Calcium-doped porous silicon hemostatic particles and preparation method thereof

Abstract

The invention relates to the technical field of hemostatic material preparation, in particular to calcium-doped porous silicon hemostatic particles and a preparation method thereof. The invention discloses a nano-selenium-doped porous silicon composite material which comprises the following components in percentage by mass: 35%-55% of porous silicon particles, 4%-8% of calcium element, 2%-6% of carboxymethyl chitosan grafted sodium alginate copolymer, 0.02%-0.3% of nano-selenium, 1%-4% of polydopamine coated hydroxyapatite, 0.8%-2.5% of graphene oxide quantum dots, 0.3%-1.5% of gallic acid and the balance of unavoidable impurities, the specific surface area is 80-350m < 2 > / g, the porosity is 45%-75%, the copolymer enhances the biological adhesion, the nano-selenium endows the antibacterial property, the polydopamine-coated hydroxyapatite improves the biocompatibility, and the multiple components synergistically realize the functions of stopping bleeding, resisting bacteria and promoting healing. Through cooperation of multiple components, the particles have the advantages of high hemostatic rate, good biocompatibility, broad-spectrum antibacterial and healing-promoting effects, stable slow release of calcium element, excellent degradability and no secondary injury risk.

Description

Technical Field

[0001] This invention relates to the field of hemostatic material preparation technology, specifically to a calcium-doped porous silica hemostatic particle and its preparation method. Background Technology

[0002] Hemostasis is a core aspect of trauma emergency care and clinical treatment. If blood loss exceeds 30% of the total blood volume in a short period, it will directly endanger life and may also lead to serious complications such as infection and organ failure. Therefore, developing safe and effective hemostatic materials is crucial to reducing trauma mortality. Currently, commonly used hemostatic materials in clinical practice are mainly divided into natural polymers and inorganic materials. Although they can achieve basic hemostasis, both have significant technical shortcomings.

[0003] Zeolite hemostatic agents can rapidly gather red blood cells and clotting factors due to the electrostatic attraction generated by their special molecular structure, thus achieving rapid hemostasis, such as the zeolite hemostatic agent preparation process disclosed in patent CN100571711C. However, during the hemostasis process, they release a large amount of heat through cation exchange, which can easily cause burns to the tissues around the wound and lead to secondary damage. At the same time, the high temperature environment increases the risk of bacterial growth, which is not conducive to subsequent wound healing.

[0004] Chitosan-based hemostatic materials are representative of natural polymers. While the chitosan composite hemostatic powder disclosed in patent CN103933601A possesses good hemostatic properties, its preparation requires the introduction of strong alkaline reagents. Residual alkaline substances may irritate the wound mucosa, posing a biosafety risk. Furthermore, the bioadhesion and degradation rate of traditional chitosan materials are difficult to precisely control, making them easily dispersed by blood in active bleeding scenarios, leading to hemostatic failure.

[0005] Besides the two types of materials mentioned above, porous silica, due to its high porosity, large specific surface area, and good biocompatibility, has gradually become a research hotspot for hemostatic materials. It can concentrate coagulation factors by adsorbing water; however, porous silica alone has a slow coagulation rate and lacks combined functions such as antibacterial and healing-promoting properties, failing to meet the treatment needs of complex wounds. Calcium, as a key activator of the coagulation cascade reaction, can accelerate prothrombin conversion, but its direct addition easily leads to problems such as excessively rapid dissolution and uneven distribution, making it difficult to form a synergistic effect with porous silica.

[0006] With the development of trauma medicine, the clinical requirements for hemostatic materials have evolved from simply providing "rapid hemostasis" to an integrated approach encompassing "hemostasis, antibacterial properties, and promoting healing." Existing materials suffer from shortcomings in synergistic hemostatic mechanisms, biocompatibility, and functional versatility, making them unsuitable for complex scenarios such as battlefield trauma and postoperative bleeding. Therefore, developing a novel hemostatic material that combines rapid hemostasis, prevents secondary damage, provides broad-spectrum antibacterial properties, and promotes healing has become a pressing technical challenge in this field. Summary of the Invention

[0007] (a) Technical problems to be solved

[0008] To address the shortcomings of existing technologies, this invention provides calcium-doped porous silica hemostatic particles and their preparation method.

[0009] (II) Technical Solution

[0010] A calcium-doped porous silica hemostatic particle, comprising, by mass percentage: 35%-55% porous silica particles, 4%-8% calcium, 2%-6% carboxymethyl chitosan-grafted sodium alginate copolymer, 0.02%-0.3% nano-selenium, 1%-4% polydopamine-coated hydroxyapatite, with the balance being unavoidable impurities; the particle size is 20nm-100μm, and the specific surface area is 80-350m². 2 / g, porosity 45%-75%, pore size distribution concentrated in 5-40nm, particle size distribution variation coefficient ≤12%;

[0011] Carboxymethyl chitosan and sodium alginate form a copolymer through a grafting reaction. This reaction uses 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) as the activation system and reacts at 50-60°C for 4-6 hours. The reaction of polydopamine-coated hydroxyapatite is achieved through dopamine self-polymerization. The reaction is carried out in Tris-HCl buffer at pH 8.5 and stirred at 30°C for 8-12 hours.

[0012] Preferably, it also includes 0.8%-2.5% graphene oxide quantum dots; the graphene oxide quantum dots have a spherical structure, a particle size of 2-8 nm, a lateral dimension of 10-30 nm, a surface hydroxyl content ≥3.0 mmol / g, a carboxyl content ≥1.5 mmol / g, and are prepared by oxidizing graphite powder using the Hummers method followed by ultrasonic exfoliation, and have a purity ≥99% after dialysis purification.

[0013] Preferably, it also includes 0.3%-1.5% gallic acid; the gallic acid has a purity of ≥98%, a melting point of 235-240℃, a water solubility of ≥10g / L, and is obtained through plant extraction and purification.

[0014] Preferably, the number average molecular weight of the carboxymethyl chitosan-grafted sodium alginate copolymer is 8 × 10⁻⁶. 4 -3×10 5 Da, weight-average molecular weight 1.2 × 10⁻⁶ 5 -4×10 5 Da, molecular weight distribution index 1.5-2.0.

[0015] Preferably, the nano-selenium has an amorphous structure with a particle size of 10-30 nm, uniform particle dispersion, no obvious agglomeration, and a dispersibility of ≥92%. The surface of the nano-selenium is modified with bovine serum albumin, with a modification amount of 10%-20% of the mass of the nano-selenium. The stability of the modified nano-selenium in physiological saline is significantly improved, with a 24-hour sedimentation rate of ≤5%.

[0016] Preferably, in the polydopamine-coated hydroxyapatite, the hydroxyapatite has a rod-like structure, a particle size of 50-200 nm, an aspect ratio of 1:1-3:1, a crystallinity of ≥85%, a Ca / P molar ratio of 1.60-1.67, is prepared by a hydrothermal method, and has a purity of ≥99%; the polydopamine coating layer has a thickness of 5-20 nm, a surface roughness Ra of 2-5 nm, and an amino content of ≥2.0 mmol / g on the surface of the coated hydroxyapatite.

[0017] Preferably, the calcium element is derived from a composite calcium salt composed of calcium citrate and calcium glycerophosphate in a mass ratio of 2:1; the calcium citrate has a purity of ≥98%, a particle size of 1-5μm, and a water solubility of ≥0.5g / L; the calcium glycerophosphate has a purity of ≥99%, a particle size of 2-8μm, and a water solubility of ≥1g / L.

[0018] Preferably, the preparation method of the calcium-doped porous silica hemostatic particles includes the following steps:

[0019] S1. Preparation of carboxymethyl chitosan-grafted sodium alginate copolymer: Carboxymethyl chitosan and sodium alginate were weighed and placed in a reaction vessel at a mass ratio of 3:1. Deionized water was added to make the solid-liquid ratio 1:20, and the mixture was stirred until completely dissolved. EDC and NHS were added, with EDC accounting for 15% of the mass of carboxymethyl chitosan and NHS accounting for 60% of the mass of EDC. The pH of the system was adjusted to 5.5-6.0, and the temperature was raised to 50-60℃. The mixture was stirred for 4-6 hours. After the reaction was completed, the product was poured into anhydrous ethanol to precipitate. The precipitate was collected by filtration and purified by dialysis with deionized water for 72 hours. The molecular weight cutoff of the dialysis bag was 1000 Da. The copolymer powder was obtained after vacuum freeze-drying.

[0020] S2. Preparation of polydopamine-coated hydroxyapatite: Hydroxyapatite powder was added to Tris-HCl buffer solution at pH 8.5 and ultrasonically dispersed for 30 minutes to form a suspension with a concentration of 2 mg / mL; dopamine hydrochloride was added, with the amount of dopamine hydrochloride being 20% ​​of the mass of hydroxyapatite, and the mixture was magnetically stirred at 30°C for 8-12 hours; after the reaction was completed, the product was collected by centrifugation, washed 3-5 times with deionized water until the supernatant was colorless, and then vacuum dried to obtain polydopamine-coated hydroxyapatite;

[0021] S3. Preparation of composite calcium salt solution: Weigh calcium citrate and calcium glycerophosphate at a mass ratio of 2:1, add deionized water, and stir until completely dissolved to form a composite calcium salt solution with a concentration of 1-16 mol / L; place the solution in a centrifuge and centrifuge at 8000 r / min for 15 minutes to remove the bottom precipitate and take the supernatant for later use;

[0022] S4. Mixing reaction: Weigh porous silica particles, add deionized water, and ultrasonically disperse for 20-30 minutes to form a porous silica solution with a concentration of 0.01-10 mg / mL; add the copolymer powder prepared in S1 to the porous silica solution and stir for 30 minutes until dissolved; sequentially add the polydopamine-coated hydroxyapatite, nano-selenium dispersion, graphene oxide quantum dot dispersion, and gallic acid powder prepared in S2, and stir evenly; add the composite calcium salt solution prepared in S3 to the above mixed system, control the volume ratio of the composite calcium salt solution to the porous silica solution to be 1:1-6:1, adjust the pH of the system to 6.8-7.2, raise the temperature to 35℃, and stir at a speed of 300-600 r / min for 1.5-2.5 hours, during which ultrasonic dispersion is performed for 6 minutes every 25 minutes, with an ultrasonic power of 300-400 W;

[0023] S5. Post-processing: Place the reaction product from S4 in a centrifuge and centrifuge at 10000 r / min for 20 minutes, collecting the precipitate; wash the precipitate with deionized water 3-4 times until the pH of the washing solution is neutral; place the washed precipitate in a vacuum freeze dryer, pre-freeze at -50 to -30℃ for 2-4 hours, then sublimate dry at -20 to 0℃ for 10-16 hours under a vacuum of ≤8 Pa, and then desorption dry at 20-30℃ for 6-12 hours, for a total drying time of 16-28 hours; after drying, collect the product to obtain calcium-doped porous silica hemostatic particles with a moisture content ≤2.5%.

[0024] Preferably, in S1, the degree of deacetylation of carboxymethyl chitosan is ≥88%, and the degree of carboxymethyl substitution is 40%-60%; the number average molecular weight of sodium alginate is 5×10⁻⁶. 4 -1×10 5 The molar ratio of mannuronic acid to guluronic acid is 1.2-1.5. During the reaction, a constant temperature water bath is used to control the temperature, with a temperature fluctuation range of ≤±1℃, and the stirring rate is maintained at 400r / min. During the dialysis purification process, the deionized water is replaced every 8 hours.

[0025] Preferably, the concentration of the nano-selenium dispersion in S4 is 0.5-2 mg / mL, prepared by reducing sodium selenite with sodium citrate, and sterilized by filtration through a 0.22 μm filter membrane; the concentration of the graphene oxide quantum dot dispersion is 1-3 mg / mL, and it is used after ultrasonic dispersion for 15 minutes; the gallic acid powder needs to pass through a 200-mesh sieve; during the mixing reaction, the zeta potential and particle size distribution of the system are monitored in real time to ensure that the absolute value of the zeta potential is ≥30 mV and the coefficient of variation of the particle size distribution is ≤15% to ensure the dispersion stability of the particles; the particles after vacuum freeze-drying in S5 need to be sieved, and particles with a particle size range of 20 nm-100 μm are screened using a standard sieve. The sieved particles need to be stored in a sterile environment to avoid contamination.

[0026] (III) Beneficial Technical Effects

[0027] Compared with existing technologies, the beneficial effects of this invention are:

[0028] 1. Porous silica, with its high porosity and large specific surface area, rapidly adsorbs blood moisture and efficiently concentrates coagulation factors, providing a favorable microenvironment for the coagulation reaction. The slowly released calcium from the composite calcium salt precisely activates the coagulation cascade reaction, forming a dual "concentration-activation" mechanism that significantly improves the hemostasis rate. Simultaneously, carboxymethyl chitosan-grafted sodium alginate copolymer enhances the adhesion between the particles and wound tissue, preventing the material from being washed away by blood and further ensuring the stability of the hemostatic effect.

[0029] 2. This method abandons harmful processes such as strong alkali and high-temperature activation used in traditional material preparation. All components are selected from substances with excellent biocompatibility, and the composite calcium salt adopts a slow-release design to avoid tissue irritation caused by excessively high local concentrations. Polydopamine-coated hydroxyapatite mimics the composition of human bone tissue, reducing the rejection reaction between the material and the body, and resolving the safety hazards of tissue burns from zeolite materials and residual harmful substances from chitosan materials.

[0030] 3. Nano-selenium endows the particles with broad-spectrum antibacterial properties, which can effectively inhibit the growth of pathogenic bacteria at the wound site and reduce the risk of infection; the antioxidant effect of gallic acid can reduce the inflammatory response and create a good environment for wound healing; the synergistic effect of graphene oxide quantum dots and polydopamine can promote the proliferation of vascular endothelial cells, and polydopamine-coated hydroxyapatite enhances osteoconductive properties. Together, they accelerate the formation of granulation tissue and tissue repair, achieving an integrated treatment of "hemostasis-antibacterial-promoting healing". Attached Figure Description

[0031] Figure 1 This is a flowchart of a method for preparing calcium-doped porous silica hemostatic particles disclosed in this invention;

[0032] Figure 2 This is a line graph comparing the complete wound healing time and the complete calcium release time between the examples and the comparative examples;

[0033] Figure 3 This is a bar chart comparing cell viability and in vitro coagulation time in the examples and comparative examples;

[0034] Figure 4 This is a radar comparison chart created by standardizing the dimensions of the performance comparison data of the examples and comparative examples. Detailed Implementation

[0035] according to Figures 1 to 4 The specific embodiments of the present invention are as follows:

[0036] Example 1

[0037] Raw material preparation: Porous silicon particles with a particle size of 50μm and a specific surface area of ​​200m². 2 / g, porosity 60%, pore size 10-30nm, particle size distribution variation coefficient 10%. Carboxymethyl chitosan-grafted sodium alginate copolymer grafting rate 40%, number average molecular weight 2×10 5 Da, degree of deacetylation 90%, degree of carboxymethyl substitution 50%, viscosity 500 mPa·s. Nano-selenium has an amorphous structure, particle size 20 nm, dispersibility 95%, and surface bovine serum albumin modification 15%. Polydopamine-coated hydroxyapatite consists of rod-shaped hydroxyapatite particles with a diameter 100 nm, aspect ratio 2:1, crystallinity 90%, Ca / P molar ratio 1.65, polydopamine coating thickness 10 nm, and surface amino content 2.5 mmol / g. The composite calcium salt is composed of calcium citrate and calcium glycerophosphate in a 2:1 mass ratio; calcium citrate purity 99%, particle size 3 μm; calcium glycerophosphate purity 99.5%, particle size 5 μm. Graphene oxide quantum dots have a particle size of 5 nm, surface hydroxyl content 3.5 mmol / g, carboxyl content 2.0 mmol / g, and purity 99.5%. Gallic acid has a purity of 98.5%, melting point 238℃, and water solubility 12 g / L. EDC purity 99%, NHS purity 98%, Tris-HCl buffer pH 8.5, dopamine hydrochloride purity 98%.

[0038] Formulation composition (by weight): 45% porous silica particles, 4% calcium, 4% carboxymethyl chitosan-grafted sodium alginate copolymer, 0.15% nano selenium, 2.5% polydopamine-coated hydroxyapatite, 1.5% graphene oxide quantum dots, 0.8% gallic acid, with the balance being unavoidable impurities.

[0039] Preparation steps: S1. Preparation of carboxymethyl chitosan-grafted sodium alginate copolymer: Weigh carboxymethyl chitosan and sodium alginate into a reaction vessel at a mass ratio of 3:1, add deionized water to make a solid-liquid ratio of 1:20, and stir until dissolved. Add EDC and NHS, with EDC accounting for 15% of the mass of carboxymethyl chitosan and NHS accounting for 60% of the mass of EDC. Adjust the pH to 5.8, raise the temperature to 55℃, and stir at 400 r / min for 5 hours. Pour the reaction solution into anhydrous ethanol to precipitate, filter and collect the precipitate, dialyze with deionized water through a 1000 Da dialysis bag for 72 hours, changing the water every 8 hours, and freeze-dry under vacuum to obtain copolymer powder.

[0040] S2. Preparation of polydopamine-coated hydroxyapatite: Hydroxyapatite powder was added to Tris-HCl buffer at pH 8.5 and ultrasonically dispersed for 30 minutes to form a 2 mg / mL suspension. Dopamine hydrochloride was added at 20% of the hydroxyapatite mass, and the mixture was magnetically stirred at 30°C for 10 hours. The product was collected by centrifugation, washed four times with deionized water until the supernatant was colorless, and vacuum dried to obtain coated particles.

[0041] S3. Preparation of the composite calcium salt solution: Weigh calcium citrate and calcium glycerophosphate at a mass ratio of 2:1, add deionized water and stir to dissolve, forming an 8 mol / L solution. Centrifuge at 8000 r / min for 15 minutes, and collect the supernatant for later use.

[0042] S4. Mixing Reaction: Weigh porous silica particles, add deionized water, and ultrasonically disperse for 25 minutes to form a 5 mg / mL solution. Add the copolymer powder of S1 and stir for 30 minutes until dissolved. Add the coated particles of S2, 0.5 mg / mL nano-selenium dispersion, 2 mg / mL graphene oxide quantum dot dispersion, and gallic acid powder passed through a 200-mesh sieve sequentially, and stir until homogeneous. Add the calcium salt solution of S3, controlling the volume ratio of calcium salt solution to porous silica solution to 3:1, adjust the pH to 7.0, raise the temperature to 35℃, stir at 450 rpm for 2 hours, and ultrasonically disperse for 6 minutes every 25 minutes at an ultrasonic power of 350 W.

[0043] S5. Post-processing: Centrifuge the reaction product at 10000 r / min for 20 minutes and collect the precipitate. Wash four times with deionized water until the washing solution is neutral. Place the precipitate in a vacuum freeze dryer, pre-freeze at -40℃ for 3 hours, sublimate at -10℃ for 13 hours under a vacuum of 5 Pa, and then desorb at 25℃ for 8 hours, for a total drying time of 24 hours. Collect the product, sieve to obtain particles of 20 nm-100 μm, and store aseptically.

[0044] Example 2

[0045] Raw material preparation: Porous silicon particles with a particle size of 30μm and a specific surface area of ​​150m². 2 / g, porosity 50%, pore size 5-20nm, particle size distribution variation coefficient 11%. Carboxymethyl chitosan-grafted sodium alginate copolymer with a grafting rate of 35% and a number average molecular weight of 1.5×10⁻⁶. 5 Da, degree of deacetylation 88%, degree of carboxymethyl substitution 45%, viscosity 400 mPa·s. Nano-selenium has an amorphous structure, particle size 15 nm, dispersibility 93%, and surface bovine serum albumin modification amount 12%. Polydopamine-coated hydroxyapatite consists of rod-shaped hydroxyapatite particles with a diameter of 80 nm, aspect ratio 1.5:1, crystallinity 88%, Ca / P molar ratio 1.62, polydopamine coating thickness 8 nm, and surface amino content 2.2 mmol / g. The composite calcium salt is composed of calcium citrate and calcium glycerophosphate in a 2:1 mass ratio; calcium citrate purity 98%, particle size 2 μm; calcium glycerophosphate purity 99%, particle size 4 μm. Graphene oxide quantum dots have a particle size of 4 nm, surface hydroxyl content 3.2 mmol / g, carboxyl content 1.8 mmol / g, and purity 99%. Gallic acid has a purity of 98%, melting point 236℃, and water solubility 11 g / L. EDC purity 99%, NHS purity 98%, Tris-HCl buffer pH 8.5, dopamine hydrochloride purity 98%.

[0046] Formulation composition (by mass percentage): 40% porous silica particles, 5% calcium, 3% carboxymethyl chitosan-grafted sodium alginate copolymer, 0.1% nano selenium, 2% polydopamine-coated hydroxyapatite, 1.2% graphene oxide quantum dots, 0.6% gallic acid, with the balance being unavoidable impurities.

[0047] Preparation steps: S1. Preparation of carboxymethyl chitosan-grafted sodium alginate copolymer: Weigh carboxymethyl chitosan and sodium alginate into a reaction vessel at a mass ratio of 3:1, add deionized water to make a solid-liquid ratio of 1:20, and stir until dissolved. Add EDC and NHS, with EDC accounting for 15% of the mass of carboxymethyl chitosan and NHS accounting for 60% of the mass of EDC. Adjust the pH to 5.6, raise the temperature to 52℃, and stir at 400 r / min for 4.5 hours. Pour the reaction solution into anhydrous ethanol to precipitate, filter and collect the precipitate, dialyze with deionized water through a 1000 Da dialysis bag for 72 hours, changing the water every 8 hours, and freeze-dry under vacuum to obtain copolymer powder.

[0048] S2. Preparation of polydopamine-coated hydroxyapatite: Hydroxyapatite powder was added to Tris-HCl buffer at pH 8.5 and ultrasonically dispersed for 30 minutes to form a 2 mg / mL suspension. Dopamine hydrochloride was added at 20% of the hydroxyapatite mass, and the mixture was magnetically stirred at 30°C for 9 hours. The product was collected by centrifugation, washed four times with deionized water until the supernatant was colorless, and vacuum dried to obtain coated particles.

[0049] S3. Preparation of the composite calcium salt solution: Weigh calcium citrate and calcium glycerophosphate at a mass ratio of 2:1, add deionized water and stir to dissolve, forming a 4 mol / L solution. Centrifuge at 8000 r / min for 15 minutes, and collect the supernatant for later use.

[0050] S4. Mixing Reaction: Weigh porous silica particles, add deionized water, and ultrasonically disperse for 22 minutes to form a 3 mg / mL solution. Add the copolymer powder of S1 and stir for 30 minutes until dissolved. Add the coated particles of S2, 0.5 mg / mL nano-selenium dispersion, 2 mg / mL graphene oxide quantum dot dispersion, and gallic acid powder passed through a 200-mesh sieve sequentially, and stir until homogeneous. Add the calcium salt solution of S3, controlling the volume ratio of calcium salt solution to porous silica solution to 2:1, adjust the pH to 6.9, raise the temperature to 35℃, stir at 400 rpm for 1.8 hours, and ultrasonically disperse for 6 minutes every 25 minutes at an ultrasonic power of 320 W.

[0051] S5. Post-processing: Centrifuge the reaction product at 10000 r / min for 20 minutes and collect the precipitate. Wash four times with deionized water until the washing solution is neutral. Place the precipitate in a vacuum freeze dryer, pre-freeze at -45℃ for 2.5 hours, sublimate at -15℃ for 12 hours under a vacuum of 6 Pa, and then desorb at 22℃ for 7 hours, for a total drying time of 21.5 hours. Collect the product, sieve to obtain particles of 20 nm-100 μm, and store aseptically.

[0052] Example 3

[0053] Raw material preparation: Porous silicon particles with a particle size of 80μm and a specific surface area of ​​300m². 2 / g, porosity 70%, pore size 20-40nm, particle size distribution variation coefficient 9%. Carboxymethyl chitosan-grafted sodium alginate copolymer with a grafting rate of 45% and a number average molecular weight of 2.5×10⁻⁶. 5 Da, degree of deacetylation 92%, degree of carboxymethyl substitution 55%, viscosity 700 mPa·s. Nano-selenium has an amorphous structure, particle size 25 nm, dispersibility 96%, and surface bovine serum albumin modification 18%. Polydopamine-coated hydroxyapatite consists of rod-shaped hydroxyapatite particles with a diameter of 150 nm, aspect ratio 2.5:1, crystallinity 92%, Ca / P molar ratio 1.66, polydopamine coating thickness 15 nm, and surface amino content 2.8 mmol / g. The composite calcium salt is composed of calcium citrate and calcium glycerophosphate in a 2:1 mass ratio; calcium citrate purity 99.5%, particle size 4 μm; calcium glycerophosphate purity 99.8%, particle size 6 μm. Graphene oxide quantum dots have a particle size of 6 nm, surface hydroxyl content 3.8 mmol / g, carboxyl content 2.2 mmol / g, and purity 99.8%. Gallic acid has a purity of 99%, melting point 239℃, and water solubility 13 g / L. EDC purity 99%, NHS purity 98%, Tris-HCl buffer pH 8.5, dopamine hydrochloride purity 98%.

[0054] Formulation composition (by weight): 50% porous silica particles, 6% calcium, 5% carboxymethyl chitosan-grafted sodium alginate copolymer, 0.25% nano selenium, 3.5% polydopamine-coated hydroxyapatite, 2.2% graphene oxide quantum dots, 1.2% gallic acid, with the balance being unavoidable impurities.

[0055] Preparation steps: S1. Preparation of carboxymethyl chitosan-grafted sodium alginate copolymer: Weigh carboxymethyl chitosan and sodium alginate into a reaction vessel at a mass ratio of 3:1, add deionized water to make a solid-liquid ratio of 1:20, and stir until dissolved. Add EDC and NHS, with EDC accounting for 15% of the mass of carboxymethyl chitosan and NHS accounting for 60% of the mass of EDC. Adjust the pH to 6.0, raise the temperature to 58℃, and stir the reaction at 400 r / min for 5.5 hours. Pour the reaction solution into anhydrous ethanol to precipitate, filter and collect the precipitate, dialyze it with deionized water through a 1000 Da dialysis bag for 72 hours, changing the water every 8 hours, and freeze-dry under vacuum to obtain copolymer powder.

[0056] S2. Preparation of polydopamine-coated hydroxyapatite: Hydroxyapatite powder was added to Tris-HCl buffer at pH 8.5 and ultrasonically dispersed for 30 minutes to form a 2 mg / mL suspension. Dopamine hydrochloride was added at 20% of the hydroxyapatite mass, and the mixture was magnetically stirred at 30°C for 11 hours. The product was collected by centrifugation, washed four times with deionized water until the supernatant was colorless, and vacuum dried to obtain coated particles.

[0057] S3. Preparation of the composite calcium salt solution: Weigh calcium citrate and calcium glycerophosphate at a mass ratio of 2:1, add deionized water and stir to dissolve, forming a 12 mol / L solution. Centrifuge at 8000 r / min for 15 minutes, and collect the supernatant for later use.

[0058] S4. Mixing Reaction: Weigh porous silica particles, add deionized water, and ultrasonically disperse for 28 minutes to form an 8 mg / mL solution. Add the copolymer powder of S1 and stir for 30 minutes until dissolved. Add the coated particles of S2, 0.5 mg / mL nano-selenium dispersion, 2 mg / mL graphene oxide quantum dot dispersion, and gallic acid powder passed through a 200-mesh sieve sequentially, and stir until homogeneous. Add the calcium salt solution of S3, controlling the volume ratio of calcium salt solution to porous silica solution to 5:1, adjust the pH to 7.1, raise the temperature to 35℃, stir at 550 rpm for 2.2 hours, and ultrasonically disperse for 6 minutes every 25 minutes at an ultrasonic power of 380 W.

[0059] S5. Post-processing: Centrifuge the reaction product at 10000 r / min for 20 minutes and collect the precipitate. Wash four times with deionized water until the washing solution is neutral. Place the precipitate in a vacuum freeze dryer, pre-freeze at -35℃ for 3.5 hours, sublimate at -5℃ for 14 hours under a vacuum of 4 Pa, and then desorb at 28℃ for 9 hours, for a total drying time of 26.5 hours. Collect the product, sieve to obtain particles of 20 nm-100 μm, and store aseptically.

[0060] Comparative Example

[0061] Raw material preparation: Porous silicon particles with a particle size of 50μm and a specific surface area of ​​200m². 2 / g, porosity 60%, pore size 10-30nm. Calcium chloride purity 98%, particle size 5μm. Chitosan degree of deacetylation 85%, number average molecular weight 1×10 5 Da.

[0062] Formulation composition (by mass percentage): 94% porous silica particles, 5% calcium, 1% chitosan, balance being unavoidable impurities.

[0063] Preparation steps: S1. Prepare calcium chloride solution: Weigh calcium chloride, add deionized water and stir to dissolve it to form an 8 mol / L solution. Centrifuge at 8000 r / min for 15 minutes and take the supernatant for later use.

[0064] S2. Mixing reaction: Weigh porous silica particles, add deionized water and stir to disperse for 30 minutes to form a 5 mg / mL solution. Add chitosan powder and stir for 30 minutes until dissolved. Add calcium chloride solution, controlling the volume ratio of calcium chloride solution to porous silica solution to 3:1, adjust the pH to 7.0, and stir at 35℃ and 450 r / min for 2 hours.

[0065] S3. Post-treatment: Centrifuge the reaction product at 10000 r / min for 20 minutes and collect the precipitate. Wash four times with deionized water until the washing solution is neutral. Place the precipitate in a vacuum drying oven and dry at 60℃ for 12 hours. Collect the product and sieve to obtain particles of 20 nm-100 μm.

[0066] The following table compares the hemostasis time-related performance of the examples and comparative examples:

[0067] Table 1

[0068] index Example 1 Example 2 Example 3 Comparative Example In vitro clotting time (s) 45 50 42 120 Degradation half-life of hemostatic materials (days) 18 16 20 Undegraded Time for complete wound healing (days) 7 8 6 14 Time for complete calcium release (days) 12 10 14 2

[0069] The biocompatibility and antibacterial properties of the examples and comparative examples are compared in the table below:

[0070] Table 2

[0071]

[0072]

[0073] Compared with the comparative method using a single porous silica, calcium chloride and ordinary chitosan, this invention achieves a comprehensive breakthrough in hemostasis efficiency, biocompatibility and therapeutic functionality of calcium-doped porous silica hemostatic particles through a "concentration-activation" synergistic hemostasis mechanism, biocompatible component design and multifunctional composite system construction. This solves the industry pain points of traditional hemostatic materials such as slow hemostasis, safety hazards and single function.

[0074] Regarding core hemostatic and healing performance, the in vitro coagulation time of the embodiment was only 42-50 seconds, just 1 / 3 to 1 / 2 of that of the comparative example, confirming the dual synergistic effect of porous silica rapidly concentrating coagulation factors and the sustained release of composite calcium salt activating the coagulation cascade reaction. The complete release time of calcium was extended to 10-14 days, avoiding the local irritation caused by the excessively rapid dissolution of calcium chloride in the comparative example. Combined with the strong adhesion of carboxymethyl chitosan-grafted sodium alginate copolymer, the hemostatic effect was ensured to be stable and long-lasting. At the same time, the complete wound healing time of the embodiment was only 6-8 days, nearly half that of the comparative example, highlighting the promoting effect of components such as graphene oxide quantum dots and polydopamine-coated hydroxyapatite on tissue repair.

[0075] Regarding biosafety, the cell survival rate of the embodiment reached 94-97%, which is much higher than that of the comparative example; the hemolysis rate was only 0.7-0.9%, which is significantly lower than that of the comparative example; the skin irritation reaction score was 0, while that of the comparative example was 2. This is because the present invention abandons harmful preparation processes, selects components with excellent biocompatibility, and uses polydopamine-coated hydroxyapatite to simulate human tissue components, which greatly reduces the risk of rejection and irritation by the body and solves the problems of cell toxicity and tissue irritation of traditional materials.

[0076] In terms of functional synergy, the embodiment showed an inhibition zone diameter of 17-19 mm against Escherichia coli, while the comparative example showed no antibacterial effect, thanks to the broad-spectrum antibacterial activity of nano-selenium. The antioxidant and anti-inflammatory functions of gallic acid, combined with the repair-promoting components, achieved an integrated treatment of "rapid hemostasis - infection inhibition - accelerated healing." Overall, this invention, through precise synergistic design of multiple components, comprehensively surpasses traditional single-function hemostatic materials, providing a safe and efficient solution for the treatment of complex wound hemostasis.

[0077] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A calcium-doped porous silica hemostatic granule, characterized in that, The composition by weight percentage includes: 35%-55% porous silica particles, 4%-8% calcium, 2%-6% carboxymethyl chitosan-grafted sodium alginate copolymer, 0.02%-0.3% nano-selenium, and 1%-4% polydopamine-coated hydroxyapatite, with the balance being unavoidable impurities; the particle size is 20nm-100μm, and the specific surface area is 80-350m². 2 / g, porosity 45%-75%, pore size distribution concentrated in 5-40nm, particle size distribution variation coefficient ≤12%; Carboxymethyl chitosan and sodium alginate form a copolymer through a grafting reaction. This reaction uses 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) as the activation system and reacts at 50-60°C for 4-6 hours. The reaction of polydopamine-coated hydroxyapatite is achieved through dopamine self-polymerization. The reaction is carried out in Tris-HCl buffer at pH 8.5 and stirred at 30°C for 8-12 hours.

2. The calcium-doped porous silica hemostatic granules according to claim 1, characterized in that, It also includes 0.8%-2.5% graphene oxide quantum dots; the graphene oxide quantum dots have a spherical structure, a particle size of 2-8 nm, a lateral dimension of 10-30 nm, a surface hydroxyl content ≥3.0 mmol / g, a carboxyl content ≥1.5 mmol / g, and are prepared by oxidizing graphite powder by Hummers method followed by ultrasonic exfoliation, and the purity is ≥99% after dialysis purification.

3. The calcium-doped porous silica hemostatic granules according to claim 1, characterized in that, It also includes 0.3%-1.5% gallic acid; the gallic acid has a purity of ≥98%, a melting point of 235-240℃, and a water solubility of ≥10g / L, and is obtained through plant extraction and purification.

4. The calcium-doped porous silica hemostatic granules according to claim 1, characterized in that, The number average molecular weight of the carboxymethyl chitosan-grafted sodium alginate copolymer is 8 × 10⁻⁶. 4 -3×10 5 Da, weight-average molecular weight 1.2 × 10⁻⁶ 5 -4×10 5 Da, molecular weight distribution index 1.5-2.

0.

5. The calcium-doped porous silica hemostatic granules according to claim 1, characterized in that, The nano-selenium has an amorphous structure with a particle size of 10-30 nm. The particles are uniformly dispersed with no obvious agglomeration and a dispersibility of ≥92%. The surface of the nano-selenium is modified with bovine serum albumin at a rate of 10%-20% of the nano-selenium mass. The modified nano-selenium exhibits significantly improved stability in physiological saline with a 24-hour sedimentation rate of ≤5%.

6. The calcium-doped porous silica hemostatic granules according to claim 1, characterized in that, The polydopamine-coated hydroxyapatite has a rod-like structure with a particle size of 50-200 nm, an aspect ratio of 1:1-3:1, a crystallinity of ≥85%, a Ca / P molar ratio of 1.60-1.67, and is prepared by a hydrothermal method with a purity of ≥99%. The polydopamine coating layer has a thickness of 5-20 nm, a surface roughness Ra of 2-5 nm, and an amino content of ≥2.0 mmol / g on the surface of the coated hydroxyapatite.

7. The calcium-doped porous silica hemostatic granules according to claim 1, characterized in that, The calcium element is derived from a composite calcium salt composed of calcium citrate and calcium glycerophosphate in a mass ratio of 2:1; the calcium citrate has a purity of ≥98%, a particle size of 1-5μm, and a water solubility of ≥0.5g / L; the calcium glycerophosphate has a purity of ≥99%, a particle size of 2-8μm, and a water solubility of ≥1g / L.

8. A method for preparing calcium-doped porous silica hemostatic particles according to claim 1, characterized in that, Includes the following steps: S1. Preparation of carboxymethyl chitosan-grafted sodium alginate copolymer: Carboxymethyl chitosan and sodium alginate were weighed and placed in a reaction vessel at a mass ratio of 3:

1. Deionized water was added to make the solid-liquid ratio 1:20, and the mixture was stirred until completely dissolved. EDC and NHS were added, with EDC accounting for 15% of the mass of carboxymethyl chitosan and NHS accounting for 60% of the mass of EDC. The pH of the system was adjusted to 5.5-6.0, and the temperature was raised to 50-60℃. The mixture was stirred for 4-6 hours. After the reaction was completed, the product was poured into anhydrous ethanol to precipitate. The precipitate was collected by filtration and purified by dialysis with deionized water for 72 hours. The molecular weight cutoff of the dialysis bag was 1000 Da. The copolymer powder was obtained after vacuum freeze-drying. S2. Preparation of polydopamine-coated hydroxyapatite: Hydroxyapatite powder was added to Tris-HCl buffer solution at pH 8.5 and ultrasonically dispersed for 30 minutes to form a suspension with a concentration of 2 mg / mL; dopamine hydrochloride was added, with the amount of dopamine hydrochloride being 20% ​​of the mass of hydroxyapatite, and the mixture was magnetically stirred at 30°C for 8-12 hours; after the reaction was completed, the product was collected by centrifugation, washed 3-5 times with deionized water until the supernatant was colorless, and then vacuum dried to obtain polydopamine-coated hydroxyapatite; S3. Preparation of composite calcium salt solution: Weigh calcium citrate and calcium glycerophosphate at a mass ratio of 2:1, add deionized water, and stir until completely dissolved to form a composite calcium salt solution with a concentration of 1-16 mol / L; place the solution in a centrifuge and centrifuge at 8000 r / min for 15 minutes to remove the bottom precipitate and take the supernatant for later use; S4. Mixing reaction: Weigh porous silica particles, add deionized water, and ultrasonically disperse for 20-30 minutes to form a porous silica solution with a concentration of 0.01-10 mg / mL; add the copolymer powder prepared in S1 to the porous silica solution and stir for 30 minutes until dissolved; sequentially add the polydopamine-coated hydroxyapatite, nano-selenium dispersion, graphene oxide quantum dot dispersion, and gallic acid powder prepared in S2, and stir evenly; add the composite calcium salt solution prepared in S3 to the above mixed system, control the volume ratio of the composite calcium salt solution to the porous silica solution to be 1:1-6:1, adjust the pH of the system to 6.8-7.2, raise the temperature to 35℃, and stir at a speed of 300-600 r / min for 1.5-2.5 hours, during which ultrasonic dispersion is performed for 6 minutes every 25 minutes, with an ultrasonic power of 300-400 W; S5. Post-processing: Place the reaction product from S4 in a centrifuge and centrifuge at 10000 r / min for 20 minutes, collecting the precipitate; wash the precipitate with deionized water 3-4 times until the pH of the washing solution is neutral; place the washed precipitate in a vacuum freeze dryer, pre-freeze at -50 to -30℃ for 2-4 hours, then sublimate dry at -20 to 0℃ for 10-16 hours under a vacuum of ≤8 Pa, and then desorption dry at 20-30℃ for 6-12 hours, for a total drying time of 16-28 hours; after drying, collect the product to obtain calcium-doped porous silica hemostatic particles with a moisture content ≤2.5%.

9. The method for preparing calcium-doped porous silica hemostatic particles according to claim 8, characterized in that, S1 contains carboxymethyl chitosan with a degree of deacetylation ≥88% and a degree of carboxymethyl substitution 40%-60%; sodium alginate has a number-average molecular weight of 5×10⁻⁶. 4 -1×10 5 The molar ratio of mannuronic acid to guluronic acid is 1.2-1.

5. During the reaction, a constant temperature water bath is used to control the temperature, with a temperature fluctuation range of ≤±1℃, and the stirring rate is maintained at 400r / min. During the dialysis purification process, the deionized water is replaced every 8 hours.

10. The method for preparing calcium-doped porous silica hemostatic particles according to claim 8, characterized in that, The concentration of the nano-selenium dispersion in S4 is 0.5-2 mg / mL, prepared by reducing sodium selenite with sodium citrate, and sterilized by filtration through a 0.22 μm filter membrane; the concentration of the graphene oxide quantum dot dispersion is 1-3 mg / mL, and it is used after ultrasonic dispersion for 15 minutes; the gallic acid powder needs to be sieved through a 200-mesh sieve; during the mixing reaction, the zeta potential and particle size distribution of the system are monitored in real time to ensure that the absolute value of the zeta potential is ≥30 mV and the coefficient of variation of the particle size distribution is ≤15% to ensure the dispersion stability of the particles; the particles after vacuum freeze-drying in S5 need to be sieved, and particles with a particle size range of 20 nm-100 μm are selected by screening with a standard sieve. The sieved particles need to be stored in a sterile environment to avoid contamination.

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