Pore size adjustable natural silk nanofiber hemostatic material and preparation method and application thereof

Abstract

The application provides a natural silk nanofiber hemostatic material with adjustable pore size and a preparation method and application thereof, and the preparation process comprises the following steps: firstly, preparing a hyaluronic acid activation liquid and a silk nanofiber suspension with preset concentrations respectively; then, adding the silk nanofiber suspension into the obtained hyaluronic acid activation liquid to obtain a mixed liquid; fully stirring and reacting the mixed liquid to obtain a reaction liquid; then, adding a preset volume of an adjusting agent into the obtained reaction liquid and fully stirring to obtain a liquid to be frozen; and finally, freeze-drying the liquid to be frozen to obtain a porous sponge with ultrahigh porosity and microporous structure. The microporous structure obtained by adding the freezing adjusting agent can intercept platelets and red blood cells while concentrating blood, so that the effect of rapid hemostasis is achieved. Under the synergistic action of hyaluronic acid, silk nanofiber, the adjusting agent and a suitable freeze-drying temperature, the high-performance hemostatic material is obtained.

Description

Technical Field

[0001] This invention relates to the field of hemostatic materials technology, and in particular to a natural silk nanofiber hemostatic material with adjustable pore size, its preparation method and application. Background Technology

[0002] Developing rapid and effective hemostatic materials has long been a research hotspot in the field of hemostasis. In recent years, hemostatic materials made from nanofibers have shown great promise in this field due to their high aspect ratio, high specific surface area, high porosity, and good flexibility. Specifically, the high aspect ratio and high specific surface area of ​​nanofiber materials reproduce the fine nanoscale morphology of fibrin fibers produced during natural hemostasis; the porous structure facilitates the concentration of blood cells, platelets, and other coagulation factors; simultaneously, the high porosity and interconnected pore sizes endow the material with high and rapid water absorption efficiency, facilitating the rapid absorption of large amounts of blood and the concentration of blood cells and coagulation factors to achieve rapid hemostasis. Furthermore, hemostatic materials prepared from nanofibers have significant advantages such as low basis weight, high permeability, and controllable structural morphology, making them of significant application value and development potential in the field of hemostasis.

[0003] A study has prepared a highly absorbent nanofiber aerogel for hemostatic purposes by combining polyvinyl alcohol (PVA) and silica nanofibers (ACS nano, 2023, 17(12), 11507-11520). The nanofiber structure of this three-dimensional sponge has high porosity and water absorption, which is conducive to rapid blood absorption. However, the materials used are silica nanofibers and PVA, which have insufficient biocompatibility and are difficult to biodegrade.

[0004] Silk, as an abundant natural protein fiber, possesses excellent biocompatibility, mechanical properties, and biodegradability, and is widely used in tissue engineering and biomedical applications. At the microscopic scale, natural silk is composed of multi-layered nanofibers; the deconstructed and peeled silk nanofibers retain the original basic structure and function of fibroin fibers at the nanoscale. Natural silk nanofibers prepared from silk are natural protein nanofiber materials with good biocompatibility and biodegradability, showing great potential as hemostatic materials. Hyaluronic acid, an anionic polysaccharide found in the human body, has a high water absorption rate and can rapidly concentrate clotting factors by absorbing blood, making it widely used in the biomedical field. Simultaneously, as an anionic natural polysaccharide, hyaluronic acid can interact with positively charged amino acids on the XII chain of clotting factor, accelerating the coagulation cascade to achieve chemical hemostasis. Utilizing the excellent properties of silk nanofibers and the coagulation effect of hyaluronic acid, designing composite materials of silk nanofibers and hyaluronic acid as hemostatic materials has significant application value. Patent publication CN112316914A discloses a silk fibroin micro / nanofiber microsphere, its preparation method, and its application. The method involves mixing a prepared silk fibroin micro / nanofiber suspension with a hyaluronic acid solution, followed by a cross-linking reaction. After electrostatic spraying and freeze-drying, the silk fibroin micro / nanofiber microspheres are obtained. However, the silk fibroin micro / nanofiber microspheres prepared by this method have uncontrollable size and pore size, low yield, and are unsuitable for large-scale preparation. Furthermore, the size of these microspheres is unsuitable for reagent handling in clinical applications and makes them unsuitable for use as hemostatic materials.

[0005] In view of this, it is necessary to design an improved hemostatic material of natural silk nanofiber with adjustable pore size, as well as its preparation method and application, in order to solve the above problems. Summary of the Invention

[0006] The purpose of this invention is to provide a hemostatic material of natural silk nanofibers with adjustable pore size, its preparation method, and its application. The preparation process first involves preparing a hyaluronic acid activating solution to form an amine-reactive O-acyl isourea intermediate. Then, a silk nanofiber suspension is added to the hyaluronic acid activating solution, where the amine-reactive O-acyl isourea intermediate reacts rapidly with the amino groups in the silk nanofibers. Under the synergistic effect of bonding and entanglement, a preliminary network structure is formed. Next, a regulator is added to adjust the pore size. The pore size is further adjusted during the freeze-drying process. Under the synergistic effect of hyaluronic acid, silk nanofibers, regulator, and a suitable freeze-drying temperature, a high-performance hemostatic material with a porous structure is obtained.

[0007] To achieve the above-mentioned objectives, this invention provides a method for preparing a natural silk nanofiber hemostatic material with adjustable pore size, comprising the following steps:

[0008] S1. Prepare hyaluronic acid activation solution and silk nanofiber suspension at preset concentrations respectively;

[0009] S2. Add the silk nanofiber suspension to the hyaluronic acid activation solution obtained in step S1 to obtain a mixture; stir the mixture thoroughly to obtain a reaction solution;

[0010] S3. Add a preset volume of regulator to the reaction solution obtained in step S2 and stir thoroughly to obtain a freezing solution; freeze-dry the freezing solution to obtain the natural silk nanofiber hemostatic material with adjustable pore size.

[0011] As a further improvement of the present invention, in step S3, the volume fraction of the regulator in the liquid to be cooled is 1% to 20%.

[0012] As a further improvement of the present invention, in step S3, the regulator includes one or more of alcohols, phenols, ethers, and ketones that are miscible with water and volatile; the alcohol includes one or more of methanol, ethanol, and ethylene glycol.

[0013] As a further improvement of the present invention, in step S2, the mass of the hyaluronic acid in the mixture is 1% to 20% of the mass of the silk nanofibers.

[0014] As a further improvement of the present invention, the preparation of the hyaluronic acid activation solution specifically involves dissolving an appropriate amount of hyaluronic acid in deionized water to obtain a hyaluronic acid solution with a mass concentration of 0.1% to 10%; then adding a crosslinking agent to the hyaluronic acid solution, and after stirring and reacting thoroughly, obtaining the hyaluronic acid activation solution.

[0015] As a further improvement of the present invention, in step S3, the freeze-drying is carried out at -196 to -20°C.

[0016] As a further improvement of the present invention, in step S1, the silk is natural silk, including domestic silkworm silk or tussah silk; the diameter of the silk nanofibers is 30-1000 nm; and the mass concentration of the silk nanofiber suspension is 1%-5%.

[0017] As a further improvement of the present invention, the crosslinking agent is a mixture of morpholine ethanesulfonic acid, N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

[0018] The present invention also provides a natural silk nanofiber hemostatic material with adjustable pore size, which is prepared by the above-described preparation method of natural silk nanofiber hemostatic material with adjustable pore size; the pore size of the natural silk nanofiber hemostatic material with adjustable pore size is 0.1-20 μm.

[0019] The present invention also provides an application of a natural silk nanofiber hemostatic material with adjustable pore size, which is applied to the fields of hemostasis and skin repair.

[0020] The beneficial effects of this invention are:

[0021] (1) The method for preparing the pore size-adjustable natural silk nanofiber hemostatic material provided by the present invention firstly prepares a hyaluronic acid activating solution to form an amine-reactive O-acyl isourea intermediate, which provides favorable conditions for the subsequent reaction between hyaluronic acid and silk fibers. Then, the silk nanofiber suspension is added to the hyaluronic acid activating solution. The amine-reactive O-acyl isourea intermediate reacts rapidly with the amino groups in the silk nanofiber to form amide bonds, which allows the hyaluronic acid and the silk nanofiber molecular chains to bond together. At the same time, different silk nanofibers are intertwined with each other. Under the synergistic effect of bonding and entanglement, a network structure is formed, which endows the hemostatic material with good water resistance and mechanical properties. Next, a regulator is added to the reaction solution to adjust the pore size. As freeze-drying proceeds, on the one hand, the continuous evaporation of water and the regulator causes a fine-tuning of the network structure formed by the transparent hyaluronic acid and silk nanofibers; on the other hand, the presence of the regulator can control the ice crystal growth rate of the aqueous solution. With the synergistic effect of the network structure and a suitable ice crystal growth rate, a hemostatic material with adjustable pore size is obtained. The microporous structure can absorb blood while intercepting coagulation factors, giving the hemostatic sponge excellent hemostatic properties. Simultaneously, hyaluronic acid has a large number of carboxyl groups, which accelerate the coagulation cascade through anionic activation, achieving rapid hemostasis.

[0022] (2) This invention uses silk nanofibers as the main material and low-content hyaluronic acid as a polymeric binder with good affinity to the silk nanofibers. A cryoregulator can effectively reduce the surface tension of the natural silk nanofiber suspension, thereby reducing the growth rate and nucleation size of ice crystals during freezing, resulting in a nanofiber hemostatic material with a microporous structure. The raw materials are widely available, non-toxic, and safe. The preparation method is flexible, controllable, and simple, which is conducive to industrialization.

[0023] (3) The natural silk nanofiber hemostatic material with adjustable pore size prepared by the invention is biodegradable, has good biocompatibility (good biosafety), high porosity, large specific surface area, high water absorption rate of liquid, good biocompatibility and mechanical properties, and has good functions such as water absorption and retention, hemostasis, and wound healing promotion. It can be used as a biomedical material. Attached Figure Description

[0024] Figure 1 1a is a physical image of the natural silk nanofiber hemostatic material with adjustable pore size obtained in Example 1; 1b is a physical image of the natural silk nanofiber hemostatic material with adjustable pore size obtained in Example 2.

[0025] Figure 2 a is a physical image of the natural silk nanofiber hemostatic material with adjustable pore size obtained in Comparative Example 4; 2b is a physical image of the natural silk nanofiber hemostatic material with adjustable pore size obtained in Example 3.

[0026] Figure 3 a and Figure 3 c is a scanning electron microscope image of the pore size-tunable natural silk nanofiber hemostatic material obtained in Comparative Example 4, with scale bars of 100 μm and 20 μm, respectively; Figure 3 b and Figure 3 Image d is a scanning electron microscope image of the pore size-adjustable natural silk nanofiber hemostatic material obtained in Example 3, with scale bars of 100 μm and 20 μm, respectively.

[0027] Figure 4 4a is a stability diagram of the pore size-adjustable natural silk nanofiber hemostatic material obtained in Comparative Example 2 in water; 4b is a stability diagram of the pore size-adjustable natural silk nanofiber hemostatic material obtained in Example 3 in water.

[0028] Figure 5 The image shows a scanning electron microscope (SEM) image of the pore size-adjustable natural silk nanofiber hemostatic material obtained in Example 5. The scale bar is 20 μm.

[0029] Figure 6 The hemolysis rate diagram is shown for the pore size-adjustable natural silk nanofiber hemostatic material obtained in Example 3.

[0030] Figure 7 The hemolysis rate diagram is shown for the pore size-adjustable natural silk nanofiber hemostatic material obtained in Example 10.

[0031] Figure 8 The in vitro coagulation index diagram shows the pore size adjustable natural silk nanofiber hemostatic materials prepared in Comparative Example 4 and Example 3.

[0032] Figure 9 The hemostasis time diagram for the rat tail hemostasis model using the pore size-adjustable natural silk nanofiber hemostatic material prepared in Comparative Example 4 and Example 3.

[0033] Figure 10 The blood loss figure is shown for the pore size adjustable natural silk nanofiber hemostatic material prepared in Comparative Example 4 and Example 3, used in a rat tail hemostasis model. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0035] It should also be noted that, in order to avoid obscuring the present invention with unnecessary details, only the structures and / or processing steps closely related to the present invention are shown in the accompanying drawings, while other details that are not closely related to the present invention are omitted.

[0036] Additionally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0037] This invention provides a method for preparing a natural silk nanofiber hemostatic material with adjustable pore size, comprising the following steps:

[0038] S1. Preparation of silk nanofiber suspension and hyaluronic acid solution:

[0039] S11. Preparation of hyaluronic acid activation solution:

[0040] Dissolve an appropriate amount of hyaluronic acid in deionized water to obtain a hyaluronic acid solution with a mass concentration of 0.1% to 10%. Then add a cross-linking agent to the hyaluronic acid solution and stir for 1 to 2 hours to allow the reaction to proceed fully, thus obtaining an activated hyaluronic acid solution.

[0041] The crosslinking agent is a mixture of morpholine ethanesulfonic acid, N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

[0042] In this process, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) in the crosslinking agent reacts with the carboxyl group in hyaluronic acid to form an amine-reactive O-acyl isourea intermediate, which activates hyaluronic acid and provides favorable conditions for the subsequent reaction between hyaluronic acid and silk fibers.

[0043] S12. Preparation of silk nanofiber suspension:

[0044] After degumming, the silk was placed in a swelling solution (a mixture of calcium nitrate, ethanol, and water, with a volume ratio of ethanol to water of 1:2–6 and a mass ratio of calcium nitrate to the total volume of ethanol and water of 1–10 g:100 mL) and placed in an oven at 50–80 °C for 60–80 h to swell the silk and weaken the binding force between the nanofibers in the silk. After removal, it was washed three times with deionized water and placed in a crusher at a speed of 7000–9000 r / min with a bath ratio of 1 g:100–200 mL (i.e., 1 g silk: 100–200 mL water) for 80–100 min to mechanically crush the silk fibers. The fibers were then filtered three times and freeze-dried to obtain silk micro / nanofibers.

[0045] Take an appropriate amount of silk micro / nanofibers and add them to deionized water to obtain a silk nanofiber suspension with a mass concentration of 1% to 5%.

[0046] The silk used is natural silk, including domesticated silkworm silk or tussah silk. The diameter of the obtained silk nanofibers ranges from 30 to 1000 nm.

[0047] S2. Crosslinking:

[0048] The silk nanofiber suspension obtained in step S1 is added to the hyaluronic acid activation solution to obtain a mixture. After the mixture is stirred and reacted thoroughly, a reaction solution is obtained.

[0049] Specifically, in the mixture, the mass of hyaluronic acid is 1% to 20% of the mass of the silk nanofibers. Preferably, the mass of hyaluronic acid is 1% to 10% of the mass of the silk nanofibers.

[0050] In this process, silk nanofibers are used as the main material, and a low content of hyaluronic acid is used as a polymeric binder with good affinity to the silk nanofibers. The amine-reactive O-acyl isourea intermediate in the hyaluronic acid activation solution reacts rapidly with the amino groups in the silk nanofibers to form amide bonds, allowing the hyaluronic acid and silk nanofiber molecular chains to bond together. Simultaneously, different silk nanofibers intertwine, forming a network structure under the synergistic effect of bonding and entanglement, giving the hemostatic material good water resistance and mechanical properties. When the hyaluronic acid content is too high, it first affects the bonding between the hyaluronic acid and silk nanofiber molecular chains, resulting in uneven and insufficient bonding. Furthermore, it affects the entanglement between different silk nanofibers, thus disrupting the hemostatic network structure formed by the silk nanofibers. Simultaneously, during the subsequent freeze-drying process, it affects the interaction between the regulator and hyaluronic acid, further affecting the controllability of the silk nanofiber hemostatic network structure, thus impacting the pore structure of the hemostatic material, and reducing both mechanical strength and specific surface area.

[0051] S3. Freeze-drying:

[0052] Add a predetermined volume of regulator to the reaction solution obtained in step S2 and stir thoroughly to obtain the freezing solution. The volume fraction of the regulator in the freezing solution is 1% to 20%. The regulator includes one or more of water-miscible and volatile alcohols, phenols, ethers, and ketones; alcohols include one or more of methanol, ethanol, and ethylene glycol.

[0053] After freeze-drying the liquid to be frozen at -196 to -20°C for 60 to 80 hours, a natural silk nanofiber hemostatic material with adjustable pore size is obtained.

[0054] In this process, a regulator is added to adjust the microporous structure of natural silk nanofibers; specifically, the regulator adjusts the pore size. As freeze-drying proceeds, on the one hand, the continuous evaporation of water and the regulator fine-tunes the network structure formed by the transparent hyaluronic acid and silk nanofibers. Under suitable concentrations of regulator and freezing temperatures, a network structure in which transparent hyaluronic acid and silk nanofibers are bonded together is formed. On the other hand, the presence of the regulator can control the ice crystal growth rate of the aqueous solution. With the synergistic effect of the network structure and a suitable ice crystal growth rate, a hemostatic material with adjustable pore size is obtained, improving its water absorption and hemostatic performance. Low concentrations of regulator are more effective in controlling the network structure of the hemostatic material and can form a more stable hemostatic network structure.

[0055] This invention also provides a natural silk nanofiber hemostatic material with adjustable pore size, prepared using the aforementioned method for preparing a natural silk nanofiber hemostatic material with adjustable pore size. The microporous structure on the upper, middle, and lower surfaces of this natural silk nanofiber hemostatic material has a pore size of 0.1–20 μm, and the pores are interconnected.

[0056] The present invention also provides an application of a natural silk nanofiber hemostatic material with adjustable pore size, which is applied to the fields of hemostasis and skin repair.

[0057] The present invention will now be described in detail through specific embodiments.

[0058] Example 1

[0059] A method for preparing a hemostatic material made of natural silk nanofibers with adjustable pore size includes the following steps:

[0060] S1. Preparation of silk nanofiber suspension and hyaluronic acid solution:

[0061] S11. Preparation of hyaluronic acid activation solution:

[0062] Dissolve an appropriate amount of hyaluronic acid in deionized water to obtain a hyaluronic acid solution with a mass concentration of 1% (i.e., 1g of hyaluronic acid dissolved in 100mL of water). Then, add a cross-linking agent to the hyaluronic acid solution and stir for 2 hours to allow the reaction to proceed fully, thus obtaining an activated hyaluronic acid solution.

[0063] The crosslinking agent is a mixture of 1 mg morpholine ethanesulfonic acid, 4 mg N-hydroxysuccinimide and 4 mg 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

[0064] S12. Preparation of silk nanofiber suspension:

[0065] 5g of degummed silk was placed in a swelling solution (a mixture of calcium nitrate, ethanol, and water, with a volume ratio of ethanol to water of 1:3 and a calcium nitrate content of 5g:100mL) and placed in an oven at 60℃ for 72h. After removal, it was washed three times with deionized water and placed in a crusher with a blade speed of 8000r / min at a bath ratio of 1g:120mL for 90min of mechanical treatment. After filtering three times with a filter screen, the silk micro-nano fibers were obtained by freeze drying.

[0066] Take 1g of silk micro / nanofibers and add them to 100mL of deionized water to obtain a silk nanofiber suspension with a mass concentration of 1%.

[0067] The silk used is domesticated silkworm silk, and the diameter of the obtained silk nanofibers is 30-1000 nm.

[0068] S2. Crosslinking:

[0069] Add 100 mL of silk nanofiber suspension obtained in step S1 to 1 mL of hyaluronic acid activation solution to obtain a mixture. After the mixture is stirred and reacted thoroughly, 101 mL of reaction solution is obtained.

[0070] Specifically, in the mixture, the mass of hyaluronic acid is 1% of the mass of silk nanofibers. The mass concentration of the total solute (i.e., silk nanofibers, hyaluronic acid, and cross-linking agent) after mixing is 1.0089%.

[0071] S3. Freeze-drying:

[0072] Add 5.3 mL of anhydrous ethanol as a modifier to 101 mL of the reaction solution obtained in step S2 and stir thoroughly to obtain the cryogenic solution. The volume fraction of the modifier in the cryogenic solution is 5%. Then, freeze-dry the cryogenic solution at -80℃ for 72 h to obtain a natural silk nanofiber hemostatic material with adjustable pore size.

[0073] The physical image of the natural silk nanofiber hemostatic material with adjustable pore size obtained in Example 1 is shown below. Figure 1 As shown in a.

[0074] Example 2

[0075] A method for preparing a hemostatic material with adjustable pore size natural silk nanofibers differs from Example 1 in that, in step S2, 100 mL of the silk nanofiber suspension obtained in step S1 is added to 5 mL of hyaluronic acid activating solution to obtain a mixture. After thorough stirring and reaction, 105 mL of reaction solution is obtained. In the mixture, the mass of hyaluronic acid is 5% of the mass of the silk nanofibers. The mass concentration of the total solute after mixing is 1.0428%. In step S3, 5.5 mL of anhydrous ethanol regulator is added to the 105 mL reaction solution obtained in step S2 and stirred thoroughly to obtain a freezing solution. In the freezing solution, the volume fraction of the regulator is 5%. Other steps are largely the same as in Example 1 and will not be repeated here.

[0076] The physical image of the natural silk nanofiber hemostatic material with adjustable pore size obtained in Example 2 is shown below. Figure 1 As shown in b.

[0077] Example 3

[0078] A method for preparing a hemostatic material with adjustable pore size natural silk nanofibers differs from Example 1 in that, in step S2, 100 mL of the silk nanofiber suspension obtained in step S1 is added to 10 mL of hyaluronic acid activating solution to obtain a mixture. After thorough stirring and reaction, 110 mL of reaction solution is obtained. In the mixture, the mass of hyaluronic acid is 10% of the mass of the silk nanofibers. The mass concentration of the total solute after mixing is 1.0818%. In step S3, 5.8 mL of anhydrous ethanol regulator is added to the 110 mL reaction solution obtained in step S2 and stirred thoroughly to obtain a freezing solution. The volume fraction of the regulator in the freezing solution is 5%. Other steps are largely the same as in Example 1 and will not be repeated here.

[0079] The physical image of the natural silk nanofiber hemostatic material with adjustable pore size obtained in Example 3 is shown below. Figure 2 As shown in b, the scanning electron microscope image is as follows. Figure 3 As shown in images b and 3d (the prepared hemostatic material was cut to a suitable size, attached to a sample stage coated with conductive adhesive, sputter-coated with gold for 2 minutes, and observed using a scanning electron microscope), the hemostatic material exhibits a dense structure. The electron microscope images reveal that the network structure of the obtained hemostatic material has small pore sizes, a relatively dense network structure, and more compact and uniform cross-linking and entanglement between nanofibers. Furthermore, it possesses a highly interconnected network structure, significantly improving its water absorption capacity.

[0080] Hemolysis tests were performed on the hemostatic material prepared in Example 3. Specifically, (1) the prepared hemostatic material was disinfected in 75% alcohol solution for 30 min, and then washed three times with PBS. 0.9 mL of PBS, 1% Triton X-100, and PBS suspension of the silkworm silk nanofiber hemostatic material were added to the three experimental groups, respectively. PBS and Triton X-100 were used as the negative control group and positive control group, respectively. (2) 1 mL of 2% diluted red blood cell suspension was then added. After gentle mixing, the mixture was incubated in a 37°C water bath for 1 h. Figure 6 As shown, the silk nanofiber hemostatic material has good blood compatibility and shows no significant difference from the negative control group PBS solution. (3) Centrifuge the three groups of samples at 1000 rpm / min for 5 min, aspirate the supernatant and transfer it to a 96-well plate, and calculate the hemolysis rate by taking the average absorbance at a wavelength of 540 nm using an enzyme-linked immunosorbent assay (ELISA) reader. Figure 6 As shown in the bar chart of hemolysis rate, the hemostatic material has good blood compatibility.

[0081] Example 4

[0082] A method for preparing a hemostatic material with adjustable pore size natural silk nanofibers differs from Example 1 in that, in step S2, 100 mL of the silk nanofiber suspension obtained in step S1 is added to 20 mL of hyaluronic acid activating solution to obtain a mixture. After thorough stirring and reaction, 120 mL of reaction solution is obtained. In the mixture, the mass of hyaluronic acid is 20% of the mass of the silk nanofibers. The mass concentration of the total solute after mixing is 1.15%. In step S3, 6.3 mL of anhydrous ethanol regulator is added to the 120 mL reaction solution obtained in step S2 and stirred thoroughly to obtain a freezing solution. The volume fraction of the regulator in the freezing solution is 5%. Other steps are largely the same as in Example 1 and will not be repeated here.

[0083] Comparative Example 1

[0084] A method for preparing a hemostatic material with adjustable pore size natural silk nanofibers differs from Example 1 in that, in step S2, 100 mL of the silk nanofiber suspension obtained in step S1 is added to 25 mL of hyaluronic acid activating solution to obtain a mixture. After thorough stirring and reaction, 125 mL of reaction solution is obtained. In the mixture, the mass of hyaluronic acid is 25% of the mass of the silk nanofibers. The mass concentration of the total solute after mixing is 1.18%. In step S3, 6.6 mL of anhydrous ethanol regulator is added to the 125 mL reaction solution obtained in step S2 and stirred thoroughly to obtain a freezing solution. The volume fraction of the regulator in the freezing solution is 5%. Other steps are largely the same as in Example 1 and will not be repeated here.

[0085] Comparative Example 2

[0086] A method for preparing a hemostatic material made of natural silk nanofibers with adjustable pore size differs from Example 1 in that, in step S2, hyaluronic acid is not added; that is, the mass of hyaluronic acid in the mixture is 0% of the mass of the silk nanofibers (in this case, there is no need to prepare a hyaluronic acid activation solution). The rest is largely the same as in Example 1 and will not be repeated here.

[0087] The pore size-adjustable natural silk nanofiber hemostatic materials obtained in Example 3 and Comparative Example 2 were subjected to water stability testing. Water stability test: The hemostatic materials were prepared as follows... Figure 4 As shown in b, the hemostatic materials from Example 3 and Comparative Example 2 were placed into glass bottles filled with water and left to stand for 1 hour until fully soaked. Then, they were gently stirred with a glass rod. Figure 4 As shown in Figure a, the hemostatic material of Comparative Example 2 quickly disperses and disintegrates in water, indicating poor stability in water and thus poor mechanical properties; Figure 4 As shown in b, the morphology of the hemostatic material in Example 3 did not change, indicating that the addition of hyaluronic acid endowed the silk nanofiber hemostatic material with good water resistance (water stability).

[0088] The hemostatic materials with adjustable pore size natural silk nanofibers prepared in Examples 1-4 and Comparative Examples 1-2 were subjected to performance tests, and the results are shown in Table 1:

[0089] The hemostatic performance test is to test the hemostatic performance of a mouse tail amputation model using hemostatic materials. Specifically: (1) SD rat anesthesia experiment: The mice were fixed on a surgical board and anesthetized by intraperitoneal injection of 10% chloral hydrate (0.3 mL of 10% chloral hydrate per 100g mouse). The hair was shaved off at the surgical site and disinfected with medical iodine. (2) Weighed filter paper was placed under the tail of the mouse and cut off 4 cm from the end of the tail. After the first drop of blood dripped from the cut part, the pre-weighed hemostatic material to be tested was immediately pressed on the wound and the hemostasis time was recorded. The wound was observed every 10 seconds to see if there was bleeding. There were three parallel samples in each group. The control group was left to bleed naturally without treatment. After the bleeding stopped, the weight of the filter paper and the hemostatic material was weighed and the hemostasis time (s) and blood loss (g) were recorded.

[0090] Table 1. Performance of the hemostatic materials prepared in Examples 1-4 and Comparative Examples 1-2

[0091]

[0092] As shown in Table 1, with the increase of hyaluronic acid content, the hemostatic effect of the hemostatic material first increases and then decreases, while the mechanical properties gradually increase and the porosity gradually decreases. This is mainly because the increase of hyaluronic acid content enhances the network structure of the porous material, making its mechanical properties stronger and its porosity lower. After the proportion of hyaluronic acid reaches 10%, its hemostatic effect weakens. Excess hyaluronic acid adheres to the surface of the porous material, destroying its interconnected porous structure, reducing porosity, and weakening its water and blood absorption effects, thereby reducing its ability to absorb blood and concentrate clotting factors.

[0093] Example 5

[0094] A method for preparing a hemostatic material with adjustable pore size using natural silk nanofibers differs from Example 3 in that, in step S1, a 2% (w / w) silk nanofiber suspension is prepared; in step S2, 100 mL of the 2% (w / w) silk nanofiber suspension obtained in step S1 is added to 20 mL of a 1% (w / w) hyaluronic acid activating solution to obtain a mixture, which is then stirred thoroughly to obtain 120 mL of reaction solution. In the mixture, the mass of hyaluronic acid is 10% of the mass of the silk nanofibers. The total solute concentration after mixing is 1.9833%. In step S3, 6.3 mL of anhydrous ethanol as a modifier is added to the 120 mL reaction solution obtained in step S2 and stirred thoroughly to obtain a freezing solution. The volume fraction of the modifier in the freezing solution is 5%. Other steps are largely the same as in Example 3 and will not be repeated here.

[0095] Depend on Figure 5 It can be seen that the pore size of the natural silk nanofiber hemostatic material with adjustable pore size obtained in Example 5 is more compact than that in Example 3, indicating that the increase in the content of silk nanofiber and hyaluronic acid can improve the density of the network structure of the hemostatic material.

[0096] Example 6

[0097] A method for preparing a hemostatic material of natural silk nanofibers with adjustable pore size, compared with Example 3, differs in that, in step S3, 12.2 mL of anhydrous ethanol as a regulator is added to 110 mL of the reaction solution obtained in step S2 and stirred thoroughly to obtain a freezing solution. The volume fraction of the regulator in the freezing solution is 10%, and the other steps are largely the same as in Example 3, and will not be repeated here.

[0098] Example 7

[0099] A method for preparing a hemostatic material of natural silk nanofibers with adjustable pore size, compared with Example 3, differs in that, in step S3, 27.5 mL of anhydrous ethanol as a modifier is added to 110 mL of the reaction solution obtained in step S2 and stirred thoroughly to obtain a freezing solution. The volume fraction of the modifier in the freezing solution is 20%, and the other steps are largely the same as in Example 3, and will not be repeated here.

[0100] Comparative Example 3

[0101] A method for preparing a hemostatic material with adjustable pore size natural silk nanofibers differs from Example 3 in that, in step S3, 110 mL of anhydrous ethanol as a regulator is added to the 110 mL reaction solution obtained in step S2 and stirred thoroughly to obtain a freezing solution. The volume fraction of the regulator in the freezing solution is 50%, and other aspects are largely the same as in Example 3, and will not be repeated here.

[0102] Comparative Example 4

[0103] A method for preparing a natural silk nanofiber hemostatic material with adjustable pore size differs from Example 3 in that, in step S3, no ethanol regulator is added; that is, the volume fraction of the regulator in the freezing liquid is 0%. The rest is largely the same as in Example 3 and will not be repeated here.

[0104] The physical image of the natural silk nanofiber hemostatic material with adjustable pore size obtained in Comparative Example 4 is shown below. Figure 2 As shown in Figure a, the scanning electron microscope image is as follows: Figure 3 As shown in a and 3c, the physical images reveal that the hemostatic material obtained in Comparative Example 4 has a rough surface and large, uneven pore size. Electron microscopy images show that the network structure of the obtained hemostatic material has large, uneven pore size, and fewer cross-linking entanglements between nanofibers, resulting in a less dense network structure.

[0105] The hemostatic materials prepared in Comparative Example 4 and Example 3 were subjected to in vitro coagulation performance tests. Specifically, (1) the prepared hemostatic materials were cut into 2*2cm pieces. 2 The samples were arranged in squares, with three parallel samples per group. Before the experiment, the samples were sterilized by UV irradiation for 3 hours and incubated in a 37℃ oven for 1 hour. (2) 5 mg of hemostatic material was placed in a culture dish, and then 200 μL of anticoagulated whole blood was slowly added to the well plate. Then, 10 μL of 0.2 mol / L CaCl2 was added to the well plate to activate the coagulation cascade reaction and induce the formation of blood clots. The well plate was then allowed to stand at 37℃ for 1 minute. (3) 10 mL of deionized water was added to a centrifuge tube to rinse the blood clots and a digital photograph was taken. Finally, 200 μL of the deionized water used to rinse the blood clots was transferred to a 96-well plate. (4) The absorbance of hemoglobin was measured at 544 nm using an ELISA reader. Figure 8As shown in the in vitro coagulation index diagrams of the pore size-adjustable natural silk nanofiber hemostatic materials prepared in Comparative Example 4 and Example 3, the coagulation index of the hemostatic material obtained in Example 3 is significantly lower, indicating that its coagulation effect is significantly better than that of Comparative Example 4.

[0106] The hemostatic properties of the hemostatic materials prepared in Example 3 and Comparative Example 4 were tested using a mouse tail amputation model. The results are as follows: Figure 9 and Figure 10 As shown, the control group received no hemostatic treatment, the gelatin sponge was commercially available gelatin sponge for hemostasis, Comparative Example 4 was used without adding a cryostat, and Example 3 was used with a cryostat. The hemostasis time and blood loss figures of the pore-adjustable natural silk nanofiber hemostatic materials prepared in Comparative Example 4 and Example 3 show that the hemostatic performance of the hemostatic material obtained in Example 3 is significantly better than that of Comparative Example 4.

[0107] The hemostatic materials with adjustable pore size natural silk nanofibers prepared in Examples 6-7 and Comparative Examples 3-4 were subjected to performance tests, and the results are shown in Table 2:

[0108] Table 2. Performance of the hemostatic materials prepared in Examples 6-7 and Comparative Examples 3-4

[0109]

[0110] As shown in Table 2, with the increase of ethanol dosage, the hemostatic performance of the hemostatic material first increases and then decreases, the mechanical properties gradually decrease, the pore size of the porous material decreases significantly, and the pore size first increases and then decreases. This indicates that the amount of ethanol will affect the structure of the hemostatic material, and thus affect its performance.

[0111] Example 8

[0112] A method for preparing a natural silk nanofiber hemostatic material with adjustable pore size is different from that in Example 3, the freeze-drying temperature in step S3 is -40℃. The other steps are roughly the same as in Example 3 and will not be repeated here.

[0113] Example 9

[0114] A method for preparing a natural silk nanofiber hemostatic material with adjustable pore size is different from that in Example 3, the freeze-drying temperature in step S3 is -196℃. The other steps are roughly the same as in Example 3 and will not be repeated here.

[0115] Comparative Example 5

[0116] A method for preparing a natural silk nanofiber hemostatic material with adjustable pore size is different from that in Example 3, the freeze-drying temperature in step S3 is -210℃. The other steps are roughly the same as in Example 3 and will not be repeated here.

[0117] The hemostatic materials with adjustable pore size natural silk nanofibers prepared in Examples 8-9 and Comparative Example 5 were subjected to performance tests, and the results are shown in Table 3:

[0118] Table 3. Performance of the hemostatic materials prepared in Examples 8-9 and Comparative Example 5

[0119]

[0120] As shown in Table 3, as the freezing temperature decreases, the hemostatic performance of the obtained hemostatic material first increases and then decreases, the mechanical strength gradually decreases, the porosity varies within a certain range, and the pore size of the hemostatic material decreases. This indicates that the change in freezing temperature affects the structure of the obtained hemostatic material, and thus affects its performance.

[0121] Example 10

[0122] A method for preparing a natural silk nanofiber hemostatic material with adjustable pore size is different from that in Example 3, in step S1, silkworm silk is replaced with tussah silk. The rest is roughly the same as in Example 3 and will not be described again here.

[0123] The hemolytic material prepared in Example 10 was subjected to a hemolysis test. The test method was the same as in Example 3, except that the PBS suspension of the silkworm silk nanofiber hemolytic material was replaced with the PBS suspension of the tussah silk nanofiber hemolytic material. The results are as follows: Figure 7 As shown, by Figure 7 The hemolysis rate bar chart shows that the hemostatic material prepared in Example 10 has good blood compatibility.

[0124] Comparative Example 6

[0125] A method for preparing a natural silk nanofiber hemostatic material with adjustable pore size is different from that in Example 3, in step S1, hyaluronic acid is replaced with chitosan. The rest is roughly the same as in Example 3 and will not be described again here.

[0126] Comparative Example 7

[0127] A method for preparing a natural silk nanofiber hemostatic material with adjustable pore size is different from that in Example 3, in step S1, hyaluronic acid is replaced with sodium alginate. The rest is roughly the same as in Example 3 and will not be described again here.

[0128] Comparative Example 8

[0129] A method for preparing a natural silk nanofiber hemostatic material with adjustable pore size is different from that in Example 3, the ethanol regulator is replaced with glycerol in step S3. The rest is roughly the same as in Example 3 and will not be described again here.

[0130] Comparative Example 9

[0131] A method for preparing a natural silk nanofiber hemostatic material with adjustable pore size is different from that in Example 3, the ethanol regulator is replaced with tert-butanol in step S3. The rest is roughly the same as in Example 3 and will not be described again here.

[0132] The hemostatic materials with adjustable pore size natural silk nanofibers prepared in Comparative Examples 6-9 were subjected to performance tests, and the results are shown in Table 4.

[0133] Table 4 shows the properties of the hemostatic materials prepared in Comparative Examples 6-9.

[0134]

[0135] As shown in Table 4, when hyaluronic acid is replaced with other sugars or when the regulator is replaced with other alcohols, the various properties of the hemostatic material are reduced. This indicates that only through the synergistic effect of hyaluronic acid, silk nanofibers, and ethanol regulator can a high-performance hemostatic material be obtained.

[0136] In summary, this invention provides a pore size-adjustable natural silk nanofiber hemostatic material, its preparation method, and its application. The preparation process first involves preparing a hyaluronic acid activating solution to form an amine-reactive O-acylisourea intermediate. Next, a silk nanofiber suspension is added to the hyaluronic acid activating solution. The amine-reactive O-acylisourea intermediate reacts rapidly with the amino groups in the silk nanofibers, initially forming a network structure through the synergistic effects of bonding and entanglement. Then, a regulator is added to adjust the pore size. The freeze-drying process further fine-tunes the three-dimensional network structure. Under the synergistic effects of hyaluronic acid, silk nanofibers, the regulator, and a suitable freeze-drying temperature, a high-performance hemostatic material is obtained.

[0137] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for preparing a hemostatic material made of natural silk nanofibers with adjustable pore size, characterized in that, Includes the following steps: S1. Prepare hyaluronic acid activation solution and silk nanofiber suspension at preset concentrations respectively; The preparation of the hyaluronic acid activation solution specifically involves dissolving an appropriate amount of hyaluronic acid in deionized water to obtain a hyaluronic acid solution with a mass concentration of 0.1% to 10%; then adding a crosslinking agent to the hyaluronic acid solution, and stirring the reaction thoroughly to obtain the hyaluronic acid activation solution; the crosslinking agent is a mixture of morpholine ethanesulfonic acid, N-hydroxysuccinimide, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; S2. Add the silk nanofiber suspension to the hyaluronic acid activation solution obtained in step S1 to obtain a mixture; stir the mixture thoroughly to obtain a reaction solution; in the mixture, the mass of the hyaluronic acid is 1% to 20% of the mass of the silk nanofibers; S3. Add a preset volume of regulator to the reaction solution obtained in step S2 and stir thoroughly to obtain a freezing solution; freeze-dry the freezing solution to obtain the natural silk nanofiber hemostatic material with adjustable pore size; the regulator is ethanol; the freeze-drying is carried out at -196 to -20°C.

2. The method for preparing the pore size-adjustable natural silk nanofiber hemostatic material according to claim 1, characterized in that, In step S3, the volume fraction of the regulator in the liquid to be cooled is 1% to 20%.

3. The method for preparing the pore size-adjustable natural silk nanofiber hemostatic material according to claim 1, characterized in that, In step S1, the silk is natural silk, including domestic silkworm silk or tussah silk; the diameter of the silk nanofibers is 30-1000 nm; and the mass concentration of the silk nanofiber suspension is 1%-5%.

4. A natural silk nanofiber hemostatic material with adjustable pore size, characterized in that, The material is prepared by the method described in any one of claims 1 to 3; the pore size of the tunable natural silk nanofiber hemostatic material is 0.1 to 20 μm.

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