Inorganic three-dimensional composite nanofiber hemostatic sponge, preparation method and application

Abstract

The application discloses an inorganic three-dimensional composite nanofiber hemostatic sponge, a preparation method and application. The inorganic three-dimensional composite nanofiber hemostatic sponge comprises a three-dimensional interconnected network framework of SiO2 nanofibers, and kaolin particles are in-situ immobilized on the surface of the SiO2 nanofibers and the intersection nodes between the SiO2 nanofibers through chemical bonding. The inorganic three-dimensional composite nanofiber hemostatic sponge has a porosity greater than 90%, and is a full inorganic composite hemostatic sponge with an interconnected macroporous structure. The material realizes synergistic hemostasis through physical rapid blood absorption to enrich blood components and activation of endogenous and exogenous coagulation pathways mediated by kaolin, and simultaneously has good cell compatibility, blood compatibility and broad-spectrum antibacterial performance. The kaolin is uniformly immobilized in the fiber network, and there is no risk of particle shedding. The hemostatic sponge has fast hemostasis speed and low blood loss, and has simple and controllable preparation process, and is suitable for rapid hemostasis of trauma first aid, surgical operation, irregular wound surface and non-compressive bleeding.

Description

Technical Field

[0001] This application belongs to the field of medical biomaterials technology, and specifically relates to an inorganic three-dimensional composite nanofiber hemostatic sponge, its preparation method and application. Background Technology

[0002] Uncontrollable bleeding is one of the leading causes of traumatic death, and rapid and effective hemostasis measures are of crucial importance. Currently, commonly used hemostatic materials in clinical practice mainly include gauze, gelatin sponge, oxidized cellulose, chitosan-based dressings, and zeolite / kaolin-based granular hemostatic agents. However, the above-mentioned existing hemostatic materials still have several technical bottlenecks that are difficult to address in practical applications: (1) Traditional gauze achieves hemostasis only through physical compression and simple absorption, lacking active coagulation function. It has low hemostasis efficiency and long hemostasis time for moderate and severe bleeding wounds, and it is easy to adhere to the wound surface when removed, leading to secondary bleeding; (2) Although natural polymer-based hemostatic sponges (such as chitosan, gelatin, silk fibroin sponge, etc.) have certain biological activity and coagulation function, they generally have problems such as insufficient mechanical strength, poor wet stability, and difficulty in accurately controlling the degradation rate. In addition, the natural polymer materials have large batch differences and potential immunogenicity and pathogens. Risk of transmission; (3) Inorganic particulate hemostatic agents (such as zeolite particles, kaolin powder, etc.) can exert a strong procoagulant effect by activating the intrinsic coagulation pathway. However, particulate hemostatic agents have serious safety hazards during use. Free particles are easy to migrate to distal blood vessels with the blood flow, causing serious complications such as thromboembolism. In addition, the intense exothermic effect generated by the reaction of particles with water can lead to local tissue thermal damage. Moreover, powdered materials are difficult to uniformly cover irregular wounds and have poor operability. (4) Existing electrospun hemostatic sponges are mostly based on organic polymer fiber membranes, which have problems such as organic residues, acidic environment of degradation products affecting wound healing, and insufficient antibacterial properties.

[0003] Although previous studies have supported inorganic nanoparticles (such as SiO2 nanoparticles) on polymer fiber matrices, the weak organic-inorganic interfacial bonding and the easy detachment and loss of functional inorganic components in bodily fluids make it difficult to achieve sustained and stable procoagulant and antibacterial efficacy. Furthermore, existing preparation processes typically involve complex multi-step sol-gel synthesis, freeze-drying, or supercritical drying, resulting in low production efficiency, high costs, and difficulty in scaling up. In addition, techniques for uniformly and firmly immobilizing functional mineral particles in inorganic fiber matrices are still immature, and the problem of particle detachment remains unresolved. Summary of the Invention

[0004] Objective of the Invention: To address the problems of existing technologies, this application provides an inorganic three-dimensional composite nanofiber hemostatic sponge. This inorganic three-dimensional composite nanofiber hemostatic sponge is an all-inorganic hemostatic sponge, possessing rapid procoagulant activity, a three-dimensional interconnected porous structure, high liquid absorption capacity, good biocompatibility, and antibacterial function. This application also provides a method for preparing this inorganic three-dimensional composite nanofiber hemostatic sponge. This method ensures the stable immobilization of functional mineral components in the material system, and the preparation process is simple and controllable.

[0005] Technical solution: The inorganic three-dimensional composite nanofiber hemostatic sponge described in this application includes a three-dimensional interconnected network skeleton composed of SiO2 nanofibers, and the kaolin particles are chemically bonded and in situ fixed on the surface of SiO2 nanofibers and at the intersection nodes between SiO2 nanofibers.

[0006] In some embodiments, the particle size of the kaolin particles is 0.5 μm to 10 μm, preferably 1 μm to 5 μm.

[0007] In some embodiments, in the inorganic three-dimensional composite nanofiber hemostatic sponge, the mass ratio of SiO2 nanofibers to kaolin is (10~40):1.

[0008] In some embodiments, the diameter of the SiO2 nanofibers is 200 nm to 800 nm.

[0009] In some embodiments, the inorganic three-dimensional composite nanofiber hemostatic sponge is prepared by the following method: Provide a silicon source to prepare a precursor solution containing the silicon source; A solution containing PVA is mixed with a precursor solution containing a silicon source to obtain a silicon source and PVA composite spinning solution. Kaolin particles were added to the silicon source and PVA composite spinning solution, and three-dimensional spinning was performed using a conjugate electrospinning device to obtain a nanofiber sponge-like precursor. High-temperature calcination of the nanofiber sponge-like precursor yields an inorganic three-dimensional composite nanofiber hemostatic sponge.

[0010] In this application, the PVA sacrificial binder in the nanofiber sponge precursor is completely thermally decomposed and removed during the calcination process, and the TEOS hydrolysis products are transformed into SiO2 inorganic fiber skeleton through condensation. Kaolin particles form chemical bonds with the SiO2 fiber matrix through high-temperature sintering, achieving in-situ fixation. After cooling to room temperature, the sponge is taken out to obtain the inorganic three-dimensional composite nanofiber hemostatic sponge.

[0011] In some specific implementations, this application uses tetraethyl orthosilicate as the silicon source, kaolin as the coagulation active component, and polyvinyl alcohol (PVA) as the sacrificial binder to prepare a three-dimensional nanofiber sponge using conjugated electrospinning. The organic phase is removed by high-temperature calcination, resulting in a fully inorganic composite hemostatic sponge with a porosity greater than 90% and an interconnected macroporous structure. This material achieves synergistic hemostasis through rapid physical absorption and enrichment of blood components, combined with activation of intrinsic and extrinsic coagulation pathways mediated by kaolin. It also possesses good cell compatibility, blood compatibility, and broad-spectrum antibacterial properties. The kaolin is uniformly immobilized within the fiber network, eliminating the risk of particle detachment. In vivo animal experiments demonstrate that this hemostatic sponge achieves rapid hemostasis with low blood loss. Its preparation process is simple and highly controllable, making it suitable for rapid hemostasis in trauma emergency care, surgery, irregular wounds, and non-compression bleeding.

[0012] In some embodiments, the degree of polymerization of PVA is 1700–1800, the degree of alcoholysis is 87%–89%, and the mass fraction of the PVA aqueous solution is 10%.

[0013] The hemostatic sponge of this application achieves rapid hemostasis through the following synergistic mechanisms: (a) the three-dimensional interconnected macroporous structure rapidly absorbs plasma, enriching platelets and coagulation factors locally at the wound site; (b) the silanol groups (-Si-OH) on the surface of SiO2 nanofibers interact with platelet membrane receptors, activating platelets and promoting platelet adhesion and aggregation; (c) kaolin particles activate factor XII, initiating the intrinsic coagulation cascade reaction; (d) the dual synergistic effect of physical adsorption enrichment and biochemical coagulation activation significantly accelerates the coagulation process.

[0014] This application also provides a method for preparing the aforementioned inorganic three-dimensional composite nanofiber hemostatic sponge, comprising the following steps: Provide a silicon source to prepare a precursor solution containing the silicon source; A solution containing PVA is mixed with a precursor solution containing a silicon source to obtain a silicon source and PVA composite spinning solution. Kaolin particles were added to the silicon source and PVA composite spinning solution, and three-dimensional spinning was performed using a conjugate electrospinning device to obtain a nanofiber sponge-like precursor. High-temperature calcination of the nanofiber sponge-like precursor yields an inorganic three-dimensional composite nanofiber hemostatic sponge.

[0015] In some embodiments, the silicon source is tetraethyl orthosilicate (TEOS).

[0016] In this application, the tetraethyl orthosilicate used as the silicon source undergoes a hydrolysis reaction upon contact with water, generating silicic acid or silicon dioxide. The condensation product after hydrolysis has good adhesion. During the reaction, silanol groups and oligomers are formed, and the system gradually transforms from a sol to a gel with a certain viscosity. After gel formation, blending with PVA can better prepare electrospun nanofibers.

[0017] In some specific embodiments, the precursor solution containing the silicon source is prepared by the following method: tetraethyl orthosilicate (TEOS) is added to a mixed solution of water and ethanol containing an acid catalyst, and the mixture is stirred and hydrolyzed at room temperature for 2 h to 6 h to obtain a SiO2 precursor sol; wherein the molar ratio of TEOS to ethanol to water to acid catalyst is 1:(1 to 3):(1 to 3):(0.01 to 0.05).

[0018] In some specific embodiments, PVA is dissolved in deionized water to prepare a PVA aqueous solution with a mass fraction of 8% to 12%; a precursor solution containing a silicon source is mixed with the PVA aqueous solution at a volume ratio of 1:(0.5 to 2) and stirred thoroughly to obtain a SiO2 / PVA composite spinning solution; wherein PVA acts as a sacrificial binder, imparting spinnability to the spinning solution and strengthening the interfacial bonding between SiO2 fibers and kaolin particles during the spinning process.

[0019] In some embodiments, kaolin particles are added to the silicon source and PVA composite spinning solution. The amount of kaolin particles added is 1 wt% to 20 wt% of the theoretical SiO2 yield in the spinning solution, preferably 5 wt%. The mixture is stirred continuously at room temperature for 4 to 12 hours to uniformly disperse the kaolin particles in the spinning solution, thus obtaining the SiO2 / PVA / kaolin composite spinning solution. Theoretical SiO2 yield = amount of TEOS × 60.08 g / mol (SiO2 molecular weight).

[0020] In some embodiments, the precursor solution containing the silicon source further includes ethanol, water and an acid catalyst, wherein the molar ratio of the silicon source, ethanol, water and acid catalyst is 1:(1-3):(1-3):(0.01-0.05).

[0021] In some embodiments, the parameters of the three-dimensional spinning are: applied voltage of 15kV to 30kV, receiving distance of 15cm to 25cm, and spinning propulsion rate of 0.5mL / h to 3mL / h.

[0022] In this application, the parameters of three-dimensional spinning can be appropriately adjusted according to the viscosity of the spinning solution and the content of kaolin, including voltage and distance.

[0023] In some embodiments, the spinning environment temperature is 20°C to 30°C and the relative humidity is 30% to 60%. During the spinning process, the fibers self-assemble under the action of an electric field to form a three-dimensional fluffy nanofiber sponge precursor. The obtained precursor sponge is collected and dried at room temperature.

[0024] In some embodiments, the three-dimensional spinning employs a conjugate electrospinning device, which includes two or more sets of nozzles arranged symmetrically, forming a symmetrical electric field between the nozzles, causing the fibers to collide, intertwine, and self-assemble into a three-dimensional fluffy structure in space.

[0025] In some embodiments, the calcination parameters are: heating to 600℃ to 1000℃ at a heating rate of 1℃ / min to 5℃ / min, and holding at that temperature for 2h to 4h.

[0026] In some embodiments, the calcination temperature is 800 °C, the heating rate is 2 °C / min, and the holding time is 2 hours.

[0027] In some embodiments, the acid catalyst is hydrochloric acid or phosphoric acid.

[0028] In some embodiments, the acid catalyst is phosphoric acid with a concentration of 0.01 mol / L to 0.1 mol / L.

[0029] In some embodiments, the precursor solution containing a silicon source is mixed with a solution containing PVA at a volume ratio of 1:(0.5 to 2).

[0030] This application also provides the application of the above-mentioned inorganic three-dimensional composite nanofiber hemostatic sponge in the preparation of hemostatic dressings.

[0031] In some embodiments, the hemostatic dressing is used for trauma first aid hemostasis, surgical hemostasis, or non-compression hemostasis.

[0032] Beneficial effects: Compared with existing technologies, the inorganic three-dimensional composite nanofiber hemostatic sponge of this application has the following advantages: First, kaolin is in-situ immobilized, firmly immobilized in the SiO2 nanofiber matrix through Si-O-Al chemical bonding, eliminating the risk of particle detachment. Particle detachment does not occur under liquid immersion and blood flushing conditions, eliminating the risk of distal embolism associated with traditional granular kaolin hemostatic agents; Second, the SiO2 nanofiber matrix has a three-dimensional interconnected macroporous structure with excellent liquid absorption performance; Third, the inorganic three-dimensional composite nanofiber hemostatic sponge of this application has a dual synergistic hemostatic mechanism, resulting in high hemostatic efficiency. At the physical level, the three-dimensional macroporous structure rapidly absorbs liquid and enriches coagulation components; at the biochemical level, the silanol groups on the surface of SiO2 fibers activate platelet adhesion and aggregation, and kaolin particles activate factor XII to initiate the intrinsic coagulation pathway. Simultaneously, both PT and APTT are significantly shortened, indicating that both intrinsic and extrinsic coagulation pathways are effectively activated, achieving rapid hemostasis through a synergistic physical-biochemical process. The inorganic three-dimensional composite nanofiber hemostatic sponge of this application exhibits excellent biocompatibility and blood compatibility: cytotoxicity experiments show that the material has no significant toxicity to L929 cells, with a hemolysis rate of less than 5%, meeting the blood compatibility standards for medical materials; the inorganic three-dimensional composite nanofiber hemostatic sponge of this application also exhibits significant antibacterial properties: the hemostatic sponge is resistant to Staphylococcus aureus (… S. aureus ) and Escherichia coli ( E. coli All of them showed significant antibacterial activity, which helps prevent wound infection and reduce post-hemostasis complications. The inorganic three-dimensional composite nanofiber hemostatic sponge of this application has excellent hemostatic effect in vivo: in the mouse tail amputation hemostasis model, the hemostasis time of the optimized formula (S-SiO2-5K) was only 32±7s and the blood loss was only 10.8±0.6mg, which was significantly better than the fiber membrane control and medical gauze control with the same composition, verifying the hemostatic advantages of the three-dimensional sponge structure.

[0033] Furthermore, the inorganic three-dimensional composite nanofiber hemostatic sponge of this application is constructed in one step through conjugated electrospinning to form a three-dimensional fluffy nanofiber network with a porosity exceeding 90%. It possesses an extremely high specific surface area and liquid absorption capacity (greater than 8 times its own dry weight), enabling it to rapidly absorb plasma upon contact with the wound surface and efficiently enrich platelets and coagulation factors locally, forming a procoagulant microenvironment. The inorganic three-dimensional composite nanofiber hemostatic sponge of this application is entirely inorganic, eliminating the risk of organic residues: PVA is used as a sacrificial binder, and after high-temperature calcination, the PVA is completely removed, resulting in a hemostatic sponge composed entirely of inorganic components (SiO2 and kaolin). This eliminates the acidic microenvironment and potential immunogenicity issues caused by organic polymer degradation products, making its biocompatibility significantly superior to hemostatic sponges containing organic components. The preparation process of the inorganic three-dimensional composite nanofiber hemostatic sponge of this application is simple and can be mass-produced. This invention employs a strategy combining one-step conjugate electrospinning with single-step calcination, eliminating the need for complex processes such as freeze drying or supercritical drying. The process is simple, parameters are controllable, and equipment requirements are low, making it suitable for industrial-scale production. Attached Figure Description

[0034] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0035] Figure 1 The preparation and reaction mechanism of SiO2 / kaolin nanofiber membranes and sponges are shown in Figure A, which shows the hydrolysis, condensation and cross-linking reaction of TEOS; Figure B shows the nanofiber membrane preparation process; Figure C shows the three-dimensional nanofiber sponge assembly process; Figure D shows the morphology of the sponge during the preparation process; and Figure E shows the morphology of the sponge after calcination. Figure 2 The morphology of SiO2 / kaolin nanofiber membrane and three-dimensional sponge is characterized. In the figure, A is the SEM of the sample before calcination; B is the SEM of the sample after calcination; C is the elemental distribution of O, Si and Al in the sample after calcination. Figure 3 The physicochemical properties of SiO2 / kaolin nanofiber membranes and sponges are characterized. Figure A shows the pore size distribution of the nanofiber membrane prepared in Example 8; Figure B shows the pore size distribution of the nanosponge prepared in Example 2; Figure C shows the FTIR spectrum; Figure D shows the XRD spectrum; Figure E shows the in vitro degradation curve; Figure F shows the water absorption rate; Figure G shows the bulk density; and Figure H shows the porosity. Figure 4 The biocompatibility of SiO2 / kaolin nanofiber membranes with sponge cells in vitro is shown in Figure A, where cell live / dead staining is shown, and Figure B shows the quantitative analysis of CCK-8 cell proliferation. Figure 5 The in vitro antibacterial properties of SiO2 / kaolin nanofiber membranes and sponges are shown in Figure A, which is a colony photograph; Figure B shows the antibacterial rate against Staphylococcus aureus; and Figure C shows the antibacterial rate against Escherichia coli. Figure 6 To illustrate the in vitro hemostasis and blood compatibility of SiO2 / kaolin nanofiber membranes with sponges, Figure A shows whole blood coagulation test; Figure B shows coagulation index (BCI); Figure C shows coagulation photographs; Figure D shows coagulation time; Figure E shows hemolysis photographs; Figure F shows hemolysis rate; Figure G shows erythrocyte adhesion SEM; Figure H shows prothrombin time (PT); and Figure I shows activated partial thromboplastin time (APTT). Figure 7 To evaluate the hemostatic effect of SiO2 / kaolin nanofibers in a mouse tail amputation model, Figure A shows the mouse tail amputation hemostasis model; Figure B shows a photograph of the hemostasis process; Figure C shows the blood loss; and Figure D shows the hemostasis time. Detailed Implementation

[0036] The technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application. In addition, in the description of this application, the term "comprising" means "including but not limited to". The terms first, second, third, etc. are used only as illustrative purposes and do not impose numerical requirements or establish an order. Various embodiments of this application may exist in the form of a range; it should be understood that the description in the form of a range is only for convenience and conciseness and should not be construed as a hard limitation on the scope of this application; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single values ​​within that range. For example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which applies regardless of the range. Additionally, whenever a numerical range is specified in this document, it means that any referenced number (fraction or integer) within the range is included.

[0037] Example 1: Preparation of SiO2 precursor sol (1) At room temperature, take 5 mL of tetraethyl orthosilicate (TEOS, analytical grade) and add it to a beaker containing 5 mL of anhydrous ethanol and 5 mL of deionized water; (2) Add an appropriate amount of phosphoric acid solution as an acid catalyst to the above mixture and adjust the pH of the system to 2-3; (3) Stir continuously at room temperature for 3-4 hours under magnetic stirring to fully hydrolyze TEOS and obtain a transparent and uniform SiO2 precursor sol for later use.

[0038] Example 2: Preparation of pure SiO2 three-dimensional nanofiber sponge (S-SiO2-0K, control group) (1) Weigh an appropriate amount of PVA powder (degree of polymerization 1750, degree of alcoholysis 88%), add it to deionized water, and stir and dissolve it in a water bath at 80℃~90℃ until it is completely transparent, and prepare a PVA aqueous solution with a mass percentage of 10%. (2) The SiO2 precursor sol obtained in Example 1 was mixed with PVA aqueous solution at a volume ratio of 1:1 and magnetically stirred at room temperature for more than 2 hours to ensure that the two components were fully mixed and homogeneous, thus obtaining SiO2 / PVA composite spinning solution. (3) The above composite spinning solution is loaded into a 10mL plastic syringe and installed on the injection pump of the conjugate electrospinning device; the spinning parameters are set as follows: applied voltage 10kV, receiving distance 10cm, feed rate 1.5mL / h, ambient temperature 25℃, relative humidity 40%~50%; (4) Start the conjugate electrospinning device to spin. Under the action of a symmetrical electric field, the fibers collide, intertwine and self-assemble in the collection area to form a three-dimensional fluffy white sponge-like precursor. Spin continuously for 4h to 8h and collect the obtained precursor sponge (S-SiO2 / PVA). (5) The precursor sponge was placed in a muffle furnace and heated to 800°C at a heating rate of 2°C / min, and kept at the temperature for 2 hours. PVA was completely removed by pyrolysis at high temperature, and the TEOS hydrolysis condensation product was converted into SiO2 inorganic fiber skeleton. (6) Cool naturally to room temperature with the furnace, take out the sample, and you will get pure SiO2 three-dimensional nanofiber sponge (S-SiO2-0K or S-SiO2).

[0039] Example 3: Preparation of SiO2 / kaolin three-dimensional composite nanofiber sponge (S-SiO2-1K) SiO2 / PVA composite spinning solution was prepared according to the method in Example 2; kaolin particles were added to the spinning solution, and the amount of kaolin added was 1 wt% of the theoretical SiO2 yield in the spinning solution; the kaolin was stirred at room temperature for more than 6 hours to ensure uniform dispersion; the remaining spinning parameters and calcination process were the same as in Example 2, and a three-dimensional composite nanofiber sponge was obtained, which was named S-SiO2-1K.

[0040] Example 4: Preparation of SiO2 / kaolin three-dimensional composite nanofiber sponge (S-SiO2-3K) SiO2 / PVA composite spinning solution was prepared according to the method in Example 2; kaolin particles were added to the spinning solution, and the amount of kaolin added was 3wt% of the theoretical SiO2 yield in the spinning solution; the kaolin was stirred at room temperature for more than 6 hours to ensure uniform dispersion; the remaining spinning parameters and calcination process were the same as in Example 2, and a three-dimensional composite nanofiber sponge was obtained, which was named S-SiO2-3K.

[0041] Example 5: Preparation of SiO2 / kaolin three-dimensional composite nanofiber sponge (S-SiO2-5K) (1) Prepare SiO2 precursor sol according to the method of Example 1; (2) Prepare a 10% PVA aqueous solution according to the method in step (1) of Example 2; (3) Mix the SiO2 precursor sol and PVA aqueous solution at a volume ratio of 1:1 and stir until homogeneous; (4) Weigh kaolin particles (particle size 1μm~5μm), add 5wt% of the theoretical SiO2 yield in the spinning solution, add to the above mixed spinning solution, and continuously stir magnetically at room temperature for more than 8 hours to make the kaolin particles fully and evenly dispersed in the spinning solution to obtain SiO2 / PVA / kaolin composite spinning solution. (5) The above composite spinning solution was spun in three dimensions using a conjugate electrospinning device. The spinning parameters were: applied voltage 10kV, receiving distance 10cm, feed rate 1.5mL / h, ambient temperature 25℃, relative humidity 40%~50%; continuous spinning for 6 hours, and the resulting three-dimensional fluffy precursor sponge was collected. (6) Place the precursor sponge in a muffle furnace and heat it to 800°C at a heating rate of 2°C / min. Keep it heated for 2 hours. After cooling to room temperature, take it out to obtain an all-inorganic three-dimensional SiO2 / kaolin composite nanofiber sponge (S-SiO2-5K).

[0042] Example 6: Preparation of SiO2 / kaolin three-dimensional composite nanofiber sponge (S-SiO2-7K) SiO2 / PVA composite spinning solution was prepared according to the method in Example 2; kaolin particles were added to the spinning solution, and the amount of kaolin added was 7wt% of the theoretical yield of SiO2 in the spinning solution; the remaining steps were the same as in Example 5, and a three-dimensional composite nanofiber sponge was obtained, and the obtained sample was named S-SiO2-7K.

[0043] Example 7: Preparation of SiO2 / kaolin three-dimensional composite nanofiber sponge (S-SiO2-10K) SiO2 / PVA composite spinning solution was prepared according to the method in Example 2; kaolin particles were added to the spinning solution, and the amount of kaolin added was 10 wt% of the theoretical SiO2 yield in the spinning solution; the remaining steps were the same as in Example 5, and a three-dimensional composite nanofiber sponge was obtained, and the obtained sample was named S-SiO2-10K.

[0044] Example 8: Preparation of SiO2 fiber membrane (M-SiO2, two-dimensional membrane control group) Using the same spinning solution formulation as in Example 2 (SiO2 / PVA composite spinning solution), but employing a conventional flat electrospinning apparatus (non-conjugate spinning), fibers were collected on a planar aluminum foil to obtain a two-dimensional fiber membrane precursor (M-PVA / SiO2); subsequently, it was treated according to the same calcination process (800℃, 2h) as in Example 2 to obtain a SiO2 / kaolin two-dimensional fiber membrane (M-SiO2), which served as a control group to verify the advantages of the three-dimensional structure.

[0045] Example 9: Characterization of the physicochemical properties of materials (1) Figure 3 Figure A shows the pore size distribution of the calcined SiO2 fiber membrane (M-SiO2) prepared in Example 8. Figure 3 Figure B in the middle is a pore size distribution diagram of the calcined SiO2 nanofiber sponge (S-SiO2) prepared in Example 2. Figure 3 A comparison of the pore size distribution in Figures A and B shows that the pore size of the three-dimensional nanofiber sponge prepared in this application is significantly increased, forming a more fluffy nanofiber network.

[0046] (2) The SiO2 / kaolin nanofiber sponges with different formulations obtained in Examples 2 to 7 were characterized as follows. 1) Microstructure: Field emission scanning electron microscopy (FE-SEM) was used to observe the fiber morphology, diameter distribution, and dispersion state of kaolin particles. Results are as follows: Figure 2 As shown in Figures A and B: the fiber surface of pure SiO2 sponge (S-SiO2) is smooth, with a diameter of about 300nm to 500nm; as the amount of kaolin added increases, it can be observed that kaolin particles are uniformly attached to the fiber surface and fiber intersections, and the fiber diameter increases slightly. Figure 2 The EDS elemental surface scan results in the C-figure confirm that Si, O, and Al elements are uniformly distributed in the material, indicating that kaolinite achieves uniform loading in the sponge.

[0047] 2) Crystal structure: such as Figure 3 The XRD pattern in the middle D figure shows that SiO2 is in an amorphous state after calcination (with a wide scattering peak in the range of 2θ = 20° to 25°). Kaolinite is transformed into metakaolinite phase after calcination at 800℃, and its characteristic diffraction peaks are consistent with those of standard metakaolinite.

[0048] 3) Chemical composition: such as Figure 3 As shown in the FTIR spectrum in Figure C, the Si-O-Si antisymmetric stretching vibration absorption peak (approximately 1070 cm⁻¹) can be observed. -1 The presence of Si-OH bending vibration peaks and characteristic absorption peaks related to Al-O confirmed the coexistence of SiO2 and kaolin components. The complete disappearance of characteristic absorption peaks (CH stretching, OH stretching, etc.) of PVA after calcination confirmed that PVA had been thoroughly removed.

[0049] 4) Liquid absorption performance: such as Figure 3 As shown in Figure F, the absorbance ratio of dried sponge samples was calculated by immersing them in deionized water or simulated body fluid (SBF) until saturation. The absorbance ratio of S-SiO2-5K can reach more than 8 times its own dry weight, which is significantly higher than that of the two-dimensional fiber membrane control (M-SiO2-5K) and medical gauze.

[0050] 5) The results of bulk density and porosity are as follows: Figure 3 China G map and Figure 3 As shown in Figure H, the three-dimensional nanofiber sponge samples (S-SiO2 series) have lower bulk density and higher porosity than the fiber membrane (M-SiO2) samples. The bulk density of the sponges ranges from approximately 5-9 mg / cm³, while the bulk density of the membranes is approximately 18 mg / cm³. In different sponge groups, the bulk density increases slightly with increasing kaolin loading. Meanwhile, the porosity of all sponge samples is higher than 90%, and the porosity increases with increasing kaolin loading, with the optimal formulation S-SiO2-5K achieving a porosity of over 93%. This low-density, high-porosity structure endows them with excellent liquid absorption capacity.

[0051] Example 10: Cell compatibility evaluation The cell compatibility of the material was evaluated using mouse fibroblast L929 cells. (1) Samples such as S-SiO2-0K and S-SiO2-5K were co-cultured with L929 cells for 24h and 48h respectively in accordance with ISO 10993 standard using material extraction solution (0.1g / mL, 37℃ for 24h). (2) Cell viability was detected by CCK-8 assay, and Calcein-AM / PI live / dead cell fluorescence staining was performed simultaneously. (3) Results showed that the cell compatibility of the samples was assessed using L929 fibroblasts by live / dead cell staining and CCK-8 assay. Live / dead cell staining results showed that after 1, 3, and 5 days of culture, all groups had a large number of live cells (green) and a small number of dead cells (red). Figure 4(As shown in Figure A). CCK-8 assays showed that cell viability increased in a time-dependent manner for all samples. The S-SiO2-5K group consistently exhibited the highest OD values ​​at all test time points, indicating that the kaolin-containing formulation possesses good cell compatibility. Figure 4 (As shown in Figure B).

[0052] Example 11: Blood compatibility evaluation (hemolysis test) (1) Take fresh anticoagulated whole blood from healthy individuals and prepare a red blood cell suspension with a mass percentage concentration of 2%; (2) Each group of sponge samples (M-SiO2, S-SiO2, S-SiO2-1K, S-SiO2-3K, S-SiO2-5K) were incubated with red blood cell suspension at 37°C for 1 hour; deionized water was used as a positive control (complete hemolysis) and normal saline was used as a negative control. (3) Centrifuge and collect the supernatant. Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance at a wavelength of 540 nm and calculate the hemolysis rate. (4) The results showed that the hemolysis rate of all sample groups ranged from 0.9±0.2% to 2.8±0.4%. With the increase of kaolin loading, the hemolysis rate increased slightly, but was still significantly lower than the safety threshold of 5.0% (all p <0.05 (as shown in Figures E and F in Figure 6), meeting the requirements of the national standard GB / T 16886.4 for the blood compatibility of medical materials, indicating that the material will not cause significant red blood cell rupture and has good blood compatibility.

[0053] Example 12: Evaluation of in vitro coagulation performance (1) Whole blood clotting time test Fresh anticoagulated whole blood was taken and dropped onto the surface of each group of samples (medical gauze was used as a control). After standing in a 37°C water bath for different times, deionized water was added to terminate the reaction. The absorbance of the supernatant at 540 nm was measured to evaluate the amount of uncoagulated free hemoglobin.

[0054] Results showed that whole blood clotting time was measured to evaluate the in vitro hemostatic performance of the samples. After testing, no residual blood was observed in the three-dimensional composite nanofiber sponge group, while a large amount of unclotted blood remained in the two-dimensional fiber membrane (M-SiO2) group and the gauze group (Figure 6, C). The clotting time of the three-dimensional composite nanofiber sponge group was significantly faster (90s ± 10s to 122s ± 8s), compared to the M-SiO2 group (237s ± 8s) and the gauze group (285s). The difference was statistically significant compared to ±5s. p<0.01 (as shown in Figure D in Figure 6). This advantage may stem from the three-dimensional structure of the three-dimensional composite nanofiber sponge, which has high porosity and a large specific surface area, enabling it to enhance liquid absorption and accelerate blood clotting.

[0055] (2) Measurement of prothrombin time (PT) and activated partial thromboplastin time (APTT) Fresh platelet-poor plasma (PPP) was collected and mixed with the extracts of each group of sample materials in a specified ratio. PT and APTT were then measured using a fully automated coagulation analyzer.

[0056] The results show that: Figure 6 As shown in Figures H and I, the PT and APTT values ​​of all three-dimensional composite nanofiber sponge groups were shorter than those of the blank group and the M-SiO2 group. p <0.05). Among them, S-SiO2-5K had the shortest PT and APTT values, at 16s±0.8s and 25s±1.2s, respectively. These results indicate that the three-dimensional composite nanofiber sponge containing kaolin can effectively activate extrinsic and intrinsic coagulation pathways.

[0057] (3) Platelet adhesion test After incubating each group of samples with platelet-rich plasma (PRP) at 37°C, the samples were fixed, dehydrated, and sputter-coated with gold. The adhesion of platelets on the material surface was then observed using SEM.

[0058] The results show that: Figure 6 As shown in Figure G, a large number of platelets adhered to the surface of the S-SiO2-5K sponge and exhibited an activated morphology with pseudopodia extension. The number of platelets adhering and the degree of activation were significantly better than those in the control group, which confirmed the synergistic promoting effect of silanol groups on the surface of SiO2 fibers and kaolin particles on platelet adhesion and activation.

[0059] Example 13: Evaluation of antibacterial properties Plate colony counting method After co-culturing each group of samples with a certain concentration of bacterial suspension, an appropriate amount was diluted and plated. After incubation at 37°C for 24 hours, the number of colonies was counted and the antibacterial rate was calculated (the Control group was the culture medium without antibacterial agent).

[0060] The results show that: by the plate diffusion method ( Figure 5 (As shown in Figure A) The sample was evaluated for its resistance to Staphylococcus aureus (Staphylococcus aureus). S.aureus ) and Escherichia coli ( E.Coli The antibacterial activity of kaolin was observed. The number of bacterial colonies gradually decreased with increasing kaolin content. Quantitative analysis ( Figure 5Figures B and C show that the S-SiO2-5K group exhibited the best performance, achieving an antibacterial rate of 98.8% ± 0.4% against Staphylococcus aureus and 93.2% ± 2.5% against Escherichia coli. These values ​​are higher than those of the S-SiO2 group (33.6% ± 1.7% and 52.0% ± 6.4%, respectively) and the two-dimensional fiber membrane (M-SiO2) group (15.2% ± 2.6% and 44.0% ± 6.8%, respectively) (all p-values ​​for comparisons are less than 0.05). These findings indicate that the loading of kaolinite endows the nanofiber sponge with strong broad-spectrum antibacterial properties.

[0061] Example 14: Evaluation of in vivo hemostatic performance (mouse tail amputation hemostasis model) (1) Laboratory animals and grouping Healthy adult Kunming mice (weighing 30g–35g) were randomly divided into the following experimental groups (n ≥ 5 per group): The study group was divided into a blank control group (no treatment), a medical gauze group, a two-dimensional SiO2 fiber membrane group (M-SiO2), a pure SiO2 three-dimensional sponge group (S-SiO2-0K), and a SiO2 / kaolin three-dimensional sponge group (S-SiO2-5K).

[0062] (2) Experimental procedures After mice were anesthetized by inhalation of isoflurane, their tails were transversely severed with a scalpel about 1 cm from the tip of the tail, and the wound was immediately brought into contact with the corresponding hemostatic sponge. The time from when the material came into contact with the wound until the bleeding completely stopped was recorded as the hemostasis time. Blood during the hemostasis process was collected on pre-weighed filter paper, and the amount of blood loss was calculated.

[0063] (3) Experimental results The in vivo hemostatic effects of the three-dimensional composite nanofiber sponge and the two-dimensional fiber membrane were evaluated using a mouse tail truncation model (Figure 7A). Measurements of bleeding time and blood loss are shown below. Figure 7 As shown in Figures B, C, and D, the S-SiO2-5K sponge exhibited the shortest hemostasis time and the lowest blood loss, achieving hemostasis within 32s ± 7s with a blood loss of 10.8mg ± 0.6mg. These values ​​were significantly superior to the control group gauze (285s ± 5s; 46.8mg ± 2.7mg), M-SiO2 membrane (240s ± 10s; 38.4mg ± 2.1mg), and S-SiO2 sponge (152s ± 12s; 27.3mg ± 1.9mg), with highly significant differences. p<0.01). Although the M-SiO2 membrane reduced bleeding compared to the control group, its performance was still lower than that of the sponge group. Overall, these in vivo results indicate that the combination of the three-dimensional porous nanofiber structure and kaolinite synergistically improves hemostatic efficiency, highlighting the potential of the S-SiO2-5K sponge in clinical applications.

[0064] In the above embodiments, the descriptions of each embodiment have different focuses. For parts not described in detail in a certain embodiment, please refer to the relevant descriptions in other embodiments.

[0065] The present application provides a detailed description of an inorganic three-dimensional composite nanofiber hemostatic sponge, its preparation method, and its application. Specific examples have been used to illustrate the principles and implementation methods of the present application. The descriptions of the above embodiments are only for the purpose of helping to understand the methods and core ideas of the present application. At the same time, those skilled in the art will recognize that there will be changes in the specific implementation methods and application scope based on the ideas of the present application. Therefore, the content of this specification should not be construed as a limitation of the present application.

Claims

1. An inorganic three-dimensional composite nanofiber hemostatic sponge, characterized in that, The inorganic three-dimensional composite nanofiber hemostatic sponge includes a three-dimensional interconnected network framework composed of SiO2 nanofibers and kaolin. The kaolin is chemically bonded and in situ fixed on the surface of SiO2 nanofibers and at the intersection nodes between SiO2 nanofibers.

2. The inorganic three-dimensional composite nanofiber hemostatic sponge according to claim 1, characterized in that, The mass ratio of the SiO2 nanofibers to the kaolin is (10~40):

1.

3. The inorganic three-dimensional composite nanofiber hemostatic sponge according to claim 1, characterized in that, The diameter of the SiO2 nanofibers is 200 nm to 800 nm.

4. The inorganic three-dimensional composite nanofiber hemostatic sponge according to claim 1, characterized in that, The inorganic three-dimensional composite nanofiber hemostatic sponge is prepared by the following method: Provide a silicon source to prepare a precursor solution containing the silicon source; A solution containing PVA is mixed with a precursor solution containing a silicon source to obtain a silicon source and PVA composite spinning solution. Kaolin particles were added to the silicon source and PVA composite spinning solution, and three-dimensional spinning was performed using a conjugate electrospinning device to obtain a nanofiber sponge-like precursor. High-temperature calcination of the nanofiber sponge-like precursor yields an inorganic three-dimensional composite nanofiber hemostatic sponge.

5. A method for preparing the inorganic three-dimensional composite nanofiber hemostatic sponge as described in claim 1, characterized in that, Includes the following steps: Provide a silicon source to prepare a precursor solution containing the silicon source; A solution containing PVA is mixed with a precursor solution containing a silicon source to obtain a silicon source and PVA composite spinning solution. Kaolin particles were added to the silicon source and PVA composite spinning solution, and three-dimensional spinning was performed using a conjugate electrospinning device to obtain a nanofiber sponge-like precursor. High-temperature calcination of the nanofiber sponge-like precursor yields an inorganic three-dimensional composite nanofiber hemostatic sponge.

6. The method for preparing the inorganic three-dimensional composite nanofiber hemostatic sponge according to claim 5, characterized in that, The precursor solution containing the silicon source also includes ethanol, water and an acid catalyst, and the molar ratio of the silicon source, ethanol, water and acid catalyst is 1:(1~3):(1~3):(0.01~0.05).

7. The method for preparing the inorganic three-dimensional composite nanofiber hemostatic sponge according to claim 5, characterized in that, The parameters for the three-dimensional spinning are: applied voltage of 15kV to 30kV, receiving distance of 15cm to 25cm, and spinning propulsion rate of 0.5mL / h to 3mL / h.

8. The method for preparing the inorganic three-dimensional composite nanofiber hemostatic sponge according to claim 5, characterized in that, The calcination parameters are as follows: heating to 600℃~1000℃ at a heating rate of 1℃ / min~5℃ / min, and holding at that temperature for 2h~4h.

9. The method for preparing the inorganic three-dimensional composite nanofiber hemostatic sponge according to claim 5, characterized in that, The acid catalyst is hydrochloric acid or phosphoric acid; and / or, the precursor solution containing a silicon source is mixed with a solution containing PVA at a volume ratio of 1:(0.5-2).

10. The application of the nanofiber hemostatic sponge prepared by the preparation method of the inorganic three-dimensional composite nanofiber hemostatic sponge as described in any one of claims 1 to 4 or the inorganic three-dimensional composite nanofiber hemostatic sponge as described in any one of claims 5 to 9 in the preparation of hemostatic dressings.

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