Kaolin-based multifunctional hemostatic microspheres and preparation method thereof

Abstract

This invention relates to the field of hemostatic materials technology, and more particularly to a kaolin-based multifunctional hemostatic microsphere and its preparation method. The preparation method includes the following steps: kaolin-calcium peroxide composite material and sodium alginate are mixed evenly in water, then added dropwise to a zinc chloride solution. After separation and freeze-drying, the kaolin-based multifunctional hemostatic microspheres are obtained. The kaolin-calcium peroxide composite material is prepared by the following method: kaolin, ammonia, polyethylene glycol, and hydrogen peroxide are added sequentially to a calcium chloride aqueous solution with stirring. After the reaction is complete, the mixture is centrifuged, washed, and dried to obtain the kaolin-calcium peroxide composite material. The kaolin-based multifunctional hemostatic microspheres prepared by this invention have antibacterial properties, and their hemostatic performance, applicability, and stability are enhanced.

Description

Technical Field

[0001] This invention relates to the field of hemostatic materials technology, and in particular to a kaolin-based multifunctional hemostatic microsphere and its preparation method. Background Technology

[0002] In trauma care and surgery, the development of efficient and multifunctional hemostatic materials is crucial for managing life-threatening hemorrhage. Modern hemostatic material design has shifted from passive coverage to active regulation, aiming for a synergistic effect of rapid hemostasis, infection control, and wound healing promotion. Against this backdrop, kaolin, a natural silicate mineral, has attracted significant attention due to its unique biological activity. Its hemostatic mechanism is not simply physical adsorption, but rather based on contact activation: the permanent negative charge and Si-OH and Al-OH on its layered surface can efficiently adsorb and activate coagulation factor XII, strongly initiating the intrinsic coagulation pathway, while simultaneously providing a large specific surface area for platelet adhesion and aggregation, achieving synergistic hemostasis through physical and biochemical processes.

[0003] Based on this mechanism, mature products such as QuikCkot@Combat Gauze (kaolin-coated gauze) have emerged in the market and are recommended by multiple guidelines. Related patents, such as US20040171577A1 (regarding zeolite hemostasis), also verify the general principle of inorganic mineral particles activating coagulation through water absorption and surface activation. However, through in-depth analysis of existing technologies (including academic literature and patents), we have found that current kaolin-based hemostatic materials have a series of key defects caused by the material itself: First, the morphology and size of kaolin particles are uncontrollable, posing potential safety and efficacy risks. Existing technologies (such as early QuikCkot powder and its similar patent WO2008094193A1) mostly use mechanically pulverized kaolin, resulting in a wide particle size distribution and irregular morphology. This directly causes significant batch-to-batch differences in hemostatic efficacy, and excessively small or sharp particles pose potential biosafety risks of microvascular embolism and chronic inflammation, reflecting that the processing of mineral raw materials is still in a crude stage.

[0004] Secondly, in terms of functionality, existing materials are too simplistic and unable to cope with the complex wound microenvironment. Most products (such as patent CN102553000A) only utilize the inherent coagulation activity of kaolin, completely lacking intrinsic antibacterial capabilities. Although there have been attempts, such as patent US20110190617A1, to introduce silver ions through physical mixing, this method easily leads to the burst release and uneven distribution of antibacterial components, failing to provide lasting protection. Furthermore, existing materials typically exist as bio-inert foreign bodies after hemostasis, unable to actively guide tissue repair and regeneration, contradicting the concept of integrated wound management.

[0005] Therefore, there is an urgent need in this field to fundamentally address the core issues of kaolin's particle uniformity, safety, multifunctional integration, and biodegradability through interdisciplinary material design, while inheriting the highly efficient hemostatic activity of kaolin. Developing a structurally controllable and functionally integrated kaolin-based multifunctional hemostatic material has become a clear innovative direction for next-generation mineral-based biomaterials. Summary of the Invention

[0006] The purpose of this invention is to address the aforementioned shortcomings of existing technologies by proposing a kaolin-based multifunctional hemostatic microsphere and its preparation method. These composite microspheres exhibit excellent hemostatic and antibacterial effects.

[0007] The first objective of this invention is to provide a method for preparing kaolin-based multifunctional hemostatic microspheres, wherein kaolin calcium peroxide composite material and sodium alginate are mixed evenly in water, then added dropwise to zinc chloride solution, and after separation and freeze-drying, kaolin-based multifunctional hemostatic microspheres are obtained. The kaolin calcium peroxide composite material is prepared by the following method: kaolin, ammonia, polyethylene glycol and hydrogen peroxide are added sequentially to a calcium chloride aqueous solution by stirring. After the reaction is completed, the mixture is centrifuged, washed and dried to obtain the kaolin calcium peroxide composite material.

[0008] Furthermore, the mass of the kaolin-calcium peroxide composite material is 10%-90% of the mass of sodium alginate.

[0009] Furthermore, the crosslinking agent is one of calcium salts, zinc salts, strontium salts, barium salts, iron salts, and aluminum salts; the concentration of the crosslinking agent solution is 1%. Specifically, the types of crosslinking agents include, but are not limited to: calcium salts (calcium chloride, calcium nitrate, calcium acetate, calcium lactate, calcium gluconate, calcium sulfate); zinc salts (zinc chloride, zinc sulfate); strontium salts (strontium chloride, strontium nitrate); barium salts (barium chloride, barium nitrate); iron salts (ferric chloride, etc.); and aluminum salts (aluminum chloride, etc.).

[0010] Furthermore, the mass ratio of calcium chloride to kaolin is 6:0.5-15.

[0011] Furthermore, the molar ratio of ammonia to calcium ions in the ammonia solution is greater than 6.

[0012] Furthermore, the polyethylene glycol specifically refers to polyethylene glycol 200.

[0013] Furthermore, the amount of needle used for adding the solution is less than or equal to 27g.

[0014] Furthermore, the concentration of the calcium chloride aqueous solution is 0.8-1.5 g / ml.

[0015] Furthermore, the volume ratio of polyethylene glycol to calcium chloride aqueous solution is 18-22:6.

[0016] Furthermore, the molar ratio of hydrogen peroxide to calcium ions is greater than 5.

[0017] Furthermore, the drying temperature is 50-80℃.

[0018] The first objective of this invention is to provide a kaolin-based multifunctional hemostatic microsphere prepared using the above-described preparation method.

[0019] This invention employs a room-temperature precipitation method to prepare a kaolin-calcium peroxide composite material with integrated hemostatic and antibacterial functions. First, calcium ions are enriched on the surface or edge sites of kaolin. Then, hydrogen peroxide is introduced under alkaline conditions to promote the nucleation and growth of calcium peroxide at the interface, forming a composite rather than physically mixed kaolin-calcium peroxide composite material. In the kaolin-calcium peroxide composite material, CaO2 is uniformly loaded onto kaolin in the form of nanodomains. Through Si-O-Ca bridging and interface-induced strain, a stable heterogeneous structure of "uniform loading-lattice micro-shrinkage-local amorphous" is constructed. The purity, phase interface are clear, and the surface negative potential is enhanced, laying the structural and interface foundation for subsequent electronic structure regulation, oxygen release / antibacterial and hemostatic performance improvement.

[0020] This invention utilizes the large specific surface area, good adsorption properties, and biocompatibility of kaolin to uniformly distribute nano-calcium peroxide on the surface of kaolin, achieving a better antibacterial effect. It also reduces powder dispersion and erosion loss, improves effective contact time and utilization rate. Furthermore, the characteristics of kaolin itself in activating coagulation factors and initiating its own coagulation pathway, along with the calcium ion precipitation of calcium peroxide, synergistically improve the hemostatic performance of the kaolin-calcium peroxide composite material.

[0021] The ammonia water used in this invention is used to provide alkalinity and enhance adsorption; PEG200 is used to improve dispersion, inhibit violent reactions and agglomeration, and improve batch consistency.

[0022] The kaolin-based multifunctional hemostatic microspheres prepared in this invention adhere more easily to and fill irregular wounds, achieving sustained release of active components while balancing rapid hemostasis with continuous seepage control. They possess antibacterial properties and exhibit improved hemostatic performance, applicability, and stability. Kaolin itself has a procoagulant effect (promoting coagulation cascades and platelet-related processes). The composite microspheres can create a local microenvironment conducive to coagulation on the wound surface, resulting in faster clot / clot formation and a more stable hemostatic coating. This makes them suitable for rapid initial hemostasis in open wounds and bleeding wounds. In a mouse model of coagulation dysfunction, they achieve a faster hemostatic response and reduce persistent bleeding and secondary hemorrhage.

[0023] The kaolin-based multifunctional hemostatic microspheres prepared in this invention exhibit excellent antibacterial capabilities while achieving hemostasis, reducing the bacterial load on the wound surface and thus minimizing infection-induced inflammation and secondary bleeding. In the wound exudate environment, the kaolin-based multifunctional hemostatic microspheres gradually release their active components, creating local conditions unfavorable to bacterial survival, thereby inhibiting common pathogens and reducing early bacterial load and the risk of secondary infection. They are particularly suitable for contaminated wounds or wounds with a high risk of infection.

[0024] This invention involves thoroughly mixing kaolin calcium peroxide composite material with sodium alginate to ensure consistent distribution of active components in the matrix, avoiding agglomeration and sedimentation. The mixture is then added dropwise to a crosslinking agent solution using a microsphere preparation device for crosslinking and molding. The key control measures include particle size, crosslinking density, and structural strength to achieve good conformability and retention.

[0025] The raw materials used in this invention are readily available and have good biocompatibility. At the same time, the preparation process is relatively simple, highly operable, and easy to produce on a large scale.

[0026] The kaolin-based multifunctional hemostatic microspheres prepared by this invention can be directly applied to wounds. The microsphere formulation is easier to adhere to and fill irregular wound surfaces, has stronger resistance to erosion and retention, and can be further loaded, coated, or filled into common medical carriers (such as gauze, hemostatic sponges, patches, gels, spray coatings, etc.) to achieve usability and processability in different scenarios.

[0027] The preparation process of this invention is relatively simple, highly operable, has controllable quality, acceptable cost, and is easy to mass-produce. Attached Figure Description

[0028] Figure 1 Scanning electron microscope images of CaO2 prepared in Comparative Example 2 and K-CaO2 prepared in Example 1; Figure 2 HRTEM image of K-CaO2 prepared in Example 1; Figure 3 HRTEM image of CaO2 prepared in Comparative Example 2; Figure 4 X-ray diffraction patterns of K, CaO2, and K-CaO2; Figure 5 The antibacterial properties of K, CaO2, and K-CaO2; Figure 6 To assess the antibacterial properties of 3K-CaO2 prepared in Comparative Example 3; Figure 7 The BCI values ​​are for K, CaO2, and K-CaO2. Figure 8 Photographs of the microsphere products prepared for comparative examples and embodiments; Figure 9 The particle size distribution of the microspheres prepared for comparative examples and embodiments; Figure 10 The microstructures of SA, KSA, and KCSA are shown. Figure 11 , 12 BCI values ​​for SA, KSA, KCSA, and 60% KCSA; Figure 13 In vitro coagulation properties of microspheres with different particle sizes; Figure 14 The antibacterial properties of SA, KSA, and KCSA. Detailed Implementation

[0029] The following are specific embodiments of the present invention, which are described in conjunction with the accompanying drawings. However, the present invention is not limited to these embodiments.

[0030] The kaolin ore sample used was provided by Jiangxi Chongyi County Huaming Kaolin Co., Ltd. The kaolin was first ground and crushed, and then sieved through a 200-mesh standard sieve. The kaolin obtained was named K.

[0031] Example 1 A multifunctional hemostatic microsphere with both antibacterial and hemostatic properties is produced by following these steps: Preparation of kaolin-calcium peroxide composite material: 6 g of anhydrous calcium chloride and 60 ml of deionized water were added to a beaker and stirred until dissolved. Then, 6 g of kaolin K was added and stirred until homogeneous. Next, 30 mL of ammonia water (15 mol / L) was added and stirred for 10 min. Then, 200 mL of polyethylene glycol 200 (PEG200) was added and stirred at 600 rpm for 10 min. Finally, 30 mL of 30% hydrogen peroxide was quickly added and stirring continued for 6 h. After the reaction was complete, the mixture was centrifuged at 10,000 rpm, washed three times with anhydrous ethanol, and dried in a forced-air drying oven. The resulting kaolin-calcium peroxide composite material was ground into powder and named K-CaO2.

[0032] Preparation of crosslinking agent: Taking zinc chloride as an example, prepare a 1 w / w% zinc chloride solution. Preparation method: Weigh 5.0 g of zinc chloride and add it to 495.0 mL of deionized water. Add a magnetic rotor and stir with a stirrer until completely dissolved. If it cannot be dissolved, add 0.5~1.0 mL of concentrated hydrochloric acid as needed and continue stirring until completely dissolved.

[0033] Add 0.675 g K-CaO2 and 37.5 ml deionized water to a beaker, stir rapidly for 30 min, and slowly add 0.75 g sodium alginate (SA) in batches, stirring rapidly until SA is completely dissolved and KC is evenly dispersed. Then, add it dropwise to 1% zinc chloride (ZnCl2) solution through a microsphere preparation device (using a 24G needle). After separation and freeze-drying, kaolin-based multifunctional hemostatic microspheres are obtained and named KCSA.

[0034] Example 2 This embodiment reduces the amount of K-CaO2 added compared to embodiment 1.

[0035] 0.075 g K-CaO2 was placed in a beaker containing 37.5 mL of deionized water and stirred rapidly for 30 min. 0.75 g sodium alginate (SA) was added slowly in batches and stirred rapidly until SA was completely dissolved and KC was evenly dispersed. Then, it was added dropwise to 1% zinc chloride (ZnCl2) solution through a microsphere preparation device (using a 24G needle). After separation and freeze-drying, kaolin-based multifunctional hemostatic microspheres were obtained and named 10%KCSA.

[0036] Example 3 This method reduces the amount of K-CaO2 added.

[0037] 0.45 g K-CaO2 was placed in a beaker containing 37.5 mL of deionized water and stirred rapidly for 30 min. 0.75 g sodium alginate (SA) was added slowly in batches and stirred rapidly until SA was completely dissolved and K-CaO2 was evenly dispersed. Then, it was added dropwise to 1% zinc chloride (ZnCl2) solution through a microsphere preparation device (using a 24G needle). After separation and freeze-drying, kaolin-based multifunctional hemostatic microspheres were obtained and named 60%KCSA.

[0038] Example 4 This solution uses a needle with a smaller inner diameter.

[0039] Add 0.675 g K-CaO2 and 37.5 ml deionized water to a beaker, stir well, and then slowly add 0.75 g sodium alginate in batches. Stir with a high-speed stirrer until the sodium alginate solution is completely dissolved. Then, add the solution dropwise to a 1% zinc chloride (ZnCl2) solution using a microsphere preparation device (27G needle). After separation and freeze-drying, kaolin-based multifunctional hemostatic microspheres are obtained and named KCSA. 27 .

[0040] Example 5 This solution uses a needle with a larger inner diameter.

[0041] Add 0.675 g K-CaO2 and 37.5 ml deionized water to a beaker, stir well, and then slowly add 0.75 g sodium alginate in batches. Stir with a high-speed stirrer until the sodium alginate solution is completely dissolved. Then, add the solution dropwise to a 1% zinc chloride (ZnCl2) solution using a microsphere preparation device (18G needle). After separation and freeze-drying, kaolin-based multifunctional hemostatic microspheres are obtained and named KCSA. 18 .

[0042] Example 6 Expand production.

[0043] Add 36 g K-CaO2 and 1964 ml deionized water to a beaker, stir well, and then slowly add 40 g sodium alginate in batches. Stir with a high-speed stirrer until the sodium alginate solution is completely dissolved and mixed evenly with kaolin. Then, add it dropwise to a 1% zinc chloride (ZnCl2) solution through a microsphere preparation device (using a 24G needle). After separation and freeze-drying, kaolin-based multifunctional hemostatic microspheres are obtained and named KD-KCSA.

[0044] Comparative Example 1 6 g of anhydrous calcium chloride and 60 ml of deionized water were added to a beaker and stirred until dissolved. Then, 6 g of kaolin was added and stirred until homogeneous. Next, 30 mL of ammonia water was added and stirred for 10 min. Then, 200 mL of polyethylene glycol 200 (PEG200) was added and stirred at 600 rpm for 10 min. Finally, 30 mL of hydrogen peroxide was quickly added and stirring continued for 6 h. After the reaction was complete, the mixture was centrifuged at 10,000 rpm, washed three times with anhydrous ethanol, and dried in a forced-air drying oven. The resulting kaolin-calcium peroxide composite material was ground into powder and named K-CaO2.

[0045] Comparative Example 2 This solution does not include kaolin.

[0046] 0.75 g of SA was placed in a beaker containing 37.5 mL of deionized water and stirred rapidly until the SA was completely dissolved. Then, it was added dropwise to a 1% zinc chloride (ZnCl2) solution using a microsphere preparation device. After separation and freeze-drying, sodium alginate microspheres were obtained and named SA.

[0047] Comparative Example 3 This scheme increases the amount of K-CaO2 added.

[0048] 0.75 g of K-CaO2 kaolin-calcium peroxide composite material was placed in a beaker containing 37.5 mL of deionized water and stirred rapidly for 30 min. 0.75 g of sodium alginate (SA) was then slowly added in batches, with rapid stirring until the SA was completely dissolved and the K-CaO2 was uniformly dispersed. This mixture was then added dropwise to a 1% zinc chloride (ZnCl2) solution using a microsphere preparation device. After separation and freeze-drying, kaolin-based multifunctional hemostatic microspheres were obtained and named 100% KCSA. The concentration was too viscous to be extruded continuously in droplets and it was very easy to clog the needle.

[0049] Comparative Example 4 This solution uses a needle with a smaller inner diameter.

[0050] Add 0.675 g K-CaO2 and 37.5 ml deionized water to a beaker, stir well, and then slowly add 0.75 g sodium alginate in batches. Stir with a high-speed stirrer until the sodium alginate solution is completely dissolved and evenly mixed with kaolin. Then, add the mixture dropwise to a 1% zinc chloride (ZnCl2) solution using a microsphere preparation device (30G needle). After separation and freeze-drying, kaolin-based multifunctional hemostatic microspheres are obtained and named KCSA. 30 However, the needle's inner diameter is too small, making it easy to clog the needle tip and hindering continuous preparation.

[0051] Comparative Example 5 The solution involves adding CaO2.

[0052] 0.675 g CaO2 and 37.5 ml deionized water were added to a beaker and stirred until homogeneous. Then, 0.75 g sodium alginate was slowly added in batches and stirred with a high-speed stirrer until a complete sodium alginate solution was obtained. The solution was then added dropwise to a 1% zinc chloride (ZnCl2) solution using a microsphere preparation device (24G needle). After separation and freeze-drying, kaolin-based multifunctional hemostatic microspheres were obtained and named CSA. However, due to the precipitation of calcium ions, the mixed solution cross-linked prematurely, making it difficult to extrude into microspheres.

[0053] Comparative Example 6 0.675 g of K was placed in a beaker containing 37.5 mL of deionized water and stirred rapidly for 30 min. 0.75 g of sodium alginate (SA) was added slowly in batches and stirred rapidly until SA was completely dissolved and K was evenly dispersed. Then, it was added dropwise to 1% zinc chloride (ZnCl2) solution through a microsphere preparation device (using a 24G needle). After separation and freeze-drying, kaolin-based multifunctional hemostatic microspheres were obtained and named KSA.

[0054] Comparative Example 7 Prepare pure CaO2.

[0055] Add 6 g of anhydrous calcium chloride and 60 ml of deionized water to a beaker, stir to dissolve, then add 30 mL of ammonia (15 mol / L), stir for 10 min, then add 200 mL of polyethylene glycol 200 (PEG200), stir at 600 rpm for 10 min, then quickly add 30 mL of 30% hydrogen peroxide, and continue stirring for 6 h. After the reaction is complete, centrifuge at 10000 rpm, wash three times with anhydrous ethanol, and dry in a forced-air drying oven. Grind into powder to obtain the precursor powder, named CaO2.

[0056] Morphological and structural characterization analysis of kaolin-calcium peroxide composite materials: Pure CaO2 is densely aggregated into granular particles, but after the introduction of kaolin, CaO2 nanoparticles are uniformly anchored on the surface of the lamellae and in the interlayer structure, significantly improving particle dispersibility and interfacial stability. Figure 1 ).

[0057] HRTEM images of K-CaO2 show clear lattice striations within the kaolin-loaded CaO2 grains. Figure 2 The interplanar spacings of the (110) and (002) crystal planes were measured to be d = 0.239 nm and d = 0.278 nm, respectively, which are slightly smaller than those of the pure CaO2 sample (d = 0.241 nm and d = 0.284 nm). Figure 3 This indicates that the introduction of kaolin induced lattice compression and strain effects. This lattice contraction is attributed to Ca. 2+ During nucleation and growth, CaO2 undergoes coordination and electrostatic interactions with hydroxyl groups and oxygen bridges on the kaolin surface, resulting in restricted growth of CaO2 at the heterogeneous interface and micro-distortion of the unit cell. Furthermore, amorphous regions are visible in the central areas of some CaO2 grains, indicating that strong interfacial constraints and localized strain lead to localized disorder in the crystal structure. This structural feature may increase oxygen vacancies and defect density, thereby promoting the generation of reactive oxygen species and surface reactivity.

[0058] X-ray diffraction (XRD) showed diffraction peaks consistent with CaO2 (JCPDS 03-0865) at 2θ≈30.1°, 35.6°, 47.3°, and 53.1°, with symmetrical peak shapes and high signal-to-noise ratio. Figure 4No CaO (32–37°) or CaCO3 (29.4°) signals were observed. The slight positive shift in peak position is consistent with the d-spacing contraction of HRTEM, indicating that the interface constraint induces the average compressive strain. In summary, CaO2 in K-CaO2 is uniformly loaded onto kaolinite in the form of nanodomains. Through Si-O-Ca bridging and interface-induced strain, a stable heterostructure of "uniform loading-lattice micro-contraction-local amorphous" is constructed. The purity, phase interface are clear, and the surface negative potential is enhanced, laying the structural and interface foundation for subsequent electronic structure regulation, oxygen release / antibacterial and hemostatic performance improvement.

[0059] Analysis of the antibacterial properties of kaolin-calcium peroxide composite materials: Preparation of LB (Luria-Bertani) medium: Accurately weigh 20 mg of LB broth powder, add 1000 mL of distilled water, heat in an autoclave to dissolve, and cool to obtain a liquid culture medium for bacteria.

[0060] Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 25923) were used as Gram-negative and Gram-positive bacteria models, respectively. The antibacterial properties of the samples were evaluated using the plate count method. 10 μL of bacterial cryopreservation solution was added to 10 mL of liquid culture medium, sealed with a bacterial breathable membrane, and incubated at 37 °C and 180 rpm for 12 h in a constant temperature shaker. 500 μL of the bacteria incubated for 12 h was then diluted to a final concentration of 10. 5~6 CFU / mL. A certain amount of sample powder was weighed and dispersed into 10 mL of liquid culture medium. 500 μL of diluted bacterial solution was added, and the mixture was sealed with a bacterial breathable membrane and incubated in a constant temperature shaker at 37 ℃ and 180 rpm for 4 h. The bacterial solution was then diluted 10... 4 The samples were coated and incubated upside down in a 37 ℃ constant temperature incubator for 12 h, followed by imaging and recording using a gel imaging system. The experimental samples were kaolin K, CaO2, and K-CaO2, and the experimental bacterial strains were Escherichia coli and Staphylococcus aureus.

[0061] The experimental results of the plate coating method are as follows: Figure 5 and Figure 6 As shown, the original kaolin has poor antibacterial effect. After being loaded with CaO2, the composite material has excellent antibacterial properties. However, as the amount of kaolin added increases to 18g, the antibacterial properties of the composite material disappear.

[0062] Hemostatic properties of kaolin-calcium peroxide composite materials: In this experiment, each well of a 48-well plate was filled with 10 mg of hemostatic powder (K, CaO2, and K-CaO2) and 200 μL of heavily calcified anticoagulated rabbit blood (comprising 200 μL of anticoagulated rabbit blood and 10 μL of 0.2 mol / L CaCl2). The tubes were rapidly incubated in a preheated constant-temperature bath at 37°C for 4 minutes. After incubation, samples were removed at 1, 1.5, 2, 2.5, 3, 3.5, 4, and 4.5 minutes. To prevent coagulation and dissolve coagulated blood cells, 2.0 mL of deionized water was added to each tube. Subsequently, 1 mL of the supernatant was transferred to a new centrifuge tube and centrifuged at 2000 rpm for one minute. The supernatant was then collected, and the absorbance at 545 nm was measured.

[0063] The blood coagulation index (BCI) was calculated using a formula, and a BCI close to 0 was considered the point at which clotting was complete, with the time point at which hemostasis was achieved. Kaolin can accelerate hemostasis by activating the intrinsic coagulation pathway. We hypothesize that kaolin can also impart excellent hemostatic properties to K-CaO2 in composite materials. Blood coagulation index (BCI) assessment revealed that the K-CaO2 group had the lowest BCI value, indicating that it had the fastest in vitro coagulation rate. Figure 7 ).

[0064] Appearance analysis of microspheres: First, freeze-dried microspheres of different types of mineral powders were photographed using a digital camera. Figure 8 Before and after freeze-drying, the morphology of the microspheres changed slightly, with some showing signs of shrinkage, particularly sepiolite. Different types of composite hemostatic microspheres were obtained by adjusting the amount of minerals added (e.g., kaolin) and the size of the dispensing needles. The obtained microspheres were spread evenly in petri dishes, and their morphology was photographed using a gel imaging system. The particle size distribution of the microspheres was then analyzed using software. Figure 9 The addition of kaolin can reduce the particle size distribution of the resulting microspheres, and the higher the amount of kaolin added, the smaller the particle size. Secondly, the particle size distribution can also be adjusted by changing the needle size. It was found that as the needle diameter decreases, the microsphere particle size first decreases and then increases, reaching its minimum at 24G. To evaluate the continuous, stable, and large-scale production capacity of the device, the crosslinking solution was prepared on a scale-up basis. The microspheres were continuously prepared for 24 hours using a single-channel microsphere preparation device. No clogging or stagnation was observed during this period. The mass of the freeze-dried microspheres was 45.25 g. Figure 10 ).

[0065] Hemostatic properties of microspheres In this experiment, each well of the 48-well plate was filled with 10 mg of hemostatic microspheres (SA, KSA, KCSA, 60% KCSA, KCSA). 18 KCSA 21 and KCSA27 200 μL of decalcified anticoagulated rabbit blood (containing 200 μL of anticoagulated rabbit blood and 10 μL of 0.2 mol / L CaCl2) and 200 μL of decalcified anticoagulated rabbit blood were collected. These tubes were rapidly incubated in a preheated constant-temperature bath at 37°C for 2 minutes. After incubation, samples were removed at 0.5, 1, 1.5, and 2 minutes. To prevent coagulation and dissolve coagulated blood cells, 2.0 mL of deionized water was added to each tube. Then, 1 mL of the supernatant was transferred to a new centrifuge tube and centrifuged at 2000 rpm for 1 minute. The supernatant was then collected, and the absorbance at 545 nm was measured. The coagulation index (BCI) was calculated using the formula, and a BCI close to 0 was considered the point of complete coagulation, with the time point at which hemostasis was determined. The BCI results showed that, within the same time frame, KCSA was superior to KSA and SA in hemostasis, and with increasing K-CaO2 addition, the microspheres shortened the coagulation time. Figure 11-12 Taking into account both hemostatic efficiency and formulation stability, 90% was ultimately determined as the base formulation for subsequent studies. Based on this, the microsphere particle size was adjusted by changing needles of different specifications, and their in vitro coagulation performance was compared. Figure 13 The results showed that as the particle size decreased, both indicators first increased and then decreased. Among them, the microspheres prepared with 24G needles were superior to other specifications in terms of BCI reduction and red blood cell adhesion, followed by 21G microspheres.

[0066] In vitro antibacterial properties of microspheres Preparation of LB (Luria-Bertani) medium: Accurately weigh 20 mg of LB broth powder, add 1000 mL of distilled water, heat in an autoclave to dissolve, and cool to obtain a liquid culture medium for bacteria.

[0067] Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 25923) were used as Gram-negative and Gram-positive bacteria models, respectively. The antibacterial properties of the samples were evaluated using the plate count method. 10 μL of bacterial cryopreservation solution was added to 10 mL of liquid culture medium, sealed with a bacterial breathable membrane, and incubated at 37 °C and 180 rpm for 12 h in a constant temperature shaker. 500 μL of the bacteria incubated for 12 h was then diluted to a final concentration of 10. 5~6 CFU / mL. A certain amount of sample powder was weighed and dispersed into 10 mL of liquid culture medium. 500 μL of diluted bacterial solution was added, and the mixture was sealed with a bacterial breathable membrane and incubated in a constant temperature shaker at 37 ℃ and 180 rpm for 4 h. The bacterial solution was then diluted 10... 4Plates were prepared and incubated upside down in a 37 ℃ incubator for 12 h. Image and recording were then performed using a gel imaging system. Experimental samples included SA, KSA, and KCSA; experimental bacterial species included Escherichia coli and Staphylococcus aureus. Results of the plate spreading method and bacterial viability / death staining experiments are shown below. Figure 14 As shown, all microspheres exhibit good antibacterial effects.

[0068] For any points not covered above, existing technologies shall apply.

[0069] Although specific embodiments of the present invention have been described in detail by way of examples, those skilled in the art should understand that the above examples are for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications or additions to the described specific embodiments or use similar methods to replace them, without departing from the direction of the invention or exceeding the scope defined by the appended claims. Those skilled in the art should understand that any modifications, equivalent substitutions, improvements, etc., made to the above embodiments based on the technical essence of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing kaolin-based multifunctional hemostatic microspheres, characterized in that, After the kaolin calcium peroxide composite material and sodium alginate were mixed evenly in water, the mixture was added dropwise to a crosslinking agent solution, and after separation and freeze-drying, kaolin-based multifunctional hemostatic microspheres were obtained. The kaolin calcium peroxide composite material is prepared by the following method: kaolin, ammonia, polyethylene glycol and hydrogen peroxide are added sequentially to a calcium chloride aqueous solution by stirring. After the reaction is completed, the mixture is centrifuged, washed and dried to obtain the kaolin calcium peroxide composite material.

2. The preparation method according to claim 1, characterized in that, The mass of the kaolin-calcium peroxide composite material is 10%-90% of the mass of sodium alginate.

3. The preparation method according to claim 1, characterized in that, The crosslinking agent is one of the following: calcium salt, zinc salt, strontium salt, barium salt, iron salt, and aluminum salt; the concentration of the crosslinking agent solution is 1%.

4. The preparation method according to claim 1, characterized in that, The mass ratio of calcium chloride to kaolin is 6:0.5-15.

5. The preparation method according to claim 1, characterized in that, The molar ratio of ammonia to calcium ions in ammonia water is greater than 6.

6. The preparation method according to claim 1, characterized in that, The amount of needle used for dripping is less than or equal to 27g.

7. The preparation method according to claim 1, characterized in that, The concentration of the calcium chloride aqueous solution is 0.8-1.5 g / ml; the volume ratio of polyethylene glycol to calcium chloride aqueous solution is 18-22:

6.

8. The preparation method according to claim 1, characterized in that, The molar ratio of hydrogen peroxide to calcium ions is greater than 5.

9. The preparation method according to claim 1, characterized in that, The drying temperature is 50-80℃.

10. A kaolin-based multifunctional hemostatic microsphere prepared by the preparation method according to any one of claims 1-9.

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