A high-activity stable thrombin powder preparation and a preparation method thereof

Abstract

The application relates to the field of biological medicine, and discloses a high-activity stable thrombin powder preparation and a preparation method thereof. The preparation is composed of spherical particles with a micron-nanometer hierarchical porous structure, and components include thrombin, a biocompatible skeleton material, a cryoprotective agent, a composite pore-forming agent and a surface amphiphilic modifier. The preparation method locks the activity through ultrasonic atomization and deep cold solidification of liquid nitrogen, and performs in-situ pore-forming through stepwise temperature control and sublimation drying. Through the synergistic effect of the hierarchical pore structure and the surface amphiphilic modification, the moisture resistance and flowability of the powder preparation are enhanced, the blood is rapidly perfused by utilizing capillary force, and the high-activity maintenance and second-level hemostasis response of the thrombin are realized.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a highly active and stable thrombin powder preparation and its preparation method. Background Technology

[0002] Thrombin, a key bioactive enzyme, plays an irreplaceable role in trauma treatment, surgical hemostasis, and local bleeding control. Its core pharmacological mechanism lies in its ability to specifically cleave peptide bonds in fibrinogen, converting it into insoluble fibrin polymers, thereby rapidly inducing the formation of physiological thrombi to achieve hemostasis.

[0003] In current clinical applications, thrombin is typically prepared as a dry powder to ensure the bioactivity of the protein molecules and extend the shelf life of the preparation. These powders are usually applied directly to the bleeding wound via spraying or dressing, allowing the high concentration of active ingredients to directly intervene in the coagulation cascade reaction.

[0004] Traditional thrombin powders are mostly produced using vacuum freeze-drying combined with physical mechanical pulverization, or simple spray drying processes. However, the powder particles prepared by traditional processes often exhibit irregular, dense geometric shapes. Due to the strong hydrophilicity and hygroscopicity of thrombin itself, in open wound environments or high-humidity operating room settings, the surface of ordinary powder particles easily captures moisture and becomes sticky, forming bridging phenomena between particles. This leads to clumping, blockage, or uneven distribution of the drug during spraying, and at the same time, it prevents the drug from forming a continuous and uniform effective film on the wound surface.

[0005] When traditional dense powder particles come into contact with blood, their surface rapidly swells and forms a dense, sticky coating. This prevents thrombin molecules inside the particles from diffusing outward during the critical golden window for hemostasis. It also hinders fibrinogen in the blood from penetrating into the particles, resulting in a significant decrease in the speed of hemostasis response. Consequently, even with high doses of medication, the effective concentration of the drug actually participating in the reaction is greatly reduced due to the limited diffusion. Summary of the Invention

[0006] This invention provides a highly active and stable thrombin powder formulation and its preparation method, aiming to solve the technical problems of thrombin powder in the prior art, such as moisture absorption and clumping, poor flowability, uneven distribution on bleeding wounds, and delayed hemostatic response. This invention achieves high activity retention and instantaneous hemostatic effect of thrombin under complex physiological environments by constructing a powder formulation particle with a hierarchical porous microsphere framework structure and utilizing precise microstructure design and directional surface energy modification.

[0007] To achieve the aforementioned objectives, this invention provides a highly active and stable thrombin powder formulation, comprising a plurality of spherical micron-sized particles with a hierarchical porous structure. The average particle size of the particles ranges from 20 to 80 micrometers, and their microstructure exhibits distinct secondary porosity characteristics. Specifically, the interior and surface of the spherical micron-sized particles are distributed with first-order micron-sized channels, the pore size of which is 1 to 5 micrometers. On the inner wall of the first-order micron-sized channels, second-order nano-sized channels extend and interweave, the pore size of which is 20 to 200 nanometers. This micron-nano hierarchical porous structure constitutes a highly accessible three-dimensional spatial network, significantly increasing the specific surface area of ​​the particles and providing a physical carrier for the stable loading and rapid release of thrombin molecules.

[0008] The highly active and stable thrombin powder formulation comprises a thrombin active component, a biocompatible matrix material, a cryoprotectant, a composite porogen, and a surface amphiphilic modifier. By mass percentage, the content of each component is: 5% to 15% thrombin active component, 40% to 60% biocompatible matrix material, 15% to 25% cryoprotectant, 5% to 10% composite porogen, 1% to 5% surface amphiphilic modifier, and the balance being phosphate buffer.

[0009] The thrombin active component is a high-titer bovine or porcine thrombin with a specific activity greater than 2000 units per milligram. The biocompatible framework material is selected from at least one of human serum albumin, recombinant human collagen, hydroxypropyl methylcellulose, or carboxymethyl deacetylated chitosan. These materials not only provide the necessary mechanical support strength to ensure the structural integrity of the microspheres during drying and storage, but also inhibit irreversible thermal denaturation of the protein molecules by forming hydrogen bonds with thrombin molecules through abundant amino or hydroxyl groups.

[0010] The cryoprotectant is composed of trehalose, sucrose, and mannitol in a mass ratio of 2:1:1. During freezing, trehalose can replace water molecules on the surface of thrombin molecules to form a stable glassy coating layer, effectively preventing the physical shearing damage to the protein spatial structure caused by ice crystal growth; mannitol acts as a support, raising the eutectic point temperature of the freeze-dried cake and preventing collapse during the drying process.

[0011] The composite pore-forming agent is composed of ammonium bicarbonate and polyethylene glycol 4000 in a mass ratio of 3:1. Ammonium bicarbonate, as a volatile inorganic salt, can generate gaseous products in situ during low-temperature sublimation, thereby inducing the formation of micron-sized channels. Polyethylene glycol 4000, in the early stage of ice crystal formation, promotes the growth of ice crystals in a specific direction by changing the local viscosity, providing a template for the formation of nano-sized channels.

[0012] The surface amphiphilic modifier is a bifunctional molecule with clearly defined hydrophobic and hydrophilic ends, selected from a compound of hexadecyltrimethoxysilane and polyethylene glycol monomethyl ether, or a mixture of phosphatidylcholine and fatty acid polyoxyethylene ester. The amphiphilic modifier is deposited on the inner wall of a hierarchical porous structure through physical adsorption or chemical cross-linking. Its hydrophobic end faces outwards from the pores, effectively preventing the penetration of external water vapor molecules and enhancing the powder's moisture resistance; its hydrophilic end penetrates deep into the pores or adheres to the surface of thrombin molecules, ensuring that once the particles contact the blood surface, capillary forces drive rapid blood perfusion into the pores.

[0013] This invention also provides a method for preparing the above-mentioned highly active and stable thrombin powder formulation. The method is based on the coupling of spray freeze-drying technology and in-situ pore-forming process, and specifically includes the following steps: The first step is the preparation of the precursor solution. Under light-protected conditions at a temperature of 4°C to 10°C, the biocompatible framework material is dissolved in a phosphate buffer solution with a pH of 6.8 to 7.4 to prepare a base matrix solution with a mass fraction of 5% to 12%. A cryoprotectant is then added, and the solution is stirred thoroughly until clear and transparent. Finally, the thrombin active component is added, and it is uniformly distributed using mechanical stirring or ultrasonic dispersion. The stirring speed is controlled at 100 rpm to 300 rpm to avoid generating excessive bubbles that could lead to protein denaturation.

[0014] The second step involves constructing the composite porogen and modification system. The composite porogen is added to a phosphate buffer solution at pH 6.8–7.4 and stirred until completely dissolved, preparing a 10%–15% (w / w) composite porogen solution. The composite porogen solution is then slowly added dropwise to the precursor solution, along with a surface amphiphilic modifier. Stirring continues for 30–60 minutes at a constant temperature to allow the modifier to establish a synergistic effect with the framework material at the molecular level, forming a homogeneous and stable sol-gel system.

[0015] The third step is ultrasonic atomization and cryogenic curing. The sol system described above is delivered to the atomizing nozzle via a high-frequency ultrasonic atomizing device (frequency 40kHz to 60kHz). Driven by a carrier gas (usually high-purity nitrogen) at a pressure of 0.2MPa to 0.5MPa, the sol is broken into tiny droplets with a narrow diameter distribution and high uniformity. These droplets are then directly sprayed into a cryogenic curing vessel containing liquid nitrogen. Due to the extremely low temperature environment of liquid nitrogen (-196 degrees Celsius), the droplets undergo instantaneous curing in a very short time, ensuring that the active components of thrombin are locked in situ within the glassy microspheres, avoiding solute expulsion and protein inactivation caused by slow crystallization during traditional freezing processes.

[0016] The fourth step is controlled sublimation drying. The solidified microsphere slurry is transferred to the vacuum chamber of a freeze dryer. The vacuum pump is started to reduce the pressure inside the chamber to below 10 Pa. The heating process adopts a stepped temperature program: First, in the primary drying stage from -50°C to -30°C, it is maintained for 20 to 30 hours to remove ice crystals through sublimation and initially form a porous framework; then, in the secondary drying stage from -20°C to 0°C, it is maintained for 10 to 15 hours, during which the volatile components in the composite porogen decompose and overflow, further refining the microporous structure; finally, in the desorption drying stage from 20°C to 25°C, residual physically adsorbed water is removed by heat conduction until the water content of the powder is below 3%.

[0017] Step 5: Aseptic packaging and quality control. After drying, the thrombin powder is collected under dry nitrogen protection in a Class 10,000 clean environment. It is then classified by particle size using a cyclone separator to remove excessively large or small particles, and finally aseptically packaged.

[0018] This invention employs spray freeze-drying technology, ensuring uniform heating in all directions during the curing process and balanced internal stress distribution, resulting in high crushing strength. Tests show that the average radial crushing force of a single microsphere is greater than 0.5 Newtons. This ensures that the powder does not easily break into fine powder during transportation, shaking, and spraying, thus maintaining good macroscopic flowability, with its angle of repose consistently remaining between 25 and 30 degrees.

[0019] The hydrophobic-hydrophilic amphiphilic group modification mechanism described in this invention is key to improving hemostatic efficacy. During storage, the hydrophobic end on the outer surface of the particle forms a nanoscale molecular barrier. This barrier repels water molecules in the air, resulting in a lower moisture absorption and weight gain rate compared to traditional powders. In actual use, when the powder comes into contact with blood in an aqueous environment, the hydrophilic end rapidly reduces the tension at the liquid-solid interface, triggering a strong capillary suction effect. Plasma components in the blood are forcibly drawn into the hierarchical channels inside the particle, shortening the diffusion distance between thrombin and fibrinogen. The hierarchical porous structure increases the effective collision probability at the reaction interface, thereby accelerating the generation rate of fibrin monomers and achieving a hemostatic response within seconds.

[0020] The behavior of the composite pore-forming agent during the drying process is highly deterministic. The decomposition kinetics of ammonium bicarbonate are precisely controlled by temperature. The carbon dioxide and ammonia produced move along the escape path through the micron-sized pores left after ice crystal sublimation, and the internal pressure causes a slight expansion of the micropore walls, forming highly interconnected micron-sized channels. Meanwhile, polyethylene glycol 4000 acts as a steric hindrance at the nanoscale, preventing the local aggregation and collapse of biomolecules at the end of the drying process, thus maintaining the openness of the nanoscale channels. This coexistence of multiple pore sizes results in a low-density, high-porosity (80% to 95%) powder, optimizing the aerodynamic equivalent diameter of the powder and enabling it to form a uniform drug mist cloud during spray administration, completely covering wounds with complex geometries.

[0021] The highly active and stable thrombin powder formulation described in this invention exhibits extremely high stability at the biochemical level. Due to the use of a fully enclosed liquid nitrogen spray solidification process, the thrombin molecules are kept in an extremely low-temperature environment throughout, resulting in improved enzyme activity recovery. Through precise control of the microstructure, this invention not only solves the technical challenge of long-term preservation of protein drugs but also overcomes the limitations of traditional powders in hemostatic dynamics through a cross-design combining fluid dynamics and bionics.

[0022] The core improvement of this invention—a highly active and stable thrombin powder formulation and its preparation method—lies in its focus on the microscopic morphology of the material. By introducing a hierarchical porous structure, the dynamic contact behavior of the solid-liquid interface is systematically improved. Micrometer-level pores facilitate large-scale, rapid blood absorption, while nanometer-level pores provide the microenvironment for the coagulation reaction through their extremely high specific surface area. This structural design mimics the material transport efficiency of the human capillary network. Combined with precise control of wettability through surface modification technology, it completely overcomes the slow onset of action caused by interfacial damping in ordinary powders. Furthermore, the selected biocompatible materials and cryoprotection system construct multiple protective mechanisms at the molecular level, ensuring that thrombin maintains its complex tertiary protein structure even under harsh storage and transportation conditions, thereby releasing the expected biological activity in the final application.

[0023] In terms of hemostatic kinetics, the formulation described in this invention generates a negative pressure effect upon contact with blood, utilizing its unique micron-nano hierarchical structure. This effect is induced by amphiphilic groups on the pore walls. When the hydrophobic end interacts weakly with lipid components in the blood, while the hydrophilic end forms strong hydrogen bonds with the aqueous plasma phase, blood is rapidly drawn into the particle interior. This phenomenon manifests macroscopically as the instantaneous adsorption of blood by the powder. After entering the pores, the blood flow rate changes gradient due to the decreasing pore size from micron to nanometer, increasing the contact frequency between thrombin and fibrinogen molecules. Simultaneously, due to the synergistic effect between the charge properties of the pore wall modifier and coagulation factors in the blood, the intrinsic and extrinsic coagulation pathways are further accelerated, resulting in the rapid formation of a robust fibrin network around the microparticles.

[0024] The highly active and stable thrombin powder formulation described in this invention exhibits the following microscopic physical properties: a specific surface area of ​​30 to 80 square meters per gram and a total pore volume of 0.5 to 1.2 cubic centimeters per gram. This highly developed pore structure provides a large reaction interface and also endows the powder with excellent compressive resilience, allowing it to maintain the connectivity of its pores under external pressure. This physical toughness ensures the structural stability of the drug during packaging and transportation.

[0025] To illustrate the technical solution of this invention in more detail, the key steps in the preparation process are further described below: In the precursor solution preparation stage, the selection logic for biocompatible framework materials lies in constructing a resilient protein protection matrix. Human serum albumin not only serves as a carrier, but its own colloidal osmotic pressure regulation capability helps attract wound moisture in the early stages of hemostasis, accelerating powder wetting. By compounding it with hydroxypropyl methylcellulose, the long-chain entanglement effect of cellulose is utilized to enhance the framework strength of the freeze-dried microspheres and prevent the collapse of the microporous structure.

[0026] In the construction of composite pore-forming and modifying systems, the order in which surface amphiphilic modifiers are added has a decisive influence on their distribution on the pore walls. By pre-dispersing the modifiers in a sol state, they migrate towards the gas-liquid interface due to hydrophobic interactions during droplet solidification. When the droplets cool and shrink and undergo phase separation, the modifiers are anchored at the interface between the ice crystals and the polymer matrix. As the ice crystals sublimate, these molecules remain on the inner wall surface of the pores, forming a directionally arranged molecular layer.

[0027] In the ultrasonic atomization process, frequency stability directly affects the monodispersity of droplets. The 40kHz to 60kHz frequency selected in this invention, combined with precise flow pump control, ensures that the millions of droplets generated per second have extremely high particle size uniformity. This uniformity is the foundation for achieving uniform spraying of the powder and a prerequisite for ensuring that each microsphere exhibits the same drug release kinetics.

[0028] In controlled sublimation drying with stepped temperature control, the temperature of the primary drying stage is set slightly below the glass transition temperature of the solvent system to prevent viscous flow of the microstructure. The secondary drying stage is specifically designed to remove bound water and volatile porogens. By monitoring minute fluctuations in the chamber pressure, the overflow process of porogen decomposition products can be accurately determined. When the pressure curve stabilizes, it signifies the final formation of micron-sized channels.

[0029] In the physicochemical characterization of the final product, the powder obtained by this invention exhibits unique diffraction characteristics and thermal stability. Due to the scientifically formulated cryoprotectant, thrombin exists in an amorphous state within the microspheres, without localized lattice ordering, which lowers its activation energy for chemical degradation. Thermogravimetric analysis shows that the initial decomposition temperature of this powder is more than 15 degrees Celsius higher than that of traditional powders, demonstrating its longer shelf life at room temperature.

[0030] To optimize the applicability of the formulation in special extreme environments (such as high humidity and high temperature regions), the surface amphiphilic modifier described in this invention employs a multi-layer coating strategy. At the molecular level, the first layer consists of hydrophilic phospholipid molecules tightly adhering to the inner wall of the microspheres, ensuring the environmental friendliness of the bioactivity; the second layer consists of partially cross-linked long-chain silane molecules with hydrophobic properties, forming a unique structure of a hydrophobic shell and a hydrophilic cavity. This structure causes the powder particles to exhibit extremely low surface energy in air, similar to fluorinated powder, and they do not form hydrogen bonds with water vapor molecules, thus exhibiting excellent anti-caking properties on a macroscopic scale. Once in an aqueous environment, because the wetting pressure of liquid water is much greater than the diffusion pressure of water vapor, the liquid can overcome the physical barrier of the hydrophobic layer and activate the internal hydrophilic network. This threshold-triggered wetting mechanism is one of the core original features of this invention.

[0031] The hierarchical porous structure described in this invention also exhibits a significant advantage in drug loading efficiency. Due to the high interconnectivity between micron-level and nano-level pores, thrombin molecules are not only present on the particle surface but also distributed at extremely high density on the inner walls of all effective pores within the particle. This full-space drug loading mode ensures that, during hemostasis, as the surface fibrin network forms, thrombin deep within the particle can be continuously replenished through diffusion channels, thereby achieving sustained wound closure.

[0032] The technical solution described in this invention, through deep integration of materials, structure, process, and performance, constructs a complete, high-performance thrombin powder R&D platform with independent intellectual property rights. It not only achieves a leap forward in hemostatic efficacy but also sets new technical benchmarks in formulation stability, flowability, and clinical usability. This invention provides a rapid, safe, and reliable clinical intervention for bleeding from capillaries, small blood vessels, and solid organs caused by various reasons.

[0033] The hierarchical porous microsphere framework structure proposed in this invention, while partly inspired by the biomimetic exchange mechanism between alveoli and capillaries, relies entirely on precise control of spray freeze-drying kinetics and surface chemical modification for its engineering implementation. This structure is not a simple stacking of pores, but an organic whole with specific geometric proportions, a specific wetting gradient, and specific mechanical strength. It is this holistic nature that ensures the invention maintains definite and repeatable hemostatic efficacy even in complex bleeding environments, thus meeting the stringent quality requirements of modern medical emergency medications.

[0034] Compared with the prior art, the beneficial effects of the present invention are: 1. By constructing an anti-hygroscopic barrier through surface amphiphilic modifiers, the moisture adsorption of the powder during storage and use is effectively reduced, preventing particles from sticking together and clumping, ensuring good flowability, and ensuring uniform distribution and coverage on the wound surface. 2. Relying on the micron-nano hierarchical porous structure, combined with the capillary force generated by the hydrophilic end, it drives blood to perfuse rapidly into the pores, shortens the contact distance between thrombin and fibrinogen, accelerates the coagulation reaction process, and achieves rapid hemostasis. 3. The biocompatible framework material and the cryoprotectant work synergistically to inhibit the denaturation of thrombin protein molecules, and the liquid nitrogen cryogenic solidification process locks in the activity, ensuring that the powder maintains high biological activity during long-term storage. 4. The spherical particles have good compressive strength and are not easily damaged during transportation and use. The porous structure combines toughness and breathability, and the hemostatic film formed has strong adhesion, which can effectively resist the impact of blood flow and ensure the stability of hemostasis. 5. No harmful chemical reagents are added during the preparation process. The entire process is carried out at low temperature and in the dark. The process parameters are precise and controllable. The product characteristics can be customized according to different clinical bleeding scenarios. Furthermore, the energy consumption is optimized, the environmental protection compliance is achieved, the batch consistency is good, and it is easy to carry out large-scale production. Detailed Implementation

[0035] This invention provides a highly active and stable thrombin powder preparation and its preparation method. The preparation improves the bioavailability and hemostatic response speed of thrombin in complex wound environments by constructing hierarchical porous microspheres with specific micro-geometric configuration and chemical polarity distribution.

[0036] The technical solution of the present invention will be described in detail below with reference to specific embodiments and comparative examples, so as to ensure that those skilled in the art can fully understand and implement the present invention.

[0037] Example 1: The active thrombin component was bovine thrombin with a specific activity of 2200 U / mg and a mass percentage of 10%. The biocompatible framework material is a 1:1 mixture of human serum albumin and hydroxypropyl methylcellulose, comprising 50% by mass. The cryoprotectant is a mixture of trehalose, sucrose, and mannitol in a 2:1:1 ratio, accounting for 20% by mass. The composite pore-forming agent is a mixture of ammonium bicarbonate and polyethylene glycol 4000 in a 3:1 ratio, accounting for 8% by mass. The surface amphiphilic modifier is a complex of hexadecyltrimethoxysilane and polyethylene glycol monomethyl ether, accounting for 2% by mass. The remainder is phosphate buffer; Preparation steps: S1: Preparation of precursor solution: Dissolve the biocompatible framework material in a phosphate buffer solution at pH 7.0 under a light-protected environment at 7°C to prepare an 8% mass fraction base matrix solution; add cryoprotectant and stir until clear, then add thrombin active component and stir at 200 rpm until homogeneous; S2: Construction of composite pore-forming and modification system: A composite pore-forming agent solution was added dropwise to the precursor solution, and a surface amphiphilic modifier was added simultaneously. The mixture was stirred at a constant temperature for 45 minutes to form a homogeneous sol system. S3: Ultrasonic atomization and cryogenic curing. The sol system is atomized into droplets by a 50kHz ultrasonic atomization device under the drive of 0.35MPa carrier gas, and directly sprayed into a -196℃ liquid nitrogen curing autoclave for instant curing to obtain cured microspheres. S4: Controlled sublimation drying: The solidified microspheres are transferred to a freeze dryer with a vacuum of 8 Pa and dried in a stepped temperature-controlled manner: primary drying is completed at -40℃ for 25 hours, secondary drying is completed at -10℃ for 12 hours, and residual moisture is removed at 22℃ until the moisture content is less than 3%. S5: Aseptic packaging. Particles are collected in a Class 10,000 clean environment, classified by particle size, and then aseptically packaged.

[0038] Example 2: The thrombin active component accounts for 5% by mass, and the remaining components and proportions are the same as in Example 1; Preparation steps: Same as in Example 1.

[0039] Example 3: The thrombin active component accounts for 15% by mass, and the remaining components and proportions are the same as in Example 1; Preparation steps: Same as in Example 1.

[0040] Example 4: The composite porogen accounts for 5% by mass, and the remaining components and proportions are the same as in Example 1; Preparation steps: Same as in Example 1.

[0041] Example 5: The composite porogen accounts for 10% by mass, and the remaining components and proportions are the same as in Example 1; Preparation steps: Same as in Example 1.

[0042] Example 6: The surface amphiphilic modifier was replaced with a mixture of phosphatidylcholine and fatty acid polyoxyethylene ester, accounting for 2% by mass, and the remaining components and proportions were the same as in Example 1; Preparation steps: Same as in Example 1.

[0043] Example 7: The remaining components and proportions are the same as in Example 1; Preparation steps: The ultrasonic atomizing device frequency is 40kHz, and the remaining steps are the same as in Example 1.

[0044] Example 8: The remaining components and proportions are the same as in Example 1; Preparation steps: The ultrasonic atomizing device is used at a frequency of 60 kHz, and the remaining steps are the same as in Example 1.

[0045] Comparative Example 1: The composite porogen was removed, and the remaining components were the same as in Example 1; Preparation steps: The addition of composite pore-forming agent was omitted, and there was no in-situ pore-forming step in the drying process. The remaining process parameters and steps were the same as in Example 1.

[0046] Comparative Example 2: The surface amphiphilic modifier was removed, and the remaining components were the same as in Example 1; Preparation steps: The addition of the surface amphiphilic modifier was omitted, and the remaining process parameters and steps were the same as in Example 1.

[0047] Test method: Activity and hemostatic performance test Thrombin activity retention rate test: Thrombin activity was determined after preparation and after 6 months of storage using the prothrombin time method, and the activity retention rate was calculated. Thrombin activity retention rate (%) = (Activity after 6 months of storage / Initial activity after preparation) × 100%; Hemostasis response speed test: On a standard trauma model, the time from the powder's contact with blood to the cessation of bleeding is measured; Moisture absorption resistance test: The powder was placed in an environment with a relative humidity of 75% for 48 hours, and the moisture absorption weight gain rate was measured. Flowability test: The angle of repose of the powder was determined using the fixed funnel method; Pore ​​characteristics testing: Specific surface area, total porosity, and total pore volume were determined using the nitrogen adsorption-desorption method; Moisture content test: The moisture content of the powder was determined using the Karl Fischer moisture determination method.

[0048] The test data comparisons are shown in Table 1 and Table 2.

[0049] Table 1 Comparison of thrombin activity retention rate, hemostatic response speed, and moisture absorption weight gain rate

[0050] Table 2 Comparison of Angle of Repose, Specific Surface Area, Total Porosity, and Water Content

[0051] Examples 1 to 8 utilize a three-dimensional network constructed with micron- and nano-sized pores to accelerate blood perfusion. The hydrophobic end of the amphiphilic surface modifier forms an anti-hygroscopic barrier, while the hydrophilic end enhances blood infiltration. Combined with the stabilizing effect of the biocompatible framework and cryoprotectant on thrombin, this achieves synergistic effects of activity maintenance, rapid hemostasis, and anti-hygroscopicity. Comparative Example 1, lacking a composite porogen, could not form a hierarchical porous structure, resulting in a significant decrease in hemostasis speed; Comparative Example 2, lacking the amphiphilic surface modifier, exhibited reduced anti-hygroscopic properties.

[0052] The closer the thrombin content is to 10% to 15%, the higher the content of the composite porogen is to 8% to 10%, and the higher the atomization frequency is to 50kHz to 60kHz, the higher the thrombin activity retention rate, the faster the hemostatic response, and the better the pore characteristics. Among these factors, the proportion of the composite porogen directly affects the porosity and pore connectivity, the type of surface amphiphilic modifier affects the balance between moisture absorption and blood infiltration, and the ultrasonic atomization frequency determines the particle size uniformity. These three factors work together to ensure the overall performance of the product.

[0053] Compared to Comparative Example 1 without a graded porous structure, the hemostatic response speed of the example is increased by more than 51%, the specific surface area is increased by more than 358%, and the total porosity is increased by more than 91%. Compared to Comparative Example 2 without a surface amphiphilic modifier, the moisture absorption weight gain rate is reduced by more than 65%, the angle of repose is reduced by more than 15%, the thrombin activity retention rate is increased by more than 6%, and the water content is controlled below 3%, meeting the requirements for rapid hemostasis in clinical trauma emergency care and long-term storage stability.

[0054] In summary, this invention achieves high thrombin activity retention, second-level hemostasis response, and excellent anti-hygroscopic properties through hierarchical porous structure construction coupled with surface amphiphilic modification. The particles exhibit good flowability and mechanical stability, meeting the needs of clinical emergency care and long-term storage, and possesses good potential for industrialization.

[0055] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A highly active and stable thrombin powder preparation, characterized in that, The powder preparation is composed of near-spherical micron-sized particles with a hierarchical porous structure; The spherical micron-sized particles have secondary porosity characteristics, including first-level micron-sized channels distributed inside and on the surface of the particles, and second-level nano-sized channels interwoven on the inner wall of the first-level micron-sized channels. The components of the highly active and stable thrombin powder formulation include: thrombin active component, biocompatible framework material, cryoprotectant, composite porogen, and surface amphiphilic modifier.

2. The highly active and stable thrombin powder preparation according to claim 1, characterized in that, The content of each component in the highly active and stable thrombin powder preparation, by weight percentage, is as follows: Thrombin active component: 5%~15%; Biocompatible scaffold materials: 40%~60%; Cryoprotectant: 15%~25%; Composite porogen: 5%~10%; Surface amphiphilic modifier: 1%~5%; The remainder is phosphate buffer.

3. The highly active and stable thrombin powder preparation according to claim 1, characterized in that, The thrombin active component is bovine or porcine thrombin; the thrombin active component is uniformly loaded in an amorphous state on the inner wall surface of a hierarchical porous structure made of the biocompatible framework material.

4. The highly active and stable thrombin powder preparation according to claim 1, characterized in that, The biocompatible scaffold material is selected from at least one of human serum albumin, recombinant human collagen, hydroxypropyl methylcellulose, or carboxymethyl deacetylated chitosan.

5. The highly active and stable thrombin powder preparation according to claim 1, characterized in that, The cryoprotectant is composed of trehalose, sucrose and mannitol, and the mass ratio of trehalose, sucrose and mannitol is 2:1:

1.

6. The highly active and stable thrombin powder preparation according to claim 1, characterized in that, The composite porogen is composed of ammonium bicarbonate and polyethylene glycol 4000, with a mass ratio of ammonium bicarbonate to polyethylene glycol 4000 of 3:

1. The composite porogen is added to a phosphate buffer solution with a pH of 6.8 to 7.4 and stirred until completely dissolved to prepare a composite porogen solution with a mass fraction of 10% to 15%.

7. The highly active and stable thrombin powder preparation according to claim 6, characterized in that, The ammonium bicarbonate, as a volatile component, is used to generate gaseous products in situ during the drying and sublimation process to induce the formation of the first-level micron-sized channels; the polyethylene glycol 4000 assists in the formation of the second-level nano-sized channels through steric hindrance effect.

8. The highly active and stable thrombin powder preparation according to claim 1, characterized in that, The surface amphiphilic modifier is selected from a compound of hexadecyltrimethoxysilane and polyethylene glycol monomethyl ether, or a mixture of phosphatidylcholine and fatty acid polyoxyethylene ester.

9. A method for preparing a highly active and stable thrombin powder formulation as described in any one of claims 1 to 8, characterized in that, The method includes the following steps: Step 1: Preparation of precursor solution: Dissolve the biocompatible framework material in phosphate buffer solution to prepare the basic matrix solution; add the cryoprotectant and the thrombin active component in sequence, and stir until homogeneous; Step 2: Construction of the composite pore-forming and modification system: The composite pore-forming agent solution is added dropwise to the precursor solution, and the surface amphiphilic modifier is added simultaneously. The mixture is stirred to form a homogeneous and stable sol system. Step 3: Ultrasonic atomization and cryogenic curing: The sol system is atomized into droplets by an ultrasonic atomization device and directly sprayed into a liquid nitrogen curing autoclave for instantaneous curing to obtain cured microspheres; Step 4: Controlled sublimation drying: The solidified microspheres are transferred to a freeze dryer and subjected to step-by-step temperature-controlled drying. By removing ice crystals, decomposing volatile pore-forming agents, and removing residual moisture, powder particles with a hierarchical porous structure are formed. Step 5: Aseptic packaging: The dried powder is collected, particle size classified, and aseptically packaged in a Class 10,000 clean environment.

10. The method for preparing the highly active and stable thrombin powder according to claim 9, characterized in that, In step one, the pH of the phosphate buffer solution is precisely adjusted to bring the thrombin molecules near their isoelectric point, thereby reducing the tendency of thrombin molecules to self-polymerize.

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