Camouflage starch-based porous composite particle with micro-nano hierarchical membrane structure as well as preparation method and application of camouflage starch-based porous composite particle

Abstract

The invention discloses a micro-nano graded membrane structure camouflage starch-based porous composite particle as well as a preparation method and application thereof, and relates to the technical field of biomedical materials. The porous composite particle comprises membrane-like structure camouflage bioactive glass and porous starch microspheres, wherein the membrane-like structure camouflage bioactive glass is porous bioactive glass containing chitosan / bovine serum albumin membrane-like structure camouflage; the porous composite particle provided by the invention has a relatively high liquid absorption rate, can concentrate blood coagulation factors and activate various blood coagulation mechanisms of an organism at the same time, and can safely degrade the high-efficiency hemostatic composite particle without side effects after playing a high-efficiency hemostatic role.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and in particular to a micro-nano hierarchical membrane-like structure camouflaged starch-based porous composite particle, and a preparation method and application thereof. Background Art

[0002] The dangers of uncontrolled bleeding to human life are well known. Excessive bleeding accounts for one-third of the global mortality rate. Rapidly stopping bleeding at the scene of an accident can reduce the mortality rate by half. Therefore, effective hemostasis is a crucial tool for preventing traumatic deaths. Consequently, developing effective hemostatic materials without side effects for irregular wounds, such as those involving the limbs, trunk, and internal organs, is a current research priority.

[0003] There are six common types of existing hemostatic materials: powders, sponges, bandages, gauze, hydrogels, and adhesives. However, hemostatic powders and gel adhesives are not restricted by wound surface type and are therefore suitable for a wider range of wound types. Gel adhesives such as chitosan and sodium alginate easily fall off when exposed to water and take a long time to degrade in the body, leading to inflammatory reactions such as bacterial infection. Powdered hemostatic materials, due to their excellent fluid absorption, can absorb moisture from the wound site and concentrate coagulation factors. Traditional hemostatic powders, such as zeolite and kaolin, have a highly exothermic reaction, which can cause tissue damage and body rejection.

[0004] In recent years, porous particles have become a research hotspot for hemostatic materials due to their large specific surface area, high porosity and excellent water absorption. Porous particles have a molecular sieve pore structure, which can contact and activate platelets by absorbing water and concentrating coagulation factors, thereby achieving rapid hemostasis. Hemostatic agents based on micron- and nano-scale porous particles have potential application prospects in the treatment of deep wound bleeding and irregular bleeding. Due to their small size, porous particles are easy to enter bleeding channels and contact hidden bleeding sites. Studies have shown that simple hemostatic materials cannot exert better hemostatic effects due to their single hemostatic pathway. The development trend of hemostatic materials is to use composite materials, especially composite materials of nano-inorganic materials and natural carbohydrates, to exert synergistic hemostatic effects between different materials. The prior art discloses a composite material of starch microspheres and human recombinant tissue factor ester for hemostasis, which has complex preparation steps and high costs. The prior art also discloses a calcium ion exchange porous starch hemostatic material, in which the surface of starch particles is doped with Ca 2+ , and its liquid absorption rate is generally limited in its effect on controlling bleeding. Those skilled in the art also use bioactive glass and calcium alginate fiber in combination for hemostasis and repair of wounds, but its use is limited in wounds and it cannot be quickly degraded.

[0005] It is known that bioactive glass porous nanoparticles have good coagulation function. The silicate component can significantly inhibit the release of vascular endothelial growth factor (VEGF), promoting in vitro tissue regeneration and wound healing. Due to its high specific surface area, it can quickly absorb blood and concentrate coagulation factors to stop bleeding. Among them, coagulation factor IV, namely Ca 2+ (FIV) can directly participate in the coagulation cascade reaction and promote the formation of fibrin network. The chitosan surface in the membrane-like structure carries a positive charge and can quickly aggregate red blood cells to form a hemostatic plug. The polypeptide contained in bovine serum albumin is platelet growth promoting factor, which can promote cell decomposition and thus accelerate wound hemostasis. Plant-derived corn starch has been widely used in blood loss control due to its good biocompatibility and biodegradability. Micron-sized porous microspheres are produced by enzymatic hydrolysis, which increases the specific surface area and liquid absorption rate of starch. The viscosity of porous starch microspheres increases after absorbing water, which is conducive to the aggregation of coagulation factors and accelerates platelet activation.

[0006] Therefore, there is an urgent need in this field to develop a comprehensive nano-scale and micro-scale porous composite particle with a simple preparation process, which can activate multiple coagulation pathways in the body and is biodegradable and has high efficiency and rapid coagulation. Summary of the Invention

[0007] To address the shortcomings of the aforementioned background technology, the present invention provides a micro-nano hierarchical membrane-like structure camouflaged starch-based porous composite particle, as well as its preparation method and application. These porous composite particles have a high liquid absorption rate, can concentrate coagulation factors, and simultaneously activate multiple coagulation mechanisms in the body. After exerting their highly effective hemostatic effect, they can safely degrade without side effects, resulting in highly effective hemostatic composite particles.

[0008] The first object of the present invention is to provide a micro-nano hierarchical membrane-like structure camouflaged starch-based porous composite particle, the porous composite particle comprising a membrane-like structure camouflaged bioactive glass and porous starch microspheres;

[0009] Wherein, the membrane-like structure camouflaged bioactive glass is a porous bioactive glass containing chitosan / bovine serum albumin membrane-like structure camouflage;

[0010] The weight ratio of the membrane-like structure camouflaged bioactive glass to the porous starch microspheres is 1:0.1-3.

[0011] Preferably, the porous starch microspheres are prepared by enzymatic hydrolysis with α-amylase and glucoamylase and then cross-linked with sodium trimetaphosphate.

[0012] Preferably, the porous composite particles have a diameter of 8500-13000 nm and a pore size of 800-1200 nm;

[0013] The particle size of the membrane-like structure camouflaged bioactive glass is 400-800 nm, and the pore size is 30-70 nm.

[0014] Preferably, the surface charge of the porous starch microspheres is -25 to -40 mV;

[0015] The surface charge of the membrane-like structure camouflaged bioactive glass is 28 to 35 mV.

[0016] Preferably, the membrane-like structure camouflaged bioactive glass is prepared according to the following steps:

[0017] Ethyl orthosilicate, hexadecyl ammonium bromide, and calcium nitrate tetrahydrate are uniformly dissolved in a mixed reaction solution, stirred at room temperature for 2 to 6 hours, and then calcined to remove the template to obtain porous bioactive glass, wherein the mixed reaction solution is prepared from ethanol, ether, ammonia water, and water;

[0018] performing an amination treatment on the porous bioactive glass to obtain an amination-treated porous bioactive glass;

[0019] The amino-modified porous bioactive glass is evenly dispersed in a bovine serum albumin sodium chloride aqueous solution, stirred at room temperature for 20 to 40 minutes, centrifuged, washed, and dried, and then dispersed in a chitosan sodium chloride aqueous solution, stirred at room temperature for 20 to 40 minutes to obtain a membrane-like structure camouflaged bioactive glass.

[0020] Preferably, the ratio of ethanol, ether, ammonia water and water in the mixed reaction liquid is 20:40:4:150 wt %;

[0021] The concentration of bovine serum albumin in the bovine serum albumin sodium chloride aqueous solution is 2.0 mg / mL, and the concentration of sodium chloride is 0.1 mol / L; the concentration of chitosan in the chitosan sodium chloride aqueous solution is 2.0 mg / mL, and the concentration of sodium chloride is 0.1 mol / L.

[0022] Preferably, the porous bioactive glass is subjected to an amination treatment, comprising: uniformly dispersing the porous bioactive glass in isopropanol, adding 3-aminopropyltriethoxysilane dropwise through a syringe pump, and stirring to react to obtain the amination-treated porous bioactive glass; during the stirring reaction, the stirring speed is 300-400 rpm / min, the reaction time is 12-36 hours, and the temperature is 70-90°C.

[0023] The second object of the present invention is to provide a method for preparing micro-nano hierarchical membrane-like structure camouflaged starch-based porous composite particles, comprising the following steps:

[0024] The membrane-like structure camouflaged bioactive glass and porous starch microspheres are uniformly dispersed in a water solvent, and then centrifuged to obtain micro-nano hierarchical membrane-like structure camouflaged starch-based porous composite particles.

[0025] Preferably, the weight ratio of the total weight of the membrane-like structure camouflaged bioactive glass and porous starch microspheres to the water solvent is 1:1-2.

[0026] The third object of the present invention is to provide a micro-nano graded membrane-like structure camouflaged starch-based porous composite particle for use in the preparation of hemostatic materials.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The present invention provides a micro-nano hierarchical membrane-like structure camouflaged starch-based porous composite particle, as well as its preparation method and application. The micro-nano hierarchical membrane-like structure camouflaged starch-based composite hemostatic particles of the present invention have a high liquid absorption rate and can concentrate coagulation factors by absorbing blood from the wound surface, while activating both intrinsic and extrinsic coagulation pathways. They have good blood and cell compatibility and are biodegradable in vivo, enabling rapid formation of hemostatic plugs at the wound surface, thereby improving hemostatic efficiency. These particles can be used for rapid wound hemostasis and repair, and have broad clinical application value.

[0029] The preparation method of the micro-nano hierarchical membrane-like structure-camouflaged starch-based composite hemostatic particles disclosed herein utilizes the electrostatic adsorption principle of positive and negative charges, resulting in a simple and rapid preparation method. By electrostatic adsorption, porous starch microspheres with negative surface charges after oxidative crosslinking are doped with porous bioactive glass with positive surface charges after amino treatment, thereby improving the single hemostatic pathway problem of existing silicate-based hemostatic materials. Furthermore, the porous starch microspheres are endowed with the ability to participate in the body's coagulation pathway. During the hemostatic process, the micro-nano hierarchical membrane-like structure-camouflaged starch-based composite hemostatic particles come into contact with the wound. First, the micro- and nano-scale porous particles rapidly absorb water molecules from the wound surface, concentrating coagulation factors and amplifying the coagulation cascade. Second, calcium ions on the composite particle surface participate in and activate both endogenous and exogenous coagulation pathways. Simultaneously, the membrane-like structured CS / BSA accelerates platelet activation and red blood cell aggregation. Finally, the surface charges of the two porous micro- and nano-particles: the negative charge activates FⅫ to FⅫa, triggering the endogenous coagulation pathway to exert a hemostatic effect, while the positive charge aggregates red blood cells and accelerates the formation of a hemostatic plug. Multiple hemostatic pathways work synergistically, and the micro-nano graded membrane-like structure camouflaged starch-based composite hemostatic particles can achieve rapid hemostasis in deep and irregular wounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 SEM (A, C, and D) and TEM (B) images of micro-nano hierarchical membrane-like structures camouflaged with starch-based porous composite particles. A & B: Membrane-like structures camouflaged with bioactive glass (MBC), C: Oxidatively cross-linked porous corn starch (CMS), D: Composite particles (MBC@CMS).

[0031] Figure 2Micro-nano hierarchical membrane-like structures camouflage the surface charge of starch-based porous composite particles (n=3).

[0032] Figure 3 ESD element distribution of starch-based porous composite particles camouflaged by micro-nano hierarchical membrane-like structures.

[0033] Figure 4 Contact angle of starch-based porous composite particles camouflaged by micro-nano hierarchical membrane-like structures.

[0034] Figure 5 Fourier transform infrared absorption spectrum of starch-based porous composite particles camouflaged with micro-nano hierarchical membrane-like structure.

[0035] Figure 6 Coagulation images (A) and coagulation time (B) of starch-based porous composite particles camouflaged with micro-nano hierarchical membrane-like structures.

[0036] Figure 7 Liquid absorption rate (A) and swelling rate (B) of starch-based porous composite particles camouflaged with micro-nano hierarchical membrane-like structures (n=3, ***P<0.0005).

[0037] Figure 8 APTT (A) and PT (B) of starch-based porous composite particles disguised as micro-nano hierarchical membrane structures.

[0038] Figure 9 Aggregation of red blood cells (A) and activation of platelets (B) by starch-based porous composite particles disguised as micro-nano hierarchical membrane structures.

[0039] Figure 10 Photographs of hemostasis (A), blood loss (B), and hemostasis time (C) of tail hemostasis in SD rats.

[0040] Figure 11 Photos of hemostasis of liver wound (A), blood loss (B), and hemostasis time (C).

[0041] Figure 12 Hemolysis images (A) and hemolysis results (B) of micro-nano hierarchical membrane-like structure-camouflaged starch-based porous composite particles at different concentrations (n=3).

[0042] Figure 13 SRB staining results of cell compatibility of the extract of micro-nano hierarchical membrane-like structure-camouflaged starch-based porous composite particles co-cultured with L929 for 24, 48, and 72 hours (n=6).

[0043] Figure 14 AM-PI staining results of micro-nano hierarchical membrane-structured starch-based porous composite particle extract (1000 μg / mL) co-cultured with L929 for 24, 48, and 72 hours (n=3).

[0044] Figure 15 Degradation curve (A) and Ca2+ ion release (B) of micro-nano hierarchical membrane-like structure-camouflaged starch-based porous composite particles (n=3). DETAILED DESCRIPTION

[0045] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is further described below with reference to specific embodiments and drawings, but the embodiments are not intended to limit the present invention.

[0046] The purpose of the present invention is to provide a micro-nano graded membrane structure disguised starch-based porous composite particle and its preparation method and application. The micro-nano graded membrane structure disguised starch-based porous composite particle has a high liquid absorption rate, can concentrate coagulation factors and activate multiple coagulation mechanisms of the body at the same time, and can safely degrade into high-efficiency hemostatic composite particles without side effects after exerting a high-efficiency hemostatic effect.

[0047] This invention, for the first time, utilizes the synthesis principle of electrostatic adsorption to adsorb nanoscale membrane-like structure-camouflaged bioactive glass particles onto the surface of micron-sized oxidized cross-linked porous starch microspheres, providing a novel, highly effective hemostatic porous composite particle combining nanoscale and micron-sized particles and a preparation method thereof. The particles contain micron-sized oxidized cross-linked porous starch microspheres and nanoscale membrane-like structure-camouflaged bioactive glass in weight ratios of 75:25, 67:33, 50:50, 33:67, and 25:75 (50:50 being optimal). The oxidized cross-linked porous starch microsphere-loaded membrane-like structure-camouflaged bioactive glass composite particles prepared using the optimal weight ratio have a diameter of 8500-13000 nm and a pore size distribution of 800-1200 nm. The preparation method of the oxidatively cross-linked porous starch microsphere-loaded membrane-structured camouflaged bioactive glass composite particles of the present invention does not require complex equipment, has a simple process, and uses mild conditions; and after the sample preparation is completed, only vacuum drying is required; in addition, during the preparation process of the oxidatively cross-linked porous starch microsphere-loaded membrane-structured camouflaged bioactive glass composite particles, only mixing and stirring are required, which consumes less time and energy and has a shorter product preparation cycle. The utilization rate of the equipment is improved while the time cost is reduced, and the method is environmentally friendly and does not generate toxic wastewater or waste gas.

[0048] In order to achieve the above-mentioned object, the first aspect of the present invention provides a micro-nano hierarchical membrane-like structure camouflaged starch-based porous composite particle, wherein the porous composite particle comprises a membrane-like structure camouflaged bioactive glass and porous starch microspheres;

[0049] Wherein, the membrane-like structure camouflaged bioactive glass is a porous bioactive glass containing chitosan / bovine serum albumin membrane-like structure camouflage;

[0050] The weight ratio of the membrane-like structure camouflaged bioactive glass to the porous starch microspheres is 1:0.1-3.

[0051] The porous composite particles have a diameter of 8500-13000 nm and a pore size of 800-1200 nm;

[0052] The particle size of the membrane-like structure camouflaged bioactive glass is 400-800 nm, and the pore size is 30-70 nm.

[0053] The surface charge of the porous starch microspheres is -25 to -40 mV;

[0054] The surface charge of the membrane-like structure camouflaged bioactive glass is 28 to 35 mV.

[0055] The micro-nano hierarchical membrane-like structure disguised starch-based porous composite particles have a strong liquid absorption rate, amplify the coagulation cascade reaction after aggregating coagulation factors, can rapidly aggregate red blood cells and activate platelets, and are biodegradable and biocompatible. They can efficiently and rapidly coagulate, reduce bleeding volume, and shorten bleeding time. The porous composite particles provided by the present invention participate in activating the intrinsic and exogenous coagulation pathways through the release of coagulation factor IV (calcium ions) in the process of promoting rapid coagulation. The present invention uses plant starch, which is low-cost, widely available, safe, and has no side effects. The production process and conditions provided by the present invention are simple, do not require complex large-scale equipment, and do not generate by-products.

[0056] According to the present invention, the porous starch microspheres are prepared by enzymatic hydrolysis with α-amylase and glucoamylase and then cross-linked with sodium trimetaphosphate.

[0057] Exemplarily, the method for preparing porous starch microspheres comprises the following steps:

[0058] (1) Provide the solution required for enzymatic hydrolysis of starch microspheres. α-amylase and glucoamylase are evenly dispersed in 200 mL of sodium acetate buffer.

[0059] (2) Add natural corn starch to the above buffer solution at a weight ratio of 30-50:200, and stir at 36-42°C and 250 rpm for 10 hours.

[0060] (3) Add 20 mL of sodium hydroxide solution (4%, w / w) to terminate the enzymatic reaction.

[0061] (4) The slurry was vacuum filtered, washed with deionized water three times, and then vacuum dried for 24 h to obtain porous starch microspheres.

[0062] The method for preparing porous starch microspheres further comprises the step of washing the oxidized cross-linked starch porous microspheres obtained in step (4) with deionized water.

[0063] The method for preparing oxidized cross-linked starch porous microspheres further comprises the steps of: cross-linking porous starch microspheres and sodium trimetaphosphate in a weight ratio of 20:50, and stirring at 50° C. and 250 rpm for 24 hours.

[0064] The mass ratio of α-amylase to glucose starch in the preparation of oxidative cross-linked starch porous microspheres was 160:640.

[0065] The mass ratio of enzyme to buffer in the preparation of oxidized cross-linked starch porous microspheres was 0.8:200.

[0066] The pH of the buffer solution in the preparation of oxidized cross-linked starch porous microspheres is 4-5, and the optimal pH is 4.6.

[0067] In one embodiment, oxidatively cross-linked porous starch microspheres were prepared using an enzymatic hydrolysis method and a sodium trimetaphosphate cross-linking method. The specific steps are as follows:

[0068] 160 mg of α-amylase and 640 mg of glucoamylase were evenly dispersed in 200 mL of sodium acetate buffer (pH 4.6). 40 g of native corn starch (NS) was added to the above buffer and stirred at 40°C, 250 rpm, and stirred for 10 h. 20 mL of NaOH solution (4% w / w) was then added to terminate the enzymatic reaction. The slurry was vacuum filtered, washed three times with ddH2O, and vacuum dried for 24 h to obtain microporous starch (MS). The 20 g of microporous starch obtained above was added to 50 mL of STMP coupling buffer and stirred at 50°C, 250 rpm, and stirred for 24 h. The reaction was terminated by adjusting the pH to ≈ 7 with 0.1 M HCl. The reaction mixture was vacuum filtered, washed three times with ddH2O, and vacuum dried for 24 h to obtain cross-linked porous starch microspheres (CMS).

[0069] According to the present invention, the membrane-like structure camouflaged bioactive glass is prepared according to the following steps:

[0070] Ethyl orthosilicate, hexadecyl ammonium bromide, and calcium nitrate tetrahydrate are uniformly dissolved in a mixed reaction solution, stirred at room temperature for 2 to 6 hours, and then calcined to remove the template to obtain porous bioactive glass, wherein the mixed reaction solution is prepared from ethanol, ether, ammonia water, and water;

[0071] performing an amination treatment on the porous bioactive glass to obtain an amination-treated porous bioactive glass;

[0072] The amino-modified porous bioactive glass was evenly dispersed in a bovine serum albumin (BSA) sodium chloride aqueous solution and stirred at room temperature for 20 to 40 minutes. After centrifugation, washing, and drying, it was dispersed in a chitosan (CS) sodium chloride aqueous solution and stirred at room temperature for 20 to 40 minutes to obtain a membrane-like structure camouflaged bioactive glass (MBG@BSA / CS).

[0073] The ratio of ethanol, ether, ammonia and water in the mixed reaction liquid is 20:40:4:150 wt %;

[0074] The concentration of bovine serum albumin in the bovine serum albumin sodium chloride aqueous solution is 2.0 mg / mL, and the concentration of sodium chloride is 0.1 mol / L; the concentration of chitosan in the chitosan sodium chloride aqueous solution is 2.0 mg / mL, and the concentration of sodium chloride is 0.1 mol / L.

[0075] The porous bioactive glass is subjected to an amination treatment, comprising: uniformly dispersing the porous bioactive glass in isopropyl alcohol, adding 3-aminopropyltriethoxysilane dropwise via a syringe pump, and stirring to react to obtain the amination-treated porous bioactive glass; the stirring speed during the reaction is 300-400 rpm / min, the reaction time is 12-36 hours, and the temperature is 70-90° C. The amination-treated porous bioactive glass has a surface charge of 20-30 mV.

[0076] An exemplary method for preparing a membrane-like structure camouflaged bioactive glass comprises the following steps:

[0077] Using the sol-gel method, ethyl orthosilicate, hexadecyl ammonium bromide, and calcium nitrate tetrahydrate were dissolved in a mixed reaction solution (ethanol: ether: ammonia water: water = 20:40:4:150 wt%) in sequence, stirred vigorously at room temperature for 4 h, collected by centrifugation, washed, dried, and calcined to remove the template to obtain porous bioactive glass (MBG).

[0078] The amination method of porous bioactive glass comprises the following steps: dispersing 100 mg of porous bioactive glass in 200 mL of isopropyl alcohol, adding 3-aminopropyltriethoxysilane dropwise through a syringe pump, stirring for reaction, washing, centrifuging, and drying.

[0079] The amination method of the porous bioactive glass also includes stirring at a rotation speed of 300-400 rpm / min, a reaction time of 12-36 hours, and a temperature of 70-90°C.

[0080] Membrane-like particles were prepared using the layer-by-layer self-assembly principle. Aminated MBG was uniformly dispersed in a BSA (2.0 mg / mL) sodium chloride (0.1 mol / L) aqueous solution (pH 7.5) and stirred at room temperature for 30 minutes. The particles were collected by centrifugation, washed, and dried to obtain MBG@BSA.

[0081] MBG@BSA was dispersed in a CS (2.0 mg / mL) sodium chloride (0.1 mol / L) aqueous solution (pH 7.5) and stirred at room temperature for 30 minutes. The mixture was collected by centrifugation, washed, and dried to obtain a membrane-like structure camouflaged bioactive glass.

[0082] Among them, when collecting the active glass with membrane-like structure disguise, centrifugation was performed at 10,000 rpm for 5 minutes.

[0083] When collecting the active glass with membrane-like structure disguise, it was washed three times with deionized water.

[0084] In one embodiment, a method for preparing a membrane-like structure camouflaged bioactive glass comprises the following steps:

[0085] Preparation of porous bioactive glass: 2 g of hexadecylammonium bromide and 7.5 mL of ethyl orthosilicate were dissolved in a mixed ammonia solution of ethanol and ether, wherein the volumes of water, ethanol, ether, and ammonia solution were 150 mL, 20 mL, 40 mL, and 4 mL, respectively. The pH value was 10-12, the temperature was controlled at 20-30°C, and stirring was carried out at 800-1000 rpm for 4 hours. The solution formed a gel through sol formation and then gelation. The gel was centrifuged at 8000-12000 rpm for 5 minutes to collect the sample. The wet gel was vacuum dried at 30-60°C for 12-24 hours to remove moisture, and after sufficient grinding, it was calcined at 550°C for 5 hours to remove the surfactant to form a macroporous structure.

[0086] Preparation of amino-modified porous bioactive glass: 100 mg of porous bioactive glass powder was dispersed in 200 mL of isopropanol. 0.4 mL of 3-aminopropyltriethoxysilane was added dropwise with stirring at 1 mL / min and 200-500 rpm, followed by cooling and reflux. After the reaction, the precipitate was collected by centrifugation at 10,000 rpm for 5 minutes. The sample was washed three times with anhydrous ethanol and three times with deionized water, then vacuum-dried at 50°C for 24 hours to produce the amino-modified porous bioactive glass.

[0087] Preparation of membrane-like structure camouflaged bioactive glass:

[0088] 100 mg of aminated MBG was evenly dispersed in 200 mL of BSA / NaCl aqueous solution and stirred at room temperature for 30 minutes. The MBG@BSA sample was centrifuged at 10,000 rpm for 5 minutes and washed three times with deionized water. MBG@BSA was dispersed in 200 mL of CS / NaCl aqueous solution and stirred at room temperature for 30 minutes. The MBG@BSA / CS sample was centrifuged at 10,000 rpm for 5 minutes, washed three times with deionized water, dried under vacuum for 24 hours, and stored under vacuum.

[0089] A second aspect of the present invention provides a method for preparing micro-nano hierarchical membrane-like structure camouflaged starch-based porous composite particles, comprising the following steps:

[0090] The membrane-like structure camouflaged bioactive glass and porous starch microspheres are uniformly dispersed in a water solvent, and then centrifuged to obtain micro-nano hierarchical membrane-like structure camouflaged starch-based porous composite particles.

[0091] The weight ratio of the total weight of the membrane-like structure camouflaged bioactive glass and the porous starch microspheres to the water solvent is 1:1-2.

[0092] An exemplary method for preparing micro-nano hierarchical membrane-like structure-camouflaged starch-based porous composite particles comprises:

[0093] (1) Providing a solution required for the electrostatic adsorption reaction of composite particles, wherein the solution comprises composite particles and deionized water in a weight ratio of 100:100-100:200 and a pH of 7-8.

[0094] (2) The ratios of the composite particles in step (1) were 75:25, 67:33, 50:50, 33:67, and 25:75, respectively.

[0095] (3) The reaction solution in step (1) was centrifuged at 6000-10000 rpm for 5 minutes, and the precipitate was collected, washed, and dried.

[0096] The reaction solution is a 0.5 mol / L sodium chloride aqueous solution with a pH of 7-8; the composite particle aqueous solution is ultrasonicated at 100 Hz for 2-5 minutes.

[0097] The obtained oxidative cross-linked porous starch microspheres loaded with membrane-like structure camouflaged bioactive glass porous composite particles are washed with deionized water and then dried in an oven or freeze-dried.

[0098] In one embodiment, a method for preparing micro-nano hierarchical membrane-like structure-camouflaged starch-based porous composite particles comprises:

[0099] Using the principle of electrostatic adsorption, oxidatively cross-linked porous starch microspheres and amino-modified porous bioactive glass were dispersed in deionized water (pH 7.5) to prepare a 1 mg / mL suspension. The suspension was stirred at room temperature for 12 hours. The suspension was centrifuged at 8000 rpm for 5 minutes to collect the composite microsphere sample. The collected sample was then vacuum-dried at room temperature for 24 hours.

[0100] The weight ratios of membrane-like structure camouflaged bioactive glass and oxidative cross-linked starch microspheres are 75:25, 67:33, 50:50, 33:67, and 25:75.

[0101] The pore size of the oxidatively cross-linked porous starch microspheres loaded with bioactive glass composite particles is 800-1200 nm.

[0102] A third aspect of the present invention provides an application of micro-nano graded membrane-like structure camouflaged starch-based porous composite particles in the preparation of hemostatic materials.

[0103] The oxidatively cross-linked porous starch microspheres loaded with membrane-like structures disguised as bioactive glass porous composite particles described in this invention possess strong liquid absorption and hydrophilicity. Through micro- and nano-graded absorption of water molecules at the wound surface, they activate multiple coagulation pathways in the body to achieve rapid hemostasis. They are also biodegradable and highly cytocompatible. By rapidly clotting at the wound surface, they reduce bleeding volume and duration. They can be used directly or in combination with other hemostatic materials for emergency hemostasis and skin repair, demonstrating high clinical application value.

[0104] It should be noted that the experimental methods used in the present invention are all conventional methods unless otherwise specified; the reagents and materials used are all commercially available unless otherwise specified.

[0105] Example 1

[0106] This embodiment relates to the synthesis of membrane-like structure camouflaged bioactive glass

[0107] Membrane-like camouflaged bioactive glass (MBC) was prepared using the layer-by-layer self-assembly principle. 100 mg of aminated MBG was uniformly dispersed in 200 mL of a 2.0 mg / mL solution of BSA (0.1 mol / L) in a sodium chloride (0.1 mol / L) aqueous solution (pH 7.5) and stirred at room temperature for 30 minutes. The MBG@BSA sample was centrifuged at 10,000 rpm for 5 minutes and washed three times with deionized water. The MBG@BSA was then dispersed in 200 mL of a 2.0 mg / mL solution of CS (0.1 mol / L) in a sodium chloride (0.1 mol / L) aqueous solution (pH 7.5) and stirred at room temperature for 30 minutes. The MBG@BSA / CS sample was centrifuged at 10,000 rpm for 5 minutes, washed three times with deionized water, dried under vacuum for 24 hours, and stored under vacuum.

[0108] The surface morphology and pore structure of the prepared MBC were observed using a scanning electron microscope (SEM, JSM-7500F, JEOL, Japan) and a transmission electron microscope (TEM, Sigma 300, Zeiss (Germany)). Figure 1 A and 1B). The potential distribution of the sample was measured using a laser Doppler electrophoresis instrument (Zetasizer Nano ZS, Malvern Instruments Ltd, England) ( Figure 2 ).

[0109] Example 2

[0110] This embodiment relates to the synthesis of oxidative cross-linked porous starch microspheres

[0111] 160 mg of α-amylase and 640 mg of glucoamylase were evenly dispersed in 200 mL of sodium acetate buffer (pH 4.6). 40 g of native corn starch (NS) was added to the above buffer and stirred at 40°C, 250 rpm, and stirred for 10 h. 20 mL of NaOH solution (4%, w / w) was then added to terminate the enzymatic reaction. The slurry was vacuum filtered, washed three times with deionized water, and vacuum dried for 24 h to obtain MS. 20 g of the microporous starch obtained above was added to 50 mL of sodium trimetaphosphate coupling buffer and stirred at 50°C, 250 rpm, and stirred for 24 h. The reaction was terminated by adjusting the pH to ≈ 7 with 0.1 M HCl. The reaction mixture was vacuum filtered, washed three times with deionized water, and vacuum dried for 24 h to obtain cross-linked oxidized porous starch microspheres.

[0112] The morphology and macroporous structure of the prepared porous starch microspheres (CMS) were observed using SEM. Figure 1 C) The potential distribution of CMS is shown in Figure 2 Energy dispersive spectrometer (EDS) was used to evaluate the composition of the sample ( Figure 3 ).

[0113] Example 3

[0114] This embodiment relates to the synthesis of porous bioactive glass

[0115] 150 mL of deionized water, 20 mL of ethanol, and 40 mL of ether were added sequentially to a round-bottom flask containing 2 g of CTAB. Stir at high speed at room temperature until the CTAB was completely dissolved. Subsequently, 4 mL of ammonia water was added dropwise to the above solution. After 10 minutes, 7.5 mL of TEOS was added dropwise to the above system. After 10 minutes, 1.4 g of Ca(NO3)2·4H2O was added to the above solution and reacted for 4 hours. After the reaction was completed, the precipitate was collected by centrifugation at 10,000 rpm for 5 minutes to obtain a sample. After washing with anhydrous ethanol and deionized water three times each, the sample was vacuum dried at 50°C for 24 hours. The sample was calcined at 550°C for 5 hours to remove the template. The diameter distribution of the obtained sample was 400-800 nm, the pore size distribution was 50-70 nm, and the specific surface area was 397.50 m 2 g -1 .

[0116] Example 4

[0117] This embodiment relates to the synthesis of porous bioactive glass

[0118] 150 mL of deionized water, 20 mL of ethanol, and 40 mL of ethylene glycol ethyl ether were sequentially added to a round-bottom flask containing 2 g of CTAB. Stir at high speed at room temperature until the CTAB was completely dissolved. Subsequently, 4 mL of ammonia water was added dropwise to the above solution. After 10 minutes, 7.5 mL of TEOS was added dropwise to the above system. After 10 minutes, 1.4 g of Ca(NO3)2·4H2O was added to the above solution and reacted for 4 hours. After the reaction, the precipitate was collected by centrifugation at 10,000 rpm for 5 minutes to obtain a sample. The sample was washed with anhydrous ethanol and deionized water three times each and then vacuum dried at 50°C for 24 hours. The sample was calcined at 550°C for 5 hours to remove the template. The obtained sample had a diameter distribution of 400-600 nm, a pore size distribution of 30-50 nm, and a specific surface area of 433.62 m 2 g -1 .

[0119] Example 5

[0120] This embodiment involves the amination process of porous bioactive glass

[0121] MBG (400 mg) was added to 200 mL of isopropanol and ultrasonically dispersed. Amination modification was then performed by adding 4 mL of APTES dropwise. The system was stirred at 80°C and 400 rpm under reflux for 24 hours. After the reaction, the precipitate was collected by centrifugation at 10,000 rpm for 5 minutes. The sample was washed three times with anhydrous ethanol and three times with deionized water, then dried under vacuum at 50°C for 24 hours. The resulting aminated MBG had a surface charge of 35.3 mV.

[0122] Example 6

[0123] This embodiment involves the amination process of porous bioactive glass

[0124] MBG (100 mg) was added to 200 mL of isopropanol and ultrasonically dispersed. Amination modification was then performed by adding 0.4 mL of APTES dropwise. The system was stirred at 80°C and 400 rpm under reflux for 24 hours. After the reaction, the precipitate was collected by centrifugation at 10,000 rpm for 5 minutes. The sample was washed three times with anhydrous ethanol and three times with deionized water, then dried under vacuum at 50°C for 24 hours. The resulting aminated MBG had a surface charge of 28.7 mV.

[0125] Example 7

[0126] This embodiment relates to the synthesis of micro-nano hierarchical membrane-like structure disguised starch-based porous composite particles

[0127] 75 mg of membrane-like structure camouflaged bioactive glass particles and 25 mg of oxidative cross-linked porous starch microspheres were dispersed in 200 mL of deionized water solution and stirred at room temperature at 300 rpm / min for 30 minutes. The precipitate was collected by centrifugation at 8000 rpm for 5 minutes. The diameter of the membrane-like structure camouflaged bioactive glass is known to be 580 nm, with a surface charge of 30.8 mV and a PDI of 0.256. The diameter of the oxidative cross-linked porous starch microspheres is 9500 nm, with a surface charge of negative 34.5 mV and a PDI of 0.198. The obtained nano-micrometer composite particles have a diameter of 11000 nm, a surface charge of negative 12.9 mV and a PDI of 0.298.

[0128] Example 8

[0129] This embodiment relates to the synthesis of micro-nano hierarchical membrane-like structure disguised starch-based porous composite particles

[0130] 67 mg of membrane-like camouflaged bioactive glass particles and 33 mg of oxidatively cross-linked porous starch microspheres were dispersed in 200 mL of deionized water solution and stirred at room temperature at 300 rpm / min for 30 minutes. The precipitate was collected by centrifugation at 8000 rpm for 5 minutes. The diameter of the membrane-like camouflaged bioactive glass particles is 550 nm, the surface charge is 20.3 mV, and the PDI is 0.221. The diameter of the oxidatively cross-linked porous starch microspheres is 7500 nm, the surface charge is negative 38.1 mV, and the PDI is 0.271. The resulting nano-micrometer composite particles have a diameter of 8100 nm, a surface charge of negative 18.8 mV, and a PDI of 0.213.

[0131] Example 9

[0132] This embodiment relates to the synthesis of micro-nano hierarchical membrane-like structure disguised starch-based porous composite particles

[0133] 50 mg of membrane-like structure camouflaged bioactive glass particles and 50 mg of oxidative cross-linked porous starch microspheres were dispersed in 200 mL of deionized water solution and stirred at room temperature at 300 rpm / min for 30 minutes. The precipitate was collected by centrifugation at 8000 rpm for 5 minutes. The diameter of the membrane-like structure camouflaged bioactive glass is known to be 650 nm, with a surface charge of 16.3 mV and a PDI of 0.135. The diameter of the oxidative cross-linked porous starch microspheres is 8600 nm, with a surface charge of negative 36.4 mV and a PDI of 0.167. The obtained nano-micrometer composite particles have a diameter of 9000 nm, a surface charge of negative 20.5 mV and a PDI of 0.263.

[0134] Example 10

[0135] This embodiment relates to the synthesis of micro-nano hierarchical membrane-like structure disguised starch-based porous composite particles

[0136] 33 mg of membrane-like camouflaged bioactive glass particles and 66 mg of oxidatively cross-linked porous starch microspheres were sequentially dispersed in 200 mL of deionized water solution and stirred at room temperature at 300 rpm / min for 30 minutes. The precipitate was collected by centrifugation at 8000 rpm for 5 minutes. The diameter of the membrane-like camouflaged bioactive glass is known to be 650 nm, with a surface charge of 28.3 mV and a PDI of 0.137. The diameter of the oxidatively cross-linked porous starch microspheres is 11000 nm, with a surface charge of negative 38.4 mV and a PDI of 0.131. The resulting nano-micrometer composite particles have a diameter of 12000 nm, a surface charge of negative 7.2 mV, and a PDI of 0.302.

[0137] Example 12

[0138] This embodiment relates to the synthesis of micro-nano hierarchical membrane-like structure disguised starch-based porous composite particles

[0139] 25 mg of membrane-like structure camouflaged bioactive glass particles and 75 mg of oxidative cross-linked porous starch microspheres were dispersed in 200 mL of deionized water solution and stirred at room temperature at 300 rpm / min for 30 minutes. The precipitate was collected by centrifugation at 8000 rpm for 5 minutes. The diameter of the membrane-like structure camouflaged bioactive glass is known to be 650 nm, with a surface charge of 31.2 mV and a PDI of 0.103. The diameter of the oxidative cross-linked porous starch microspheres is 9500 nm, with a surface charge of negative 37.3 mV and a PDI of 0.281. The obtained nano-micrometer composite particles have a diameter of 10000 nm, a surface charge of negative 4.1 mV and a PDI of 0.311.

[0140] Example 13

[0141] This embodiment relates to the synthesis of micro-nano hierarchical membrane-like structure disguised starch-based porous composite particles

[0142] 50 mg of membrane-like bioactive glass particles and 50 mg of oxidatively cross-linked porous starch microspheres were sequentially dispersed in 200 mL of 0.5 mol / L sodium chloride solution (pH 7.5) and stirred at 300 rpm for 30 minutes at room temperature. The precipitate was collected by centrifugation at 8000 rpm for 5 minutes and washed three times with deionized water. The membrane-like bioactive glass particles had a diameter of 800 nm, a surface charge of 25.2 mV, and a PDI of 0.114. The oxidatively cross-linked porous starch microspheres had a diameter of 11000 nm, a surface charge of -36.4 mV, and a PDI of 0.293. The resulting nano-micrometer composite particles had a diameter of 12000 nm, a surface charge of -6.9 mV, and a PDI of 0.291.

[0143] Example 14

[0144] This embodiment relates to the synthesis of micro-nano hierarchical membrane-like structure disguised starch-based porous composite particles

[0145] 50 mg of membrane-like bioactive glass particles and 50 mg of oxidatively cross-linked porous starch microspheres were sequentially dispersed in 200 mL of deionized water and ultrasonicated at 100 Hz for 2-5 minutes at room temperature. The precipitate was collected by centrifugation at 8000 rpm for 5 minutes. The precipitate was washed three times with deionized water. The known membrane-like bioactive glass particles had a diameter of 900 nm, a surface charge of 29.1 mV, and a PDI of 0.187. The oxidatively cross-linked porous starch microspheres had a diameter of 8500 nm, a surface charge of -33.4 mV, and a PDI of 0.232. The resulting nano-micrometer composite particles had a diameter of 9000 nm, a surface charge of -10.4 mV, and a PDI of 0.305.

[0146] Example 15

[0147] This embodiment relates to the synthesis of micro-nano hierarchical membrane-like structure disguised starch-based porous composite particles

[0148] 50 mg of membrane-like bioactive glass particles and 50 mg of oxidatively cross-linked porous starch microspheres were sequentially dispersed in 200 mL of 0.5 mol / L sodium chloride solution (pH 7.5) and sonicated at 100 Hz for 2-5 minutes at room temperature. The precipitate was collected by centrifugation at 8000 rpm for 5 minutes and washed three times with deionized water. The membrane-like bioactive glass particles had a diameter of 650 nm, a surface charge of 27.2 mV, and a PDI of 0.192. The oxidatively cross-linked porous starch microspheres had a diameter of 9500 nm, a surface charge of -35.8 mV, and a PDI of 0.176. The resulting nano-micrometer composite particles had a diameter of 10000 nm, a surface charge of -3.5 mV, and a PDI of 0.310.

[0149] Example 16

[0150] This example involves the morphology and structural characterization of micro-nano hierarchical membrane-like structures disguised as starch-based porous composite particles

[0151] Taking the membrane-like structure camouflaged bioactive glass prepared in Example 1 as an example, it can be observed from the scanning electron microscope image and the transmission electron microscope image that the bioactive glass has almost no pore structure and the diameter distribution of the nanoparticles is 400-800nm. Figure 1 A and Figure 1B. The specific surface area of the membrane-like structure camouflaged bioactive glass prepared in Example 1 was measured by nitrogen isothermal adsorption-desorption analyzer (Best Instrument Technology (Beijing) Co., LTD, 3H-2000PS2) to be 105.50 m 2 g -1 , the average pore size is 3.36nm.

[0152] Taking the oxidized cross-linked porous starch microspheres prepared in Example 2 as an example, the microporous structure of the oxidized cross-linked starch can be observed from the scanning electron microscope image, and the diameter size distribution of the starch microspheres is 8000-12000nm. Figure 1 As shown in C.

[0153] Taking the porous bioactive glass prepared in Example 3 as an example, the nanoparticle size distribution is 400-800 nm. The specific surface area of the macroporous bioactive glass prepared in Example 3 was measured using a nitrogen isothermal adsorption-desorption analyzer (Best Instrument Technology (Beijing) Co., LTD, 3H-2000PS2) to be 397.50 m 2 g -1 The average pore size is about 10.98nm.

[0154] Taking the porous bioactive glass prepared in Example 4 as an example, the nanoparticle size distribution is 400-600 nm. The specific surface area of the porous bioactive glass prepared in Example 4 was measured using a nitrogen isothermal adsorption-desorption analyzer (Best Instrument Technology (Beijing) Co., LTD, 3H-2000PS2) to be 433.62 m 2 g -1 The average pore size is about 3.38nm.

[0155] Taking the micro-nano hierarchical membrane-like structure camouflaged starch-based porous composite particles prepared in Example 7 as an example, the obtained nano-micron hierarchical composite particles have a diameter of 9000 nm and a surface charge of negative 12.9 mV ( Figure 3 ), PDI is 0.263. SEM results are as follows Figure 1 As shown in D. Porous bioactive glass is distributed on the surface of porous starch microspheres, and the EDS results are shown in Figure 3 shown.

[0156] Laser particle size and Zeta potential Doppler electrophoresis (Zetasizer Nano ZS, Malvern Instruments Ltd., England) were used to measure the surface charge changes of the micro-nano hierarchical membrane-like structure disguised starch-based porous composite particles prepared in Example 8 as the membrane-like structure disguised bioactive glass was deposited on the surface of the porous starch microspheres, which were 30.8, -34.5, and -12.9 mV, respectively. Figure 2 shown.

[0157] The contact angles of the samples were measured, and the contact angles of MBG and CMS were 2.3° and 4.7°, respectively. The contact angles of MBG@BSA and MBG@BSA / CS (MBC) were 12.08° and 10.8°, respectively. After doping MBC and CMS by electrostatic adsorption, the contact angle of the composite particles MBC@CMS was reduced to 8.9°. Figure 4 shown.

[0158] The micro-nano hierarchical membrane-like structure camouflaged starch-based porous composite particles prepared in Example 7 were measured using a Fourier transform infrared spectrometer (FTIR, Tensor27, Bruker Optics) at 4000-400 cm -1 Functional groups within the wavelength range of Figure 5 As shown, the deposition of membrane-like structure-camouflaged bioactive glass on the surface or pores of porous starch microspheres can be observed.

[0159] Example 17

[0160] This example involves the characterization of the in vitro accelerated coagulation performance of membrane-like structure disguised bioactive glass, oxidative cross-linked porous starch microspheres, and micro-nano hierarchical membrane-like structure disguised starch-based porous composite particles.

[0161] Taking the membrane-like structure camouflaged bioactive glass, oxidative cross-linked porous starch microspheres and porous composite particles prepared in Examples 1, 2 and 7 as an example, 1 mL of fresh SD rat anticoagulated whole blood was added to 10 mg of the above-mentioned test sample. The centrifuge tube was taken out every 10 seconds at 37°C and tilted once. When the blood was completely coagulated, the timing was stopped and the blood coagulation time of each group was recorded. A blank centrifuge tube with only blood added was used as a negative control, and Yunnan Baiyao hemostatic powder was used as a positive control. Each sample was repeated 3 times. The results showed that the in vitro blood coagulation time of the micro-nano graded membrane-like structure camouflaged starch-based porous composite particles was as short as about 37 seconds. The process and results are shown in the figure. Figure 6 shown.

[0162] Example 18

[0163] This example involves the liquid absorption rate and expansion rate of membrane-like structure disguised bioactive glass, oxidative cross-linked porous starch microspheres and micro-nano graded membrane-like structure disguised starch-based porous composite particles after contact with water.

[0164] Taking the membrane-like structure camouflaged bioactive glass, oxidatively cross-linked porous starch microspheres, and micro-nano hierarchical membrane-like structure camouflaged starch-based porous composite particles prepared in Examples 1, 2, and 7 as examples, after vacuum drying the sample at 50°C for 24 hours, 0.1g of each sample was removed, recorded as W0 g; 1mL of simulated body fluid was added, recorded as VmL (W0 / V = 1:10). The mixture was stirred at 36.5°C and 250 rpm for 10 minutes and allowed to stand for 20 minutes. The mixture was filtered until the last drop of liquid dripped into the filter flask, and the sample weight was weighed as W1. The water absorption rate was calculated using the formula: Water absorption rate (%) = (W1-W0) / W0 × 100%. The results are shown in 7A.

[0165] When measuring the swelling rate, the sample was vacuum dried at 50°C for 24 hours, 0.2 g was weighed and added to 10 mL of deionized water, and incubated at 37°C for 10, 20, 30, 40, and 50 minutes. Centrifuged at 6000 rpm for 10 minutes, the supernatant was discarded, and the residual liquid was recorded as W (g). The swelling rate formula was used to calculate the swelling rate (%) = (W-0.2) / 0.2×100%. The results are shown in the figure below. Figure 7 As shown in B.

[0166] Figure 7 The results showed that the composite MBG@CMS particles, prepared by combining oxidatively cross-linked porous starch microspheres (215±50%) with membrane-like bioactive glass (MBC) (373±10%), significantly increased their liquid absorption by 430±36%. The oxidatively cross-linked porous starch microspheres exhibited a low expansion rate, while the membrane-like bioactive glass exhibited an expansion rate of 630±6%. The composite MBG@CMS particles exhibited an expansion rate of 684±12%, while Yunnan Baiyao hemostatic powder (YB) served as a control.

[0167] Example 19

[0168] This example involves the testing of coagulation pathways involving membrane-like structure disguised bioactive glass, oxidative cross-linked porous starch microspheres, and micro-nano graded membrane-like structure disguised starch-based porous composite particles.

[0169] Taking the membrane-like structure camouflaged bioactive glass, oxidative cross-linked porous starch microspheres and composite particles prepared in Examples 1, 2 and 7 as an example, the activated partial thromboplastin time (APTT) was measured using the platelet-poor plasma (PPP) of SD rats. 50 μL APTT reagent was added to 50 μL PPP to measure APTT. For prothrombin time (PT), 50 μL PPP, 100 μL PT reagent and 2 mg sample were taken to measure PT. During the APTT and PT determination processes, a control group without sample was used, and Yunnan Baiyao hemostatic powder was used as a positive control. The test results were expressed as the percentage of time between the sample group and the control group. The results are shown in Figure 2. Figure 8 A and 8B, the results showed that micro-nano hierarchical membrane-like structure camouflaged starch-based porous composite particles can simultaneously activate the intrinsic and extrinsic coagulation pathways, and the effect is better than MBC.

[0170] Example 20

[0171] This example involves the characterization of membrane-like structure disguised bioactive glass, oxidative cross-linked porous starch microspheres and micro-nano graded membrane-like structure disguised starch-based porous composite particles in promoting platelet activation, red blood cell aggregation and fibrin formation during in vitro activated coagulation.

[0172] Taking the membrane-like structure camouflaged bioactive glass, oxidative cross-linked porous starch microspheres and composite particles prepared in Examples 1, 2 and 7 as an example, the characterization of red blood cell aggregation and fibrin formation was as follows: 10 mg of sample was added to 1 mL of SD rat anticoagulated whole blood. After the blood coagulated, it was washed 3 times with PBS to remove excess red blood cells. It was fixed in 2.5% glutaraldehyde at 4°C for 2.5 h, and dehydrated three times with gradient ethanol for 10 min each time. The in vitro red blood cell adhesion was observed with a scanning electron microscope. The results showed that the micro-nano graded membrane-like structure camouflaged starch-based porous composite particles had the best effect in aggregating red blood cells. The results are as follows Figure 9 A. Platelet activation: SD rat anticoagulated whole blood was centrifuged at 1500 rpm for 15 minutes, and platelet-rich plasma (PRP) was aspirated with a pipette. Approximately 200 μL of PRP was added to 10 mg of sample and incubated at 37°C for 1 hour. The sample was dehydrated and washed three times with a series of gradient ethanol to eliminate the adhesion of platelets on the surface of the material. Each concentration was soaked for 10 minutes. The sample was then soaked in 2.5% glutaraldehyde fixative at 4°C overnight and observed under an ordinary optical microscope after drying. The results showed that under the same conditions, the micro-nano hierarchical membrane-like structure disguised starch-based porous composite particles activated platelets to the greatest extent, such as Figure 9 B.

[0173] Example 21

[0174] This example involves the evaluation of the hemostatic effect of membrane-like structure camouflaged bioactive glass, oxidative cross-linked porous starch microspheres and micro-nano graded membrane-like structure camouflaged starch-based porous composite particles in the SD rat tail-broken bleeding model.

[0175] Taking the membrane-like structure camouflaged bioactive glass, oxidative cross-linked porous starch microspheres and composite particles prepared in Examples 1, 2 and 7 as an example, SD rats were anesthetized with 10% chloral hydrate. The surgical area was disinfected with iodine and medical alcohol, and the tail was cut off 5 cm away from the tail end. After absorbing the blood with conventional sterile gauze, the hemostatic sample was immediately applied to the wound site and gently lifted to observe the damage every 10 seconds. When the bleeding stopped, the hemostatic time and blood loss were determined. Yunnan Baiyao hemostatic powder YB was used as a positive control, and no sample was applied but only the bleeding stopped automatically as a blank control. The hemostatic effect is as follows Figure 10 The results showed that the micro-nano hierarchical membrane-like structure disguised starch-based porous composite particles could stop bleeding in as fast as 57 seconds and reduce blood loss by 228 mg.

[0176] Example 22

[0177] This example involves the evaluation of the hemostatic effect of membrane-like structure camouflaged bioactive glass, oxidative cross-linked porous starch microspheres and micro-nano graded membrane-like structure camouflaged starch-based porous composite particles in the SD rat liver bleeding model.

[0178] Taking the membrane-like structure camouflaged bioactive glass, oxidative cross-linked porous starch microspheres and composite particles prepared in Examples 1, 2 and 7 as an example, SD rats were anesthetized with 10% chloral hydrate. The abdominal surgical area was disinfected with iodine and medical alcohol, and a 1 cm long bleeding wound was prepared after exposing the liver. After absorbing the blood with conventional sterile gauze, the hemostatic sample was immediately applied to the wound site, and gently lifted to observe the damage every 10 seconds. When the bleeding stopped, the hemostasis time and blood loss were determined. Yunnan Baiyao hemostatic powder YB was used as a positive control, and the blank control was used for the self-stopping of bleeding without applying the sample. The hemostatic effect is shown in FIG. Figure 11 The results showed that the micro-nano hierarchical membrane-like structure disguised starch-based porous composite particles could stop bleeding in as fast as 45 seconds and reduce blood loss by 49 mg.

[0179] Example 23

[0180] This example involves the evaluation of the in vitro hemolytic properties of membrane-like structure disguised bioactive glass, oxidative cross-linked porous starch microspheres, and micro-nano hierarchical membrane-like structure disguised starch-based porous composite particles.

[0181] Using the membrane-like structured bioactive glass, oxidatively cross-linked porous starch microspheres, and composite particles prepared in Examples 1, 2, and 7 as examples, these samples were dispersed in 3.5 mL of PBS at varying concentrations (125, 250, 500, and 1000 μg / mL) and incubated at 37°C for 30 minutes. 0.1 mL of rat anticoagulated whole blood diluted with blood (rat anticoagulated whole blood:PBS = 4:5) was added to the PBS suspension, and the mixture was maintained at 37°C for 30 minutes, followed by 10,000 rpm for 2 minutes. The absorbance of the supernatant at 540 nm was measured. PBS solution and deionized water served as negative and positive controls, respectively, while Yunnan Baiyao hemostatic powder was also used as a control to investigate its hemolytic effect. Hemolysis rate was calculated using the formula: hemolysis rate (%) = (As - Ap) / (Ad - Ap) * 100%. As, Ap, and Ad represent the absorbance at 540 nm of the sample, PBS, and water, respectively, after the hemolysis experiment. The results showed that the hemolysis rate of each sample was below 5% as specified for hemostasis samples. Figure 12 shown.

[0182] Example 24

[0183] This example involves the cell compatibility evaluation of the co-culture of extracts of membrane-like structure camouflaged bioactive glass, oxidative cross-linked porous starch microspheres and micro-nano graded membrane-like structure camouflaged starch-based porous composite particles with SD rat fibroblasts L929.

[0184] Taking the membrane-like structure camouflaged bioactive glass, oxidative cross-linked porous starch microspheres and composite particles prepared in Examples 1, 2 and 7 as examples, the cell compatibility of the hemostatic particles was detected using the SRB staining method and the Calcein-AM / PI live / dead cell double staining kit. Mouse fibroblasts (L929) were seeded in a 96-well plate at a density of 2000 cells / well. The samples were diluted with DMEM complete culture medium to 1000, 500, 250, and 125 μg / mL. The sample dilutions were co-cultured with the cells for 24, 48, and 72 hours, and the DMEM complete culture medium group was used as the control group. The cells were stained with a 1% acetic acid solution of 0.4% SRB. The absorbance value of each well was measured at 540 nm on a microplate reader, and the cell survival rate of the sample was calculated. The results are shown in FIG. Figure 13 .

[0185] L929 cells were seeded at a density of 2000 cells / well in a 96-well plate. The sample group was cultured with 1000 μg / mL of DMEM complete medium extract for 24, 48, and 72 hours. The DMEM complete medium group served as the control group. Calcein-AM and PI dyes were used to simultaneously stain live and dead cells. Live and dead cell levels were analyzed using an inverted fluorescence microscope. The results are shown in Figure 3. Figure 14The above results show that the micro-nano hierarchical membrane-like structure camouflaged starch-based porous composite particles co-cultured with L929 have good cell compatibility and no cytotoxicity.

[0186] Example 25

[0187] This example involves the evaluation of the degradability of extracts of membrane-like structure camouflaged bioactive glass, oxidative cross-linked porous starch microspheres, and micro-nano graded membrane-like structure camouflaged starch-based porous composite particles.

[0188] Taking the membrane-like structure camouflaged bioactive glass, oxidative cross-linked porous starch microspheres and composite particles prepared in Examples 1, 2 and 7 as an example, the degradation test was carried out: the sample to be tested was dried in a vacuum at 60°C for 24 hours, and the initial weight was W0. 50.0 mg of α-amylase was added to 1 L of simulated body fluid (SBF), and then the sample to be tested was added to SBF containing α-amylase (0.2 g / mL). Stir at 37°C and 100 rpm for 1, 2, 3, 4, 5, 6 and 7 days, filter the filtrate, dry at 60°C for 24 hours, and weigh it as Wt. The formula for calculating the loss rate is as follows, and the results are as follows: Figure 15 A.

[0189]

[0190] Ca 2+ Release test: Weigh 10.0 mg of each sample to be tested, add 5 mL of hydrofluoric acid and vortex to completely dissolve the MBG shell, collect and record the volume of the supernatant. The supernatant was diluted with 1% HNO3 by an appropriate multiple and the Ca content was determined by atomic absorption spectrometry. 2+ concentration, calculate Ca 2+ Weigh 50.0 mg of each sample and add 10 mL of SBF solution. Perform an in vitro ion release test at 37°C and 100 rpm. Sampling time is: 0.5 h, 1 h, 2 h, 3 h, 6 h, 12 h, 1 d, 2 d, 3 d, 7 d. Take 1 mL of the test solution each time, dilute it with 1% HNO3, and measure the concentration using an atomic absorption spectrometer. Ca 2+ The release solution was diluted 10 times with 1% HNO3 and then the concentration was measured by atomic absorption spectrometer. The concentration points of the standard curve were set as: 2, 1, 0.5, 0.25, 0.125, 0.0625 μg / mL. 2+ The cumulative release amount is calculated as follows: Figure 15 B.

[0191]

[0192] Where: V e : Volume of supernatant removed (mL);

[0193] V0: total volume of SBF solution for drug release (mL);

[0194] C n and C i is the release concentration (μg / mL).

[0195] The above results show that the micro-nano hierarchical structure improves the agglomeration of MBC and increases its degradation rate. 2+ The release rate and relative release amount increased.

[0196] In summary, the present invention provides a micro-nano hierarchical membrane-like structure camouflaged starch-based porous composite particle and its preparation method and application. The micro-nano hierarchical membrane-like structure camouflaged starch-based porous composite particle has a strong liquid absorption rate and efficient hemostatic function. The composite particles of the present invention comprise porous starch microspheres after oxidative cross-linking, bovine serum albumin / chitosan membrane structure, and mesoporous bioactive glass. The diameter distribution of the porous composite particles of bioactive glass loaded with oxidative cross-linked porous starch microspheres and membrane-like structure camouflage is 8500-13000nm, and the pore size distribution of the porous starch microspheres is 800-1200nm. The raw materials of membrane-like structure camouflaged bioactive glass nanoparticles and oxidative cross-linked porous starch microspheres in a weight ratio of 75:25, 67:33, 50:50, 33:67, and 25:75 (optimally 50:50) are dispersed in an aqueous solution in sequence, and the oxidative cross-linked starch-loaded bioactive glass composite particles and their preparation method can be obtained by electrostatic adsorption principle. The preparation process is simple and no secondary products are generated. The composite of nano- and micron-sized porous particles can significantly increase the liquid absorption rate of bioactive glass, thereby concentrating coagulation factors and activating platelets. Compared with bioactive glass disguised as a membrane-like structure, micro- and nano-graded starch-based composite particles can increase the release rate of calcium ions, thereby accelerating the activation of coagulation factors and expanding the coagulation cascade reaction. These composite particles can simultaneously activate both intrinsic and extrinsic coagulation pathways, rapidly forming hemostatic plugs on the wound surface, sealing the wound, reducing bleeding volume, and shortening bleeding time. Micro- and nano-graded membrane-like structure-camouflaged starch-based porous composite particles have good biocompatibility and biodegradability, and can be used in areas such as rapid hemostasis and repair of surface or internal wounds, with great clinical application value.

Claims

1. A micro-nano hierarchical membrane-like structure camouflaged starch-based porous composite particle, characterized in that: The porous composite particles include membrane-like structure camouflaged bioactive glass and porous starch microspheres; Wherein, the membrane-like structure camouflaged bioactive glass is a porous bioactive glass containing chitosan / bovine serum albumin membrane-like structure camouflage; The weight ratio of the membrane-like structure camouflaged bioactive glass to the porous starch microspheres is 1:0.1-3.

2. The micro-nano hierarchical membrane-like structure camouflaged starch-based porous composite particles according to claim 1, characterized in that: The porous starch microspheres are prepared by enzymatic hydrolysis with α-amylase and glucoamylase and then cross-linking with sodium trimetaphosphate.

3. The micro-nano hierarchical membrane-like structure camouflaged starch-based porous composite particles according to claim 1, characterized in that: The porous composite particles have a diameter of 8500-13000 nm and a pore size of 800-1200 nm; The particle size of the membrane-like structure camouflaged bioactive glass is 400-800 nm, and the pore size is 30-70 nm.

4. The micro-nano hierarchical membrane-like structure camouflaged starch-based porous composite particles according to claim 1, characterized in that: The surface charge of the porous starch microspheres is -25 to -40 mV; The surface charge of the membrane-like structure camouflaged bioactive glass is 28 to 35 mV.

5. The micro-nano hierarchical membrane-like structure camouflaged starch-based porous composite particles according to claim 1, characterized in that: The membrane-like structure camouflaged bioactive glass is prepared according to the following steps: Ethyl orthosilicate, hexadecyl ammonium bromide, and calcium nitrate tetrahydrate are uniformly dissolved in a mixed reaction solution, stirred at room temperature for 2 to 6 hours, and then calcined to remove the template to obtain porous bioactive glass, wherein the mixed reaction solution is prepared from ethanol, ether, ammonia water, and water; performing an amination treatment on the porous bioactive glass to obtain an amination-treated porous bioactive glass; The amino-modified porous bioactive glass is evenly dispersed in a bovine serum albumin sodium chloride aqueous solution, stirred at room temperature for 20 to 40 minutes, centrifuged, washed, and dried, and then dispersed in a chitosan sodium chloride aqueous solution, stirred at room temperature for 20 to 40 minutes to obtain a membrane-like structure camouflaged bioactive glass.

6. The micro-nano hierarchical membrane-like structure camouflaged starch-based porous composite particles according to claim 5, characterized in that: The ratio of ethanol, ether, ammonia water and water in the mixed reaction liquid is 20:40:4:150 wt %; The concentration of bovine serum albumin in the bovine serum albumin sodium chloride aqueous solution is 2.0 mg / mL, and the concentration of sodium chloride is 0.1 mol / L; the concentration of chitosan in the chitosan sodium chloride aqueous solution is 2.0 mg / mL, and the concentration of sodium chloride is 0.1 mol / L.

7. The micro-nano hierarchical membrane-like structure camouflaged starch-based porous composite particles according to claim 5, characterized in that: The porous bioactive glass is subjected to an amination treatment, comprising: uniformly dispersing the porous bioactive glass in isopropyl alcohol, adding 3-aminopropyltriethoxysilane dropwise through a syringe pump, and reacting by stirring to obtain the amination-treated porous bioactive glass; during the stirring reaction, the stirring speed is 300-400 rpm / min, the reaction time is 12-36 hours, and the temperature is 70-90°C.

8. A method for preparing the micro-nano hierarchical membrane-like structure camouflaged starch-based porous composite particles according to any one of claims 1 to 7, characterized in that: The following steps are involved: The membrane-like structure camouflaged bioactive glass and porous starch microspheres are uniformly dispersed in a water solvent, and then centrifuged to obtain micro-nano hierarchical membrane-like structure camouflaged starch-based porous composite particles.

9. The method for preparing micro-nano hierarchical membrane-like structure camouflaged starch-based porous composite particles according to claim 8, characterized in that: The weight ratio of the total weight of the membrane-like structure camouflaged bioactive glass and the porous starch microspheres to the water solvent is 1:1-2.

10. Use of the micro-nano hierarchical membrane-like structure camouflaged starch-based porous composite particles according to any one of claims 1 to 7 in the preparation of hemostatic materials.

Images