived collagen and recombinant collagen.
[0010] Preferably, the coagulation factor molecular layer is prepared by human coagulation factor or recombinant human coagulation factor alone, or by human coagulation factor and recombinant human coagulation factor together.
[0011] According to a second aspect of this application, this application provides a method for preparing a nano-multilayer composite hemostatic material capable of slowly releasing coagulation factors, comprising:
[0012] Step S1: Prepare collagen solution, coagulation factor solution, and subphase;
[0013] Step S2: Add the subphase to a first container with a rectangular opening, such that the liquid surface of the subphase is a first preset distance from the top of the opening of the first container. After the liquid surface of the subphase settles, set two sliding push plates on opposite sides of the opening of the first container. The extension direction of the push plates is the width direction of the opening. The two push plates are arranged along the length direction of the opening. Both push plates are in contact with the liquid surface of the subphase. The two opposite edges of the two push plates, the two opposite length directions of the first container walls, and the liquid surface of the subphase together form a groove-shaped first receiving space. Slowly add the collagen solution to the groove-shaped first receiving space, let it stand, apply a pushing force to move the push plates to reduce the distance between the two push plates until the pushing force increases sharply. Stop moving the push plates and keep the push plates in their current positions. Insert the substrate sheet for the attachment of the nano-multilayer composite hemostatic material vertically downward into the first container, so that the substrate sheet is at least partially submerged in the collagen solution. Then pull the substrate sheet vertically upward and dry it to obtain a substrate sheet with a collagen fiber layer attached to its surface. Preferably, the substrate sheet is a mica sheet or a silicon sheet.
[0014] Step S3: Add the subphase to a second container with a rectangular opening, ensuring the liquid surface of the subphase is a second predetermined distance from the top of the container opening. After the subphase liquid surface settles, place two sliding push plates on opposite sides of the second container opening. The push plates extend in the width direction of the opening and are arranged along the length direction of the opening. Both push plates are in contact with the subphase liquid surface, and their opposite edges, along with the two opposite length walls of the second container and the subphase liquid surface, together form a trough-shaped second receiving space. Slowly add the coagulation factor solution into the trough-shaped second receiving space, allow it to stand, and then apply a pushing force to move the push plates. The distance between the two push plates is reduced until the pushing force increases sharply. Then, the push plates are stopped moving and their positions are kept unchanged. The substrate sheet with a collagen fiber layer attached to its surface, obtained in step S2, is inserted vertically downward into the second container, so that the substrate sheet is at least partially submerged in the coagulation factor solution. Then, the substrate sheet is pulled vertically upward and dried to obtain a substrate sheet with a coagulation factor molecular layer attached to the collagen fiber layer. Preferably, a sharp increase in pushing force means that the applied force increases several times or changes by orders of magnitude at the same push plate moving distance, for example, the applied force increases by 3 to 20 times, preferably by 8 to 15 times.
[0015] Step S4: Repeat steps S2 and S3 sequentially to obtain a multilayered nanomaterial arranged layer by layer. The arrangement of the multilayered nanomaterial is: oriented collagen fiber layer - oriented coagulation factor molecule layer - oriented collagen fiber layer - oriented coagulation factor molecule layer...
[0016] Step S5: Peel the nano-multilayer material off the substrate to obtain a thin film-like nano-multilayer composite hemostatic material. Preferably, after peeling the nano-multilayer material off the substrate, freeze-dry it to obtain a thin film-like nano-multilayer composite hemostatic material.
[0017] Preferably, step S1 includes:
[0018] Preparation of collagen solution: Add 0.01-0.05 parts of collagen to 50-200 parts of pure water, stir for 5-20 minutes, then slowly add hydrochloric acid or acetic acid to adjust the pH to 2.5-3.5, continue stirring for 1-2 hours, centrifuge at 8000-10000 r / min for 5-20 minutes, and take out the supernatant to obtain the collagen solution; preferably, the pure water is 95-105 parts.
[0019] Preparation of coagulation factor solution: Add 10-50 IU of coagulation factor to 50-200 parts of pure water, stir for 5-20 min, centrifuge at 8000-10000 r / min for 5-20 min, and take out the supernatant to obtain the coagulation factor solution; preferably, the pure water is 95-105 parts.
[0020] Preparation of the subphase: Prepare a phosphate buffer solution with a molar concentration of 0.1M-0.5M and a pH of 6.5-8.0, then add sodium chloride to prepare a sodium chloride concentration of 20%-28% as the subphase solution. Preferably, the sodium chloride concentration is 24%-26%.
[0021] Preferably, in step S2, the first container is a cuboid with an aspect ratio of 1.5-2, a first preset distance of 2-5 mm, a collagen solution level of 1-100 μm in the first container, a standing time of 5-20 min, a substrate width consistent with the first container, and a substrate length half the distance between the two push plates after the push plates stop moving. During drying, the drying temperature is controlled at 30-35℃ and the drying time at 5-24 h. After drying, the substrate is washed 5-10 times with deionized water to obtain a substrate with a collagen fiber layer attached to its surface. Preferably, the collagen solution level in the first container is 10-30 μm.
[0022] In step S3, the second container is a cuboid with an aspect ratio of 1.5-2 and a second preset distance of 2-5 mm. The liquid level of the coagulation factor solution added to the second container is 1-100 μm, and the standing time is 5-20 min. During drying, the drying temperature is controlled at 30-35℃ and the drying time is 5-24 h. After drying, the substrate is washed 5-10 times with deionized water to obtain a substrate sheet with a coagulation factor molecular layer attached to the collagen fiber layer. Preferably, the liquid level of the coagulation factor solution added to the second container is 5-10 μm.
[0023] According to a third aspect of this application, this application provides the application of the above-mentioned nano-multilayer composite hemostatic material in the preparation of in vivo and in vitro wound hemostatic materials.
[0024] Preferably, the in vivo and in vitro wound hemostatic materials are wound hemostatic materials for tissues, organs, and irregular areas of the body surface, or functional materials that promote the healing of burns and wounds.
[0025] The nano-multilayer composite hemostatic material capable of slowly releasing coagulation factors provided in this application, along with its preparation method and application, enables the slow release of coagulation factors while preserving their activity. Specifically, a sandwich-like layered preparation technique is used to prepare a layer of oriented collagen fibers that encapsulates the coagulation factors. As the collagen layer degrades, the coagulation factors are gradually released, thereby achieving a long-lasting and slow-release effect of the coagulation factors.
[0026] The nano-multilayer composite hemostatic material provided in this application combines collagen and coagulation factors through a sandwich-like structure. By utilizing the good biocompatibility of collagen and the coagulation function of coagulation factors, the two achieve a synergistic effect, which can achieve slow and continuous release of coagulation factors while preserving their activity. This helps to maintain a stable local coagulation environment at the wound site for a longer period of time and significantly reduces the risk of postoperative bleeding recurrence. Attached Figure Description
[0027] Figure 1 shows the atomic force microscopy protein orientation detection results of the nano-multilayer composite hemostatic material sample prepared in Example 1;
[0028] Figure 2 is a comparison chart of the coagulation factor content detection of the hemostatic materials prepared in Example 3 and Comparative Example 1;
[0029] Figure 3 is a curve showing the release of coagulation factors by the nano-multilayer composite hemostatic material prepared in Example 3 under a simulated wound bleeding environment.
[0030] Figure 4 shows the release curve of coagulation factors of the hemostatic material prepared in Comparative Example 1 under a simulated wound bleeding environment. Detailed Implementation
[0031] 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 embodiments of the present invention, and not all embodiments. 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. In the following embodiments, unless otherwise specified, the operating methods used are conventional operating methods, the equipment used is conventional equipment, the equipment and materials used in each embodiment are the same, and the parts specified in each embodiment and comparative example are parts by mass.
[0032] The terms "first," "second," and similar words used in this application do not indicate any order, quantity, or importance, but are merely used to distinguish different parts. Words such as "including" or "contains" mean that the element preceding the word encompasses the element listed after it, and do not exclude the possibility of encompassing other elements as well. Terms such as "above," "below," "left," and "right" are used only to indicate relative positional relationships; when the absolute position of the described object changes, this relative positional relationship may also change accordingly.
[0033] Example 1
[0034] 1) Preparation of subphase: Prepare a phosphate buffer solution with a molar concentration of 0.5M and a pH of 7.4, and then add sodium chloride to prepare a sodium chloride solution with a concentration of 25%;
[0035] 2) Preparation of collagen solution A: Add 0.02 parts of collagen to 100 parts of pure water, stir for 10 minutes, then slowly add hydrochloric acid or acetic acid to adjust the pH to 3.0, continue stirring for 2 hours, centrifuge at 10000 r / min for 10 minutes, and take out the supernatant to obtain collagen solution A;
[0036] 3) Preparation of coagulation factor solution B: Add 40 IU of coagulation factor II (lyophilized powder) to 100 parts of pure water, stir for 10 min, centrifuge at 10000 r / min for 10 min, and take out the supernatant to obtain coagulation factor II solution B;
[0037] 4) Oriented collagen fiber layer: In a rectangular container with dimensions of 10cm, 6cm, and 5cm, add the subphase liquid until the subphase liquid surface is 2mm from the top of the container. After the liquid surface is still, place rectangular plastic rods with the same width as the container at both ends as push plates. Then, slowly drip collagen solution A into the container to form a liquid surface height of 10μm. After standing for 10 minutes, slowly move the push plates towards the center of the container and monitor the force applied. Stop when the force applied increases sharply. Then, slowly insert a mica sheet with the same width as the container and a length of 4cm into the solution at the center of the container until it is completely submerged. Then, slowly lift it up and place it in a constant temperature drying oven. Dry at 30℃ for 12 hours. Then wash with 2mL of deionized water 8 times to obtain the collagen fiber layer attached to the substrate.
[0038] 5) Oriented Coagulation Factor Molecular Layer: In a cuboid container with dimensions of 10cm, 6cm, and 5cm, add subphase liquid until the subphase liquid surface is 2mm from the top of the container. After the liquid surface is still, place rectangular plastic rods with the same width as the container at both ends as push plates. Then, slowly drip coagulation factor solution B into the container to form a liquid surface height of 5μm. After standing for 10 minutes, slowly move the push plates towards the center of the container and monitor the force applied. Stop when the force applied increases sharply. Then, slowly insert the mica sheet obtained in step 4) into the solution at the center of the container until it is completely submerged. Then slowly lift it up and place it in a constant temperature drying oven. Dry at 32℃ for 12 hours. Then wash it 5 times with 2mL of deionized water to obtain an oriented coagulation factor molecular layer attached to an oriented collagen fiber layer.
[0039] 6) Repeat steps 4) and 5) 10 times to obtain a series of sequentially arranged nanolayers. After peeling off the substrate, a thin film-like nanolayered composite hemostatic material is obtained and freeze-dried. The average thickness of the nanolayered composite hemostatic material was measured to be 97 μm using atomic force microscopy, the average thickness of the collagen fiber layer was 6.3 μm, and the average thickness of the coagulation factor molecular layer was 3.4 μm.
[0040] Example 2
[0041] 1) Preparation of subphase: Prepare a phosphate buffer solution with a molar concentration of 0.3M and a pH of 7.0, and then add sodium chloride to prepare a sodium chloride solution with a concentration of 25%;
[0042] 2) Preparation of collagen solution A: Add 0.01 parts of collagen to 100 parts of pure water, stir for 10 minutes, then slowly add hydrochloric acid or acetic acid to adjust the pH to 3.0, continue stirring for 2 hours, centrifuge at 10000 r / min for 10 minutes, and take out the supernatant to obtain collagen solution A;
[0043] 3) Preparation of coagulation factor solution B: Add 30 IU of coagulation factor VII (lyophilized powder) to 100 parts of pure water, stir for 10 min, centrifuge at 10000 r / min for 10 min, and take out the supernatant to obtain coagulation factor VII solution B;
[0044] 4) Oriented collagen fiber layer: In a rectangular container with dimensions of 10cm, 6cm, and 5cm, add the subphase liquid until the subphase liquid surface is 2mm from the top of the container. After the liquid surface is still, place rectangular plastic rods with the same width as the container at both ends as push plates. Then, slowly drip collagen solution A into the container to form a liquid surface height of 10μm. After standing for 10 minutes, slowly move the push plates towards the center of the container and monitor the force applied. Stop when the force applied increases sharply. Then, slowly insert a mica sheet with the same width as the container and a length of 4cm into the solution at the center of the container until it is completely submerged. Then, slowly lift it up and place it in a constant temperature drying oven. Dry at 30℃ for 12 hours. Then wash with 2mL of deionized water 8 times to obtain the collagen fiber layer attached to the substrate.
[0045] 5) Oriented Coagulation Factor Molecular Layer: In a rectangular container with dimensions of 10cm, 6cm, and 5cm, the subphase liquid is added until the subphase liquid surface is 2mm from the top of the container. After the liquid surface is still, rectangular plastic rods with the same width as the container are placed at both ends of the container as pushers. Then, coagulation factor solution B that can form a liquid surface height of 5μm is slowly dripped into the container. After standing for 10 minutes, the rectangular pusher is slowly moved towards the center of the container, and the force applied is monitored. Stop when the force applied increases sharply. Then, the mica sheet obtained in step 4) is slowly inserted into the solution at the center of the container until it is completely submerged. Then, it is slowly lifted up and placed in a constant temperature drying oven. The temperature is controlled at 32℃ and dried for 12 hours. Then, it is washed 5 times with 2mL of deionized water to obtain an oriented coagulation factor molecular layer attached to the oriented collagen fiber layer.
[0046] 6) Repeat steps 4) and 5) 20 times to obtain a series of sequentially arranged nanolayers. After peeling them off the substrate, a thin film-like nanolayered composite hemostatic material is obtained and freeze-dried. The average thickness of the nanolayered composite hemostatic material was measured to be 116 μm using atomic force microscopy, the average thickness of the collagen fiber layer was 3.2 μm, and the average thickness of the coagulation factor molecular layer was 2.6 μm.
[0047] Example 3
[0048] 1) Preparation of subphase: Prepare a phosphate buffer solution with a molar concentration of 0.3M and a pH of 7.2, and then add sodium chloride to prepare a sodium chloride solution with a concentration of 25%;
[0049] 2) Preparation of collagen solution A: Add 0.02 parts of collagen to 100 parts of pure water, stir for 10 minutes, then slowly add hydrochloric acid or acetic acid to adjust the pH to 3.0, continue stirring for 2 hours, centrifuge at 10000 r / min for 10 minutes, and take out the supernatant to obtain collagen solution A;
[0050] 3) Preparation of coagulation factor solution B: Add 50 IU of coagulation factor II, 30 IU of coagulation factor VII, 20 IU of coagulation factor IX, and 40 IU of coagulation factor X (all lyophilized powders) to 100 parts of pure water, stir for 10 min, centrifuge at 10000 r / min for 10 min, and take out the supernatant to obtain coagulation factor solution B.
[0051] 4) Oriented collagen fiber layer: In a rectangular container with dimensions of 10cm, 6cm, and 5cm, add the subphase liquid until the subphase liquid surface is 2mm from the top of the container. After the liquid surface is still, place rectangular plastic rods with the same width as the container at both ends as push plates. Then, slowly drip collagen solution A into the container to form a liquid surface height of 20μm. After standing for 10 minutes, slowly move the push plates towards the center of the container while monitoring the force applied. Stop when the force applied increases sharply. Then, slowly insert a mica sheet with the same width as the container and a length of 4cm into the solution at the center of the container until it is completely submerged. Then, slowly lift it up and place it in a constant temperature drying oven. Dry at 30℃ for 12 hours. Then wash it 8 times with 2mL of deionized water to obtain the collagen fiber layer attached to the substrate.
[0052] 5) Oriented Coagulation Factor Molecular Layer: In a cuboid container with dimensions of 10cm, 6cm, and 5cm, add the subphase liquid until the subphase liquid surface is 2mm from the top of the container. After the liquid surface is still, place rectangular plastic rods with the same width as the container at both ends as push plates. Then, slowly drip coagulation factor solution B into the container to form a liquid surface height of 10μm. After standing for 10 minutes, slowly move the push plates towards the center of the container and monitor the force applied. Stop when the force applied increases sharply. Then, slowly insert the mica sheet obtained in step 4) into the solution at the center of the container until it is completely submerged. Then slowly lift it up and place it in a constant temperature drying oven. Dry at 32℃ for 12 hours. Then wash it 5 times with 2mL of deionized water to obtain an oriented coagulation factor molecular layer attached to the oriented collagen fiber layer.
[0053] 6) Repeat steps 4) and 5) 15 times to obtain a series of sequentially arranged nanolayers. After peeling off the substrate, a thin film-like nanolayered composite hemostatic material is obtained and freeze-dried. The average thickness of the nanolayered composite hemostatic material was measured to be 240 μm using atomic force microscopy, the average thickness of the collagen fiber layer was 6.2 μm, and the average thickness of the coagulation factor molecular layer was 9.8 μm.
[0054] Comparative Example 1
[0055] 1) Preparation of collagen solution A: Add 0.01 parts of collagen to 100 parts of pure water, stir for 10 minutes, then slowly add hydrochloric acid or acetic acid to adjust the pH to 3.0, continue stirring for 2 hours, centrifuge at 10000 r / min for 10 minutes, and take out the supernatant to obtain collagen solution A;
[0056] 2) Preparation of coagulation factor solution B: Add 50 IU of coagulation factor II, 30 IU of coagulation factor VII, 20 IU of coagulation factor IX, and 40 IU of coagulation factor X to 100 parts of pure water, stir for 10 min, centrifuge at 10000 r / min for 10 min, and take out the supernatant to obtain coagulation factor solution B.
[0057] 3) Mix the solutions obtained in steps 1) and 2) together, stir for 1 hour, and freeze-dry to obtain a collagen + coagulation factor mixed hemostatic material.
[0058] Results Analysis
[0059] I. Detection of coagulation factor levels
[0060] The hemostatic materials prepared in Example 3 and Comparative Example 1 were added to 10 mL of NH4HCO3 buffer (50 mmol / L, pH 8.0), denatured at 100 °C for 10 min, cooled to room temperature, and then 80 μg of sequence-pure trypsin was added. The mixture was then enzymatically digested at 37 °C for 16 h. The coagulation factor content of the digested products was analyzed by HPLC-MS using the characteristic peptide method.
[0061] The chromatographic column was a Zorbax SB C18 [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℃, scan range m / z 300–2000, both precise mass number scan and secondary mass spectrometry scan were data-dependent scans, collision energy 35%. The detection results are shown in Figure 2. As can be seen from Figure 2, the hemostatic material obtained by the preparation method used in the example has little effect on the content of various coagulation factors.
[0062] II. Detection of Coagulation Factor Sustained-Release Effect
[0063] Two g of the hemostatic materials prepared in Example 3 and Comparative Example 1 were injected into 4 ml of bovine serum and allowed to stand aseptically. 100 μL samples were taken at 0 h, 12 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, 168 h, and 192 h, respectively. The coagulation factor content was detected according to the method described in Part I of the Results Analysis. The detection results of the product in Example 3 are shown in Figure 3, and the detection results of the product in Comparative Example 1 are shown in Figure 4. It should be noted that, since the coagulation factor content was detected in the in vitro experiment, the coagulation factor was not consumed. Therefore, in Figure 4, the coagulation factor was released completely in a short time, and its concentration remained high for a long time. In contrast, in Figure 3, due to the slow release of the coagulation factor, it was not completely released even at 192 h, but continued to be released thereafter, increasing the concentration of the coagulation factor.
[0064] As can be seen from the comparison of Figures 3 and 4, the absorbable nano-multilayer composite hemostatic material prepared by the method of the present invention can achieve slow release of coagulation factors, thereby achieving slow and long-term release of coagulation factors, thus 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.
[0065] 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.
[0066] 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 nano-multilayer composite hemostatic material capable of slowly releasing clotting factors, characterized in that, The nano-multilayer composite hemostatic material includes multiple layers of collagen fiber arranged in different orientations and multiple layers of coagulation factor molecules arranged in different orientations, wherein the collagen fiber layers and the coagulation factor molecules are arranged alternately.
2. The nano-multilayer composite hemostatic material according to claim 1, characterized in that, The thickness of the collagen fiber layer is 3-20 μm, and the thickness of the coagulation factor molecular layer is 1-15 μm; preferably, the thickness of the collagen fiber layer is 5-10 μm, and the thickness of the coagulation factor molecular layer is 2-10 μm.
3. The nano-multilayer composite hemostatic material according to claim 1, characterized in that, The thickness of the nano-multilayer composite hemostatic material is 80-250 μm, preferably 100-200 μm.
4. The nano-multilayer composite hemostatic material according to claim 1, characterized in that, The collagen fiber layer is prepared solely from animal-derived collagen or recombinant collagen, or jointly from animal-derived collagen and recombinant collagen.
5. The nano-multilayer composite hemostatic material according to claim 1, characterized in that, The coagulation factor molecular layer is prepared from human coagulation factor or recombinant human coagulation factor alone, or from human coagulation factor and recombinant human coagulation factor together.
6. A method for preparing a nano-multilayer composite hemostatic material capable of slowly releasing coagulation factors, characterized in that, include: Step S1: Prepare collagen solution, coagulation factor solution, and subphase; Step S2: Add the subphase to a first container with a rectangular opening, such that the liquid surface of the subphase is a first preset distance from the top of the opening of the first container. After the liquid surface of the subphase settles, place two sliding push plates on opposite sides of the opening of the first container. The push plates extend in the width direction of the opening and are arranged along the length direction of the opening. Both push plates are in contact with the liquid surface of the subphase, and the two opposite edges of the two push plates, the two opposite walls of the first container in the length direction, and the liquid surface of the subphase together form a trough-shaped first receiving space. Slowly add the collagen solution into the trough-shaped first receiving space, let it stand, and apply a push plate. The force is used to move the pusher plate to reduce the distance between the two pusher plates until the force increases sharply. Then, the pusher plate is stopped, and its position is kept unchanged. The substrate sheet for the attachment of the nano-multilayer composite hemostatic material is inserted vertically downward into the first container, so that the substrate sheet is at least partially submerged in the collagen solution. Then, the substrate sheet is pulled vertically upward and dried to obtain a substrate sheet with a collagen fiber layer attached to its surface. Step S3: The subphase is added to the second container with a rectangular opening, so that the liquid surface of the subphase is a second preset distance from the top of the opening of the second container. After the liquid surface of the subphase settles, two [unclear] are placed on opposite sides of the opening of the second container. Two sliding push plates extend along the width of the opening, with both plates arranged along the length of the opening. Both push plates are in contact with the liquid surface of the subphase. The two opposite edges of the two push plates, the two opposite walls of the second container along their lengths, and the liquid surface of the subphase together form a trough-shaped second receiving space. The coagulation factor solution is slowly added to this trough-shaped second receiving space and allowed to stand. A pushing force is applied to move the push plates to reduce the distance between them until the pushing force increases sharply. At this point, the movement of the push plates is stopped, and their positions are kept unchanged. The surface with collagen attached, obtained in step S2, is then vertically inserted into the second container. A substrate sheet with a white fibrous layer is prepared by immersing at least part of the substrate sheet in a coagulation factor solution, then pulling the substrate sheet vertically upwards and drying it to obtain a substrate sheet with a coagulation factor molecular layer attached to the collagen fiber layer; Step S4: Repeat steps S2 and S3 sequentially to obtain a nano-multilayer material arranged layer by layer, the arrangement of the nano-multilayer material being oriented collagen fiber layer - oriented coagulation factor molecular layer - oriented collagen fiber layer - oriented coagulation factor molecular layer... Step S5: Peel the nano-multilayer material off the substrate sheet to obtain a thin film-like nano-multilayer composite hemostatic material.
7. The method according to claim 6, characterized in that, Step S1 includes: Collagen solution preparation: Add 0.01-0.05 parts of collagen to 50-200 parts of pure water, stir for 5-20 minutes, then slowly add hydrochloric acid or acetic acid to adjust the pH to 2.5-3.5, continue stirring for 1-2 hours, centrifuge at 8000-10000 r / min for 5-20 minutes, and take out the supernatant to obtain the collagen solution; Coagulation factor solution preparation: Add 10-50 IU of coagulation factor to 50-200 parts of pure water, stir for 5-20 minutes, then centrifuge at 8000-10000 r / min for 5-20 minutes, and take out the supernatant to obtain the coagulation factor solution; Subphase preparation: Prepare a phosphate buffer solution with a molar concentration of 0.1M-0.5M and a pH of 6.5-8.0, and then add sodium chloride to prepare a sodium chloride concentration of 20%-28% as the subphase solution.
8. The method according to claim 6, characterized in that, In step S2, the first container is a cuboid with an aspect ratio of 1.5-2 and a preset distance of 2-5 mm. The collagen solution added to the first container is 1-100 μm high, and the settling time is 5-20 min. The width of the substrate is the same as that of the first container, and the length of the substrate is half the distance between the two push plates after the push plates stop moving. During drying, the drying temperature is controlled at 30-35℃, and the drying time is 5-24 h. After drying, the surface is washed 5-10 times with deionized water to obtain the desired surface. A substrate sheet with a collagen fiber layer attached; in step S3, the second container is a cuboid with an aspect ratio of 1.5-2, a second preset distance of 2-5 mm, the liquid level of the coagulation factor solution added to the second container is 1-100 μm, the standing time is 5-20 min, the drying temperature is controlled at 30-35℃ and the drying time is 5-24 h, and after drying, it is washed with deionized water 5-10 times to obtain a substrate sheet with a coagulation factor molecular layer attached to the collagen fiber layer.
9. The application of the nano-multilayer composite hemostatic material according to any one of claims 1-5 in the preparation of in vivo and in vitro wound hemostatic materials.
10. The application according to claim 9, characterized in that, The in vivo and in vitro wound hemostatic materials are wound hemostatic materials for tissues, organs, and irregular areas of the body surface, or functional materials that promote the healing of burns and wounds.