AB-type double-microsphere powder capable of in-situ liquid absorption self-assembly, and preparation and application thereof

Abstract

This invention discloses an AB-type bimicrosphere powder capable of in-situ liquid absorption and self-assembly, as well as its preparation and application. The preparation method includes: preparing photocrosslinkable hydroxypropyl chitosan and photocrosslinkable oxidized sodium alginate, respectively; using microfluidic technology and ultraviolet light curing to obtain chitosan microspheres loaded with polyphenols and antibacterial agents, and sodium alginate microspheres, respectively; and adsorbing Fe2+ onto the sodium alginate microspheres. 2+ Two types of microspheres were freeze-dried into powder and then mixed in a specific ratio to obtain AB-type dual microsphere powder capable of in-situ liquid absorption and self-assembly. This invention utilizes microfluidic shearing and freeze-drying processes, combined with the synergistic effect of borate ester bonds and carbon-carbon double bonds, to simultaneously achieve the construction of a microsphere cross-linking network and the interaction of polyphenols and Fe. 2+ The loading allows the AB-type bimicrosphere powder, which can absorb liquid in situ and self-assemble, to rapidly absorb liquid upon contact with blood or body fluids and then self-assemble in situ through a Schiff base and a metal-polyphenol network, thereby constructing a stable microsphere scaffold.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials, specifically relating to an AB-type double microsphere powder that can self-assemble in situ by liquid absorption, and its preparation and application. Background Technology

[0002] Wound bleeding and secondary infection are common challenges in clinical surgery, emergency care, and wound treatment. Existing hemostatic materials mainly include hemostatic gauze, gelatin, or oxidized cellulose materials. These materials typically rely on physical adsorption or local pressure to achieve hemostasis. While they can shorten clotting time to some extent, their adaptability to irregular wounds or conditions with significant oozing is limited, resulting in insufficient hemostatic stability. Furthermore, the large-scale bacterial proliferation and persistent inflammatory response in wounds mean that traditional hemostatic materials generally lack antibacterial and anti-inflammatory functions, often requiring the combined use of antibiotics or antibacterial dressings. This not only increases treatment complexity but may also induce drug resistance.

[0003] In recent years, wound healing materials based on powder structures have gradually attracted attention. However, most existing powder systems are single-component and lack rich and tunable pore structures on their surfaces, making it difficult to cope with the partitioned loading and precise independent release of multiple active substances. At the same time, the powder particle structure prepared by traditional methods is uneven and difficult to control, resulting in poor uniformity of interfacial crosslinking, making it difficult to maintain good stability in multiple functions such as rapid hemostasis, antibacterial and inflammation regulation (Wenbin Zhao, Wentao Sun, Weijiang Wang, Yage Zhang & Qingming Ma. Robust and wet adhesive self-gelling powders for rapid hemostasis and efficient wound healing[J]. ACSApplied Materials&Interfaces, 2024, 16(6): 6756-6771.).

[0004] Therefore, there is an urgent need to develop a novel powder wound repair material with controllable structure and adjustable interface to achieve independent loading and precise release of multiple active substances and to rapidly form a stable interfacial cross-linking network in the body fluid environment, thereby synergistically improving hemostasis efficiency, antibacterial properties and inflammation regulation capabilities to cope with rapid hemostasis and secondary infection of complex wounds. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, the present invention aims to provide an AB-type dual microsphere powder that can self-assemble in situ by liquid absorption, as well as its preparation and application. This powder system combines droplet microfluidic technology and freeze-drying preparation, and can rapidly undergo interfacial self-assembly when exposed to blood or body fluids to form a stable microsphere scaffold structure, thereby achieving rapid hemostasis and / or antibacterial and inflammatory regulation in the repair of infected wounds.

[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a method for preparing AB-type double microsphere powder capable of in-situ liquid absorption and self-assembly, comprising the following steps: (1) Dissolve photocrosslinkable hydroxypropyl chitosan (HCM) to obtain an aqueous solution, and add a polyphenol structural unit composed of epigallocatechin gallate (EGCG) and 3-acrylamidophenylboronic acid (APBA) linked by borate ester bonds, and add a photoinitiator and an antibacterial agent to obtain a chitosan polyphenol antibacterial microsphere precursor solution. (2) Dissolve photocrosslinkable oxidized sodium alginate (OSM) to obtain an aqueous solution, and add a photoinitiator to obtain a sodium alginate microsphere precursor solution; (3) The chitosan polyphenol antibacterial microsphere precursor solution and the sodium alginate microsphere precursor solution are respectively used as the dispersed phases. In a microfluidic fluid shearing chip, they are sheared by the continuous phase to form monodisperse droplets, and chitosan polyphenol antibacterial microspheres and sodium alginate microspheres are respectively obtained under ultraviolet light curing. (4) The sodium alginate microspheres were immersed in an equal mass of FeCl2 aqueous solution to adsorb Fe. 2+ , and obtain the load Fe 2+ Sodium alginate microspheres; (5) The chitosan polyphenol antibacterial microspheres and sodium alginate iron ion microspheres were freeze-dried separately to obtain powders, and then mixed to obtain AB type double microsphere powder that can be self-assembled in situ by absorbing liquid.

[0007] Further, in steps (1) and (2), the photoinitiator is one of lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP), 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone (L2959) or 2,2'-azobis[2-methyl-N-(2-hydroxyethyl)propionamide] (VA-086); in step (1), the antibacterial agent is one of nano silver (AgNPs), zinc oxide (ZnO) or copper oxide (CuO).

[0008] Furthermore, in step (1), the preparation method of HCM is as follows: Hydroxypropyl chitosan (HCS) was prepared into an aqueous solution with a concentration of 0.5-2 wt%. The hydroxypropyl chitosan was modified with methacrylic anhydride, and the molar ratio of methacrylic anhydride to hydroxypropyl chitosan monosaccharide units was 0.1:1-0.6:1. The modification conditions were: pH value 7.0-8.5, temperature 40-60℃, reaction time 2-8 h. After the modification was completed, the mixture was cooled, dialyzed, and freeze-dried to obtain photocrosslinkable hydroxypropyl chitosan.

[0009] Furthermore, in step (2), the OSM preparation method is as follows: Sodium alginate (SA) was prepared into an aqueous solution with a concentration of 0.5-2 wt%. Sodium periodate was added to the solution at a mass ratio of sodium alginate to sodium periodate of 10:1-10:5. The reaction was carried out under light-protected conditions for 12-24 hours. The reaction was then terminated, and the solution was dialyzed in deionized water to remove small molecule impurities, thus obtaining oxidized sodium alginate (OSA). The proportion of monosaccharide units that produce aldehyde groups in OSA (degree of oxidation) was 5-40%.

[0010] OSA was prepared into an aqueous solution with a concentration of 0.5-4 wt%. OSA was modified with methacrylic anhydride (MA) at a molar ratio of MA to OSA monosaccharide units of 1:1-20:1. The modification conditions were: pH 7.0-8.5, temperature 0-15℃, and incubation for 12-24 h. After the reaction was completed, the OSA was precipitated with ethanol, washed with ethanol, and dried to obtain OSM.

[0011] Further, in step (1), HCM is dissolved in water to prepare an aqueous solution with a concentration of 2-10 wt%, the molar ratio of EGCG to APBA is 1:5-5:1, the mass fraction of polyphenol structural units is 0.1-1 wt%, the mass fraction of photoinitiator is 0.02-0.2 wt%, and the concentration of antibacterial agent is 20-200 μg / mL.

[0012] Further, in step (2), OSM is dissolved in water to prepare an aqueous solution with a concentration of 2-20 wt%, and the mass fraction of the photoinitiator is 0.02-0.2 wt%.

[0013] Further, in step (3), the continuous phase is mineral oil containing 0.5-2.0 wt% Span-80; the particle size of the chitosan polyphenol antibacterial microspheres or sodium alginate microspheres is 50-500 μm.

[0014] Furthermore, in step (4), the concentration of the FeCl2 aqueous solution is 2-20 mM, and the adsorption time is 0.5-12 h.

[0015] Further, in step (5), the chitosan polyphenol antibacterial microsphere powder and the Fe-loaded... 2+The mass ratio of sodium alginate iron ion microsphere powder is 1:9-9:1.

[0016] The present invention also provides an AB-type bimicrosphere powder that can be self-assembled in situ by liquid absorption, which is prepared by the above-described preparation method.

[0017] Furthermore, the AB-type dual microsphere powder, which can absorb liquid in situ and self-assemble, undergoes rapid interfacial cross-linking with metal-polyphenol coordination through Schiff base reaction after contact with blood or body fluids. This allows for rapid liquid absorption and interfacial self-assembly, forming a stable microsphere scaffold structure in situ on the wound surface, thereby significantly promoting blood coagulation and achieving rapid hemostasis. At the same time, this microsphere powder also has antibacterial and anti-inflammatory functions, which can inhibit bacterial growth and promote the elimination of inflammation.

[0018] Furthermore, an AB-type bimicrosphere powder capable of in-situ liquid absorption and self-assembly is composed of chitosan polyphenol antibacterial microspheres and sodium alginate iron ion microspheres, wherein: Chitosan polyphenol antibacterial microspheres are chitosan hydrogel microspheres containing carbon-carbon double bonds, amino groups, and polyphenol structural units and loaded with antibacterial agents. Sodium alginate iron ion microspheres contain carbon-carbon double bonds, aldehyde groups, and adsorb Fe. 2+ Sodium alginate hydrogel microspheres.

[0019] This invention also provides the application of the above-mentioned AB-type bimicrosphere powder that can self-assemble in situ absorb liquid in the preparation of antibacterial and anti-inflammatory products for rapid hemostasis and repair of infected wounds.

[0020] Compared with the prior art, the present invention has the following advantages: (1) This invention achieves spatial partitioning loading of active components through microfluidic technology, combining polyphenol structural units with metal ions (Fe). 2+ The polyphenols and metal ions are respectively fixed within two independent microsphere network structures, A and B. This physical isolation design effectively avoids premature coordination crosslinking between polyphenols and metal ions during preparation or storage, thus ensuring good flowability and tunable pore size of the powder system. When the AB-type dual microsphere powder, capable of in-situ liquid absorption and self-assembly, comes into contact with blood or body fluids, the instantaneous liquid absorption behavior mediated by the hierarchical pore structure triggers solute diffusion, allowing Fe... 2+ Responsive metal-polyphenol coordination occurs at the microsphere interface with chitosan. This in-situ crosslinking process synergistically works with the Schiff base reaction between chitosan and oxidized sodium alginate to construct a three-dimensional microsphere network scaffold in situ on the wound surface.

[0021] (2) The present invention prepares AB type double microsphere powder with uniform particle size and in-situ liquid absorption and self-assembly by microfluidic fluid shearing and freeze-drying process. Its uniform structure is conducive to the consistency of interfacial contact between microspheres, while the multi-level pore structure can realize rapid liquid absorption and wetting with body fluid, thereby efficiently triggering interfacial reaction and in-situ self-assembly between microspheres in body fluid environment.

[0022] (3) The present invention utilizes the fact that APBA molecules contain both phenylboronic acid structure and olefin groups that can participate in photocrosslinking reaction, so that polyphenol structural units are introduced into the microsphere network skeleton in one step during the microfluidic preparation process, and then the interfacial crosslinking between microspheres is constructed through metal polyphenols, thereby realizing the integration of structure construction and function endowment.

[0023] (4) This invention utilizes microfluidic technology to achieve precise control of microsphere size and functional component loading, and integrates hemostasis, antibacterial and inflammation regulation functions in an AB type dual microsphere powder system that can be self-assembled in situ. Compared with traditional randomly mixed powders, it significantly improves the stability and predictability of the wound repair process.

[0024] (5) The AB type double microsphere powder that can be self-assembled in situ is based on polysaccharide materials as the main skeleton. The raw materials are safe and non-toxic, have good biocompatibility, and are suitable for direct use in wound environments.

[0025] (6) The AB type double microsphere powder that can be self-assembled in situ is provided by the present invention. It exists in powder form, is flexible in use, can be adapted to irregular wounds, has a simple preparation method, low energy consumption, and has good operability and potential for large-scale application. Attached Figure Description

[0026] Figure 1 The image shows the morphology of the chitosan polyphenol antibacterial microspheres, sodium alginate iron ion microspheres, and AB-type double microsphere powder that can be self-assembled in situ under body fluid conditions, prepared in Example 1.

[0027] Figure 2 The results of the coagulation index (BCI) test for different microsphere powders prepared in Example 2 and Comparative Examples 1-3 are shown in the figure (a: Comparative Example 1; b: Comparative Example 2; c: Comparative Example 3; d: Example 2).

[0028] Figure 3 Figure 1 shows the hemostasis test results of rat femoral artery bleeding models prepared with different microsphere powders in Example 3 and Comparative Examples 4-6 (a: Comparative Example 4; b: Comparative Example 5; c: Comparative Example 6; d: Example 3).

[0029] Figure 4 The image shows the antibacterial effect of the AB-type dual microsphere powder prepared in Example 4, which can self-assemble in situ by liquid absorption, against Escherichia coli and Staphylococcus aureus.

[0030] Figure 5 The image shows the intracellular antioxidant regulation effect of the AB-type bimicrosphere powder that can be self-assembled in situ by liquid absorption prepared in Example 5. Detailed Implementation

[0031] The specific implementation of the present invention will be further described below with reference to the accompanying drawings and examples, but the implementation and protection of the present invention are not limited thereto. It should be noted that any processes not specifically described in detail below are those that can be implemented or understood by those skilled in the art by referring to the prior art. Reagents or instruments whose manufacturers are not specified are considered to be conventional products that can be purchased commercially.

[0032] Example 1 A method for preparing AB-type double microsphere powder capable of in-situ liquid absorption and self-assembly includes the following steps: (1) Hydroxypropyl chitosan (hereinafter referred to as HCS) was prepared into an aqueous solution with a concentration of 0.5 wt%. Then, methacrylic anhydride (hereinafter referred to as MA) was slowly added dropwise to the above solution for modification. The molar ratio of MA to HCS monosaccharide units was 0.3:1. The reaction was carried out at 50 °C for 5 h, and the pH value of the system was maintained at 8.0 during the reaction. After the reaction was completed, the mixture was cooled to room temperature and dialyzed in deionized water to remove unreacted methacrylic anhydride and impurities. After dialyzing, it was freeze-dried to obtain photocrosslinkable hydroxypropyl chitosan (hereinafter referred to as HCM).

[0033] (2) Prepare an aqueous solution of HCM obtained in step (1) with a concentration of 5 wt%; add a polyphenolic structural unit (EGCG) and 3-acrylamidophenylboronic acid (APBA) linked by borate ester bonds (the molar ratio of EGCG to APBA is 1:1) to make the mass fraction of the polyphenolic structural unit 0.5 wt%; add lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP) to make the mass fraction of LAP 0.1 wt%; and add silver nanoparticles (AgNPs) to make the concentration of AgNPs 100 μg / mL. Mix thoroughly and store in the dark to obtain a chitosan polyphenol antibacterial microsphere precursor solution.

[0034] (3) Prepare an aqueous solution of sodium alginate (hereinafter referred to as SA) with a concentration of 0.5wt%, add sodium periodate to it, the mass ratio of sodium alginate to sodium periodate is 10:3, react for 18h under the dark, then terminate the reaction, place it in deionized water for dialyzing to remove small molecule impurities, and obtain oxidized sodium alginate (hereinafter referred to as OSA), so that the proportion of monosaccharide units (degree of oxidation) that produce aldehyde groups in oxidized sodium alginate is 20%.

[0035] (4) Prepare an aqueous solution of OSA with a concentration of 0.5 wt%, add methacrylic anhydride (molar ratio of methacrylic anhydride to OSA monosaccharide units is 10:1), maintain the pH of the system at 8.0 during the reaction, and incubate at 10°C for 18 h. After the reaction is complete, pour the mixture into ethanol to precipitate, wash with ethanol to remove excess methacrylic anhydride, and dry to obtain photocrosslinkable oxidized sodium alginate (hereinafter referred to as OSM).

[0036] (5) Prepare an aqueous solution of OSM obtained in step (4) with a concentration of 10 wt%, and add LAP to make the mass fraction of LAP 0.1 wt%. Mix thoroughly and set aside to obtain sodium alginate microsphere precursor solution.

[0037] (6) Fluid focusing microfluidic chip is used to make balls. Mineral oil containing 1.0 wt% Span-80 is used as the continuous phase and the precursor solution obtained in step (2) or step (5) is used as the dispersed phase. Monodisperse droplets are formed at the fluid focusing junction. Then, ultraviolet light is used to photocur in the flow state to obtain chitosan polyphenol antibacterial microspheres and sodium alginate microspheres with a particle size of 200 μm.

[0038] (7) Soak the sodium alginate microspheres obtained in step (6) in an equal mass of FeCl2 aqueous solution with a concentration of 10 mM for 0.5 h for adsorption; after adsorption, wash with deionized water to obtain sodium alginate iron ion microspheres.

[0039] (8) Chitosan polyphenol antibacterial microspheres and sodium alginate iron ion microspheres were freeze-dried separately to obtain microsphere powders. The chitosan polyphenol antibacterial microsphere powder and sodium alginate iron ion microsphere powder were mixed at a mass ratio of 1:1 to obtain AB type double microsphere powder that can be self-assembled in situ by liquid absorption.

[0040] Figure 1 This paper showcases the morphological characteristics of the chitosan polyphenol antibacterial microspheres, sodium alginate iron ion microspheres, and the AB-type bimicrosphere powder that can self-assemble in situ under body fluid conditions, prepared in Example 1. At the 50µm scale, both the single chitosan polyphenol antibacterial microspheres and the sodium alginate iron ion microspheres exhibit regular spherical structures with relatively uniform surfaces, indicating that the microfluidic shearing combined with freeze-drying process can effectively achieve precise control over the size and morphology of the microspheres. Under high magnification (5µm scale), both types of microspheres exhibit interconnected porous network structures. This hierarchical pore structure facilitates rapid penetration and adsorption of body fluids, providing ample mass transfer channels for subsequent interfacial reactions.

[0041] When AB-type bimicrosphere powders capable of in-situ liquid absorption and self-assembly are combined under body fluid conditions, it can be observed that adjacent microspheres are tightly connected by numerous fibrous and lamellar structures (at the 100µm and 20µm scales), and the originally independent microsphere particles gradually form a continuous three-dimensional microsphere scaffold. This phenomenon corroborates that, in a body fluid environment, the amino groups on the surface of chitosan polyphenol antibacterial microspheres react with the sodium alginate iron ion microspheres through a Schiff base reaction and Fe... 2+ The metal-polyphenol coordination between the metal and polyphenol structural units further enhances the interfacial bonding between microspheres.

[0042] This in-situ liquid absorption self-assembly behavior driven by multiple interfacial cross-linking enables the powder system to rapidly transform into a structurally stable microsphere scaffold in the wound environment while maintaining good flowability and filling properties, providing a favorable structural basis for achieving efficient hemostasis and subsequent antibacterial and inflammatory regulation functions.

[0043] Example 2 A method for preparing AB-type double microsphere powder capable of in-situ liquid absorption and self-assembly includes the following steps: (1) HCS was prepared into an aqueous solution with a concentration of 1 wt%. MA was then slowly added dropwise to the solution for modification. The molar ratio of MA to HCS monosaccharide units was 0.1:1. The reaction was carried out at 40 °C for 8 h, and the pH of the system was maintained at 8.5 during the reaction. After the reaction was completed, the mixture was cooled to room temperature and dialyzed in deionized water to remove unreacted MA and impurities. After dialyzing, the mixture was freeze-dried to obtain HCM.

[0044] (2) Prepare an aqueous solution of HCM obtained in step (1) with a concentration of 10 wt%; add polyphenolic structural units (EGCG and APBA in a boronic acid ester bond ratio of 1:1) to make the mass fraction of polyphenolic structural units 0.5 wt%, add 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone (hereinafter referred to as L2959) to make the mass fraction of L2959 0.02 wt%, and add zinc oxide (ZnO) to make the concentration of ZnO 100 μg / mL. Mix thoroughly and store in the dark to obtain a chitosan polyphenol antibacterial microsphere precursor solution.

[0045] (3) Prepare an aqueous solution of SA with a concentration of 1 wt%, add sodium periodate to it, and the mass ratio of sodium alginate to sodium periodate is 10:5. React for 12 h under light-protected conditions, then terminate the reaction, place it in deionized water for dialyzing to remove small molecule impurities, and obtain OSA. Finally, make the proportion (degree of oxidation) of monosaccharide units that produce aldehyde groups in OSA 40%.

[0046] (4) Prepare an aqueous solution of OSA with a concentration of 2wt%, add MA (molar ratio of MA to OSA monosaccharide units of 10:1) to it, maintain the pH of the system at 8.5 during the reaction, and incubate at 15℃ for 12h. After the reaction is completed, pour the mixture into ethanol to precipitate, wash with ethanol to remove excess MA, and dry to obtain OSM.

[0047] (5) Prepare an aqueous solution of OSM obtained in step (4) with a concentration of 20 wt%, and add 0.02 wt% LAP to make the mass fraction of LAP 0.02 wt%. Mix thoroughly and set aside to obtain sodium alginate microsphere precursor solution.

[0048] (6) A fluid focusing microfluidic chip was used to make microspheres. Mineral oil containing 1.0 wt% Span-80 was used as the continuous phase, and the precursor solution obtained in step (2) or step (5) was used as the dispersed phase. Monodisperse droplets were formed at the fluid focusing junction. Then, ultraviolet light was used to photocur the microspheres in the flow state to obtain chitosan polyphenol antibacterial microspheres and sodium alginate microspheres with a particle size of 50 μm.

[0049] (7) Soak the sodium alginate microspheres obtained in step (6) in an equal mass of FeCl2 aqueous solution with a concentration of 20 mM for 12 h for adsorption; after adsorption, wash with deionized water to obtain sodium alginate iron ion microspheres.

[0050] (8) Chitosan polyphenol antibacterial microspheres and sodium alginate iron ion microspheres were freeze-dried separately to obtain microsphere powders. The chitosan polyphenol antibacterial microsphere powder and sodium alginate iron ion microsphere powder were mixed at a mass ratio of 1:9 to obtain AB type double microsphere powder that can absorb liquid in situ and self-assemble. Blood coagulation index (BCI) test was performed.

[0051] Comparative Example 1 Without adding any microsphere powder, an equal volume of fresh blood was contacted with deionized water and subjected to BCI testing under the same conditions, serving as a blank control group.

[0052] Comparative Example 2 The preparation method is basically the same as in Example 2, except that: only the chitosan polyphenol antibacterial microspheres obtained in steps (1), (2), and (6) are used and freeze-dried to obtain powder, without being combined with sodium alginate microspheres or sodium alginate iron ion microsphere powder; the obtained chitosan polyphenol antibacterial microsphere powder is used for BCI testing, and the other test conditions are the same as in Example 2.

[0053] Comparative Example 3 The preparation method is basically the same as in Example 2, except that Fe in step (7) is not performed. 2+Adsorption treatment involves directly freeze-drying the sodium alginate microspheres and chitosan polyphenol antibacterial microspheres obtained in step (6) and then mixing them in the same mass ratio to obtain Fe²-free microspheres. + Chitosan polyphenol antibacterial / sodium alginate bimicrosphere powder; the obtained powder was used for BCI testing, and the remaining conditions were the same as in Example 2.

[0054] Figure 2 The graph shows the coagulation index (BCI) test results of different microsphere powders prepared in Example 2 and Comparative Examples 1-3 (a: Comparative Example 1; b: Comparative Example 2; c: Comparative Example 3; d: Example 2). Figure 2 It can be seen that, compared with the blank control (Comparative Example 1), the single microsphere system (Comparative Example 2), or the single cross-linking system (Comparative Example 3), the AB type double microsphere powder (Example 2) containing the Schiff base reaction and metal-polyphenol coordination dual interface cross-linking network exhibits a significantly reduced BCI value, indicating that the synergistic cross-linking mechanism can effectively promote the rapid coagulation of the powder under blood contact conditions.

[0055] Example 3 A method for preparing AB-type double microsphere powder capable of in-situ liquid absorption and self-assembly includes the following steps: (1) HCS was prepared into a 2wt% aqueous solution, and MA was then slowly added dropwise to the solution for modification. The molar ratio of MA to HCS monosaccharide units was 0.6:1. The reaction was carried out at 60℃ for 2 h, and the pH of the system was maintained at 8.0 during the reaction. After the reaction was completed, the mixture was cooled to room temperature and dialyzed in deionized water to remove unreacted MA and impurities. After dialyzing, the mixture was freeze-dried to obtain HCM.

[0056] (2) Prepare an aqueous solution of HCM obtained in step (1) with a concentration of 2 wt%; add polyphenol structural units (EGCG and APBA in a molar ratio of 1:5) linked by borate ester bonds to the solution, so that the mass fraction of the polyphenol structural units is 0.1 wt%; add 2,2'-azobis[2-methyl-N-(2-hydroxyethyl)propionamide] (hereinafter referred to as VA-086) to make the mass fraction of VA-086 0.2 wt%, and add copper oxide (CuO) to make the concentration of CuO 200 μg / mL. Mix thoroughly and store in the dark to obtain a chitosan polyphenol antibacterial microsphere precursor solution.

[0057] (3) Prepare an aqueous solution of SA with a concentration of 2wt%, add sodium periodate to it, and the mass ratio of sodium alginate to sodium periodate is 10:1. React for 24 hours under light-protected conditions, then terminate the reaction, place it in deionized water for dialyzing to remove small molecule impurities, and obtain OSA. Finally, make the proportion (degree of oxidation) of monosaccharide units that produce aldehyde groups in OSA 5%.

[0058] (4) Prepare an aqueous solution of OSA with a concentration of 4 wt%, add MA (molar ratio of MA to OSA monosaccharide units of 20:1) to it, maintain the pH of the system at 8.0 during the reaction, and incubate at 10°C for 24 h. After the reaction is completed, pour the mixture into ethanol to precipitate, wash with ethanol to remove excess MA, and dry to obtain OSM.

[0059] (5) Prepare an aqueous solution of OSM obtained in step (4) with a concentration of 2wt%, and add LAP to make the mass fraction of LAP 0.2wt%. Mix thoroughly and set aside to obtain sodium alginate microsphere precursor solution.

[0060] (6) Fluid focusing microfluidic chip is used to make balls. Mineral oil containing 0.5wt% Span-80 is used as the continuous phase and the precursor solution obtained in step (2) or step (5) is used as the dispersed phase. Monodisperse droplets are formed at the fluid focusing junction. Then, ultraviolet light is used for photocuring in the flow state to obtain chitosan polyphenol antibacterial microspheres and sodium alginate microspheres with a particle size of 200μm.

[0061] (7) Soak the sodium alginate microspheres obtained in step (6) in an equal mass of FeCl2 aqueous solution with a concentration of 10 mM for 6 h for adsorption; after adsorption, wash with deionized water to obtain sodium alginate iron ion microspheres.

[0062] (8) Chitosan polyphenol antibacterial microspheres and sodium alginate iron ion microspheres were freeze-dried separately to obtain microsphere powders. The chitosan polyphenol antibacterial microsphere powder and sodium alginate iron ion microsphere powder were mixed at a mass ratio of 9:1 to obtain AB type double microsphere powder that can be self-assembled in situ by liquid absorption. The hemostatic effect of the rat femoral artery bleeding model was tested.

[0063] Comparative Example 4 Without adding any microsphere powder, the hemostatic effect of gauze alone was tested in a rat femoral artery bleeding model, serving as a blank control group.

[0064] Comparative Example 5 The preparation method is basically the same as that in Example 3, except that: only the chitosan polyphenol antibacterial microspheres obtained in steps (1), (2), and (6) are used and freeze-dried to obtain powder, without being combined with sodium alginate microspheres or sodium alginate iron ion microsphere powder; the obtained chitosan polyphenol antibacterial microsphere powder is used for hemostasis test of rat femoral artery bleeding model, and the other test conditions are the same as those in Example 3.

[0065] Comparative Example 6 The preparation method is basically the same as in Example 3, except that Fe in step (7) is not performed. 2+Adsorption treatment involves directly freeze-drying the sodium alginate microspheres and chitosan polyphenol antibacterial microspheres obtained in step (6) and then mixing them in the same mass ratio to obtain Fe²-free microspheres. + Chitosan polyphenol antibacterial / sodium alginate dual microsphere powder; the obtained powder was used for hemostasis testing in a rat femoral artery bleeding model, with the remaining conditions the same as in Example 3.

[0066] Figure 3 Figure 1 shows the hemostasis test results of a rat femoral artery bleeding model using different microsphere powders prepared in Example 3 and Comparative Examples 4-6 (a: Comparative Example 4; b: Comparative Example 5; c: Comparative Example 6; d: Example 3). Figure 3 It can be seen that, compared with the blank control (Comparative Example 4), the single microsphere system (Comparative Example 5), or the single cross-linking system (Comparative Example 6), the AB type double microsphere powder (Example 3) containing the Schiff base reaction and metal-polyphenol coordination dual interface cross-linking network, which can be self-assembled in situ by liquid absorption, showed a significant reduction in bleeding volume and hemostasis time in the rat femoral artery bleeding model. This indicates that the synergistic cross-linking mechanism can effectively promote the rapid hemostasis effect of the double powder under blood contact conditions.

[0067] Example 4 A method for preparing AB-type double microsphere powder capable of in-situ liquid absorption and self-assembly includes the following steps: (1) HCS was prepared into an aqueous solution with a concentration of 1 wt%. MA was then slowly added dropwise to the solution for modification. The molar ratio of MA to HCS monosaccharide units was 0.3:1. The reaction was carried out at 50 °C for 5 h, and the pH of the system was maintained at 8.5 during the reaction. After the reaction was completed, the mixture was cooled to room temperature and dialyzed in deionized water to remove unreacted MA and impurities. After dialyzing, the mixture was freeze-dried to obtain HCM.

[0068] (2) Prepare an aqueous solution of HCM obtained in step (1) with a concentration of 5 wt%; add polyphenol structural units (EGCG and APBA linked by borate ester bonds in a molar ratio of 5:1) to make the mass fraction of polyphenol structural units 1.0 wt%; add LAP to make the mass fraction of LAP 0.1 wt%; and add AgNPs to make the concentration of AgNPs 20 μg / mL. Mix thoroughly and store in the dark to obtain a chitosan polyphenol antibacterial microsphere precursor solution.

[0069] (3) Prepare an aqueous solution of SA with a concentration of 1 wt%, add sodium periodate to it, and the mass ratio of sodium alginate to sodium periodate is 10:3. React for 24 h under light-protected conditions, then terminate the reaction, place it in deionized water for dialyzing to remove small molecule impurities, and obtain OSA. Finally, make the proportion (degree of oxidation) of monosaccharide units that produce aldehyde groups in OSA 20%.

[0070] (4) Prepare an aqueous solution of OSA with a concentration of 2wt%, add MA (molar ratio of MA to OSA monosaccharide units of 1:1) to it, maintain the pH of the system at 7.0 during the reaction, and incubate at 0℃ for 24h. After the reaction is completed, pour the mixture into ethanol to precipitate, wash with ethanol to remove excess MA, and dry to obtain OSM.

[0071] (5) Prepare an aqueous solution of OSM obtained in step (4) with a concentration of 10 wt%, and add LAP to make the mass fraction of LAP 0.1 wt%. Mix thoroughly and set aside to obtain sodium alginate microsphere precursor solution.

[0072] (6) Fluid focusing microfluidic chip is used to make balls. Mineral oil containing 0.5wt% Span-80 is used as the continuous phase and the precursor solution obtained in step (2) or step (5) is used as the dispersed phase. Monodisperse droplets are formed at the fluid focusing junction. Then, ultraviolet light is used to cure the microspheres in the flow state to obtain chitosan polyphenol antibacterial microspheres and sodium alginate microspheres with a particle size of 500μm.

[0073] (7) Soak the sodium alginate microspheres obtained in step (6) in an equal mass of FeCl2 aqueous solution with a concentration of 2mM for 6 hours for adsorption; after adsorption, wash with deionized water to obtain sodium alginate iron ion microspheres.

[0074] (8) Chitosan polyphenol antibacterial microspheres and sodium alginate iron ion microspheres were freeze-dried separately to obtain microsphere powders. The chitosan polyphenol antibacterial microsphere powder and sodium alginate iron ion microsphere powder were mixed at a mass ratio of 1:1 to obtain AB type double microsphere powder that can be self-assembled in situ by liquid absorption.

[0075] The antibacterial effects of the AB-type dual microsphere powder prepared in Example 4, which can self-assemble in situ by liquid absorption, against *Escherichia coli* and *Staphylococcus aureus* were tested: *Escherichia coli* and *Staphylococcus aureus* were cultured separately in LB medium with shaking until the logarithmic growth phase. The bacterial solutions were diluted to 10⁻¹⁰ with physiological saline. 5 CFU / mL, take 0.01g of the AB-type dual microsphere powder prepared in Example 4, and add it to a solution containing 1mL of bacterial culture (10 5 In test tubes containing CFU / mL, and simultaneously, in the same volume of bacterial suspension (10... 5 Add an equal mass of physiological saline as a negative control to the bacterial culture (CFU / mL). Incubate the test tubes in a 37°C constant temperature shaking incubator for 30 min. After incubation, aspirate the bacterial culture from each group and dilute it 10-fold. Spread 200 μL of each solution onto a solid agar plate. Incubate the plates in a 37°C incubator for 24 hours. Observe and count the number of colonies on the plate surface.

[0076] Figure 4The image shows the antibacterial effect of the AB-type dual microsphere powder prepared in Example 4, which can self-assemble by in situ liquid absorption, against Escherichia coli and Staphylococcus aureus. Compared with the control group, the number of colonies was significantly reduced after the addition of AB-type microsphere powder, indicating that the prepared AB-type dual microsphere powder has excellent antibacterial effect against Escherichia coli and Staphylococcus aureus.

[0077] Example 5 A method for preparing AB-type double microsphere powder capable of in-situ liquid absorption and self-assembly includes the following steps: (1) HCS was prepared into an aqueous solution with a concentration of 1 wt%. MA was then slowly added dropwise to the solution for modification. The molar ratio of MA to HCS monosaccharide units was 0.6:1. The reaction was carried out at 60 °C for 8 h, and the pH of the system was maintained at 7.0 during the reaction. After the reaction was completed, the mixture was cooled to room temperature and dialyzed in deionized water to remove unreacted MA and impurities. After dialyzing, the mixture was freeze-dried to obtain HCM.

[0078] (2) Prepare an aqueous solution of HCM obtained in step (1) with a concentration of 10 wt%; add polyphenolic structural units (EGCG and APBA in a molar ratio of 1:1) linked by borate ester bonds to the solution, so that the mass fraction of the polyphenolic structural units is 1.0 wt%; add LAP to make the mass fraction of LAP 0.1 wt%; and add AgNPs to make the concentration of AgNPs 100 μg / mL. Mix thoroughly and store in the dark to obtain a chitosan polyphenol antibacterial microsphere precursor solution.

[0079] (3) Prepare an aqueous solution of SA with a concentration of 1wt%, add sodium periodate to it, and the mass ratio of sodium alginate to sodium periodate is 2:1. React for 24 hours under light-protected conditions, then terminate the reaction, place it in deionized water for dialyzing to remove small molecule impurities, and obtain OSA. Finally, the proportion of monosaccharide units that produce aldehyde groups in OSA (degree of oxidation) is 40%.

[0080] (4) Prepare an aqueous solution of OSA with a concentration of 2wt%, add MA (molar ratio of MA to OSA monosaccharide units of 10:1) to it, maintain the pH of the system at 8.0 during the reaction, and incubate at 10℃ for 24h. After the reaction is completed, pour the mixture into ethanol to precipitate, wash with ethanol to remove excess MA, and dry to obtain OSM.

[0081] (5) Prepare an aqueous solution of OSM obtained in step (4) with a concentration of 10 wt%, and add LAP to make the mass fraction of LAP 0.1 wt%. Mix thoroughly and set aside to obtain sodium alginate microsphere precursor solution.

[0082] (6) Fluid focusing microfluidic chip is used to make balls. Mineral oil containing 2.0 wt% Span-80 is used as the continuous phase and the precursor solution obtained in step (2) or step (5) is used as the dispersed phase. Monodisperse droplets are formed at the fluid focusing junction. Then, ultraviolet light is used to cure the microspheres in the flow state to obtain chitosan polyphenol antibacterial microspheres and sodium alginate microspheres with a particle size of 200 μm.

[0083] (7) Soak the sodium alginate microspheres obtained in step (6) in an equal mass of FeCl2 aqueous solution with a concentration of 10 mM for 12 h for adsorption; after adsorption, wash with deionized water to obtain sodium alginate iron ion microspheres.

[0084] (8) Chitosan polyphenol antibacterial microspheres and sodium alginate iron ion microspheres were freeze-dried separately to obtain microsphere powders. The chitosan polyphenol antibacterial microsphere powder and sodium alginate iron ion microsphere powder were mixed at a mass ratio of 1:1 to obtain AB type double microsphere powder that can be self-assembled in situ by liquid absorption.

[0085] The intracellular antioxidant regulatory effect of the AB-type dual microsphere powder prepared in Example 5, which can be self-assembled in situ by liquid absorption, was tested: Macrophages (RAW 264.7) were used based on the DCFH-DA fluorescent probe method. The probe itself is non-fluorescent; after entering the cell, it is hydrolyzed into DCFH, which is then oxidized by intracellular ROS to DCF, exhibiting strong green fluorescence. Macrophages were first treated with 10 μM H2O2 to simulate the inflammatory environment of an infected wound. Control group: only 0.01 g of PBS was added. Experimental group: 0.01 g of the AB-type dual microsphere powder prepared in Example 5 was added. After 30 min of incubation, the DCFH-DA probe was added to each group of cells, and the cells were incubated at 37°C in the dark for 30 min. The cells were then washed three times with PBS to remove any probe that had not entered the cells. Intracellular green fluorescence was observed and recorded using a fluorescence microscope.

[0086] Figure 5The images show the intracellular antioxidant regulatory effect of the AB-type dual microsphere powder prepared in Example 5, which can be self-assembled in situ by liquid absorption. These include cell morphology under bright field (top two images) and green fluorescence excitation under dark field (bottom two images). The bottom two images (green fluorescence intensity of DCF under dark field) show that the blank control group exhibits bright green fluorescence (representing high ROS levels), while the fluorescence is significantly weakened after treatment with the AB-type dual microsphere powder prepared in Example 5, indicating that the EGCG released by the microspheres effectively clears intracellular ROS. The top two images (normal bright field) show that macrophages have good morphology and distribution, serving as a control and demonstrating that the weakened fluorescence is due to reduced ROS, rather than cell death or a decrease in cell number. This indicates that the AB-type dual microsphere powder, which can be self-assembled in situ by liquid absorption, can reduce intracellular oxidative stress levels by independently loading and releasing EGCG, thereby contributing to the regulation of the inflammatory microenvironment.

[0087] The above embodiments are merely preferred embodiments of the present invention and are only used to explain the present invention, not to limit the present invention. Any changes, substitutions, modifications, etc., made by those skilled in the art without departing from the spirit and essence of the present invention should be within the protection scope of the present invention.

Claims

1. A method for preparing AB-type bimicrosphere powder capable of in-situ liquid absorption and self-assembly, characterized in that, Includes the following steps: (1) Dissolve photocrosslinkable hydroxypropyl chitosan (HCM) to obtain an aqueous solution, and add a polyphenol structural unit composed of epigallocatechin gallate (EGCG) and 3-acrylamidophenylboronic acid (APBA) linked by borate ester bonds. At the same time, add a photoinitiator and an antibacterial agent to obtain a chitosan polyphenol antibacterial microsphere precursor solution. (2) Dissolve photocrosslinkable oxidized sodium alginate OSM to obtain an aqueous solution, and add a photoinitiator to obtain a sodium alginate microsphere precursor solution; (3) The chitosan polyphenol antibacterial microsphere precursor solution and the sodium alginate microsphere precursor solution are respectively used as the dispersed phases. In a microfluidic fluid shearing chip, they are sheared by the continuous phase to form monodisperse droplets, and chitosan polyphenol antibacterial microspheres and sodium alginate microspheres are respectively obtained under ultraviolet light curing. (4) Sodium alginate microspheres were immersed in an equal mass of FeCl2 aqueous solution to adsorb Fe. 2+ Sodium alginate iron ion microspheres were obtained; (5) The chitosan polyphenol antibacterial microspheres and sodium alginate iron ion microspheres were freeze-dried separately to obtain powders, and then mixed to obtain AB type double microsphere powder that can be self-assembled in situ by absorbing liquid.

2. The method for preparing AB-type bimicrosphere powder capable of in-situ liquid absorption and self-assembly according to claim 1, characterized in that, In steps (1) and (2), the photoinitiator is one of lithium phenyl-2,4,6-trimethylbenzoylphosphonate LAP, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone L2959, or 2,2'-azobis[2-methyl-N-(2-hydroxyethyl)propionamide]VA-086; in step (1), the antibacterial agent is one of nano silver AgNPs, zinc oxide ZnO, or copper oxide CuO.

3. The method for preparing AB-type bimicrosphere powder capable of in-situ liquid absorption and self-assembly according to claim 1, characterized in that, In step (1), the preparation method of the HCM is as follows: Hydroxypropyl chitosan (HCS) was prepared into an aqueous solution with a concentration of 0.5-2 wt%. HCS was modified with methacrylic anhydride (MA), with a molar ratio of MA to HCS monosaccharide units of 0.1:1-0.6:

1. The modification conditions were: pH 7.0-8.5, temperature 40-60℃, and reaction time 2-8 h. After modification, the mixture was cooled, dialyzed, and freeze-dried to obtain HCM.

4. The method for preparing AB-type bimicrosphere powder capable of in-situ liquid absorption and self-assembly according to claim 1, characterized in that, In step (2), the OSM preparation method is as follows: Sodium alginate was prepared into an aqueous solution with a concentration of 0.5-2 wt%. Sodium periodate was added to the solution at a mass ratio of sodium alginate to sodium periodate of 10:1-2:

1. The reaction was carried out under light-protected conditions for 12-24 hours. The reaction was then terminated and dialyzed to obtain oxidized sodium alginate OSA. The proportion of monosaccharide units in OSA that oxidized to produce aldehyde groups, i.e., the degree of oxidation, was 5-40%. OSA was prepared into an aqueous solution with a concentration of 0.5-4 wt%. OSA was modified with methacrylic anhydride, and the molar ratio of methacrylic anhydride to OSA monosaccharide units was 1:1-20:

1. The modification conditions were: pH 7.0-8.5, temperature 0-15℃, and incubation for 12-24 h. After the reaction was completed, the OSA was precipitated with ethanol, washed with ethanol, and dried to obtain OSM.

5. The method for preparing AB-type bimicrosphere powder capable of in-situ liquid absorption and self-assembly according to claim 1, characterized in that, In step (1), HCM is dissolved in water to prepare an aqueous solution with a concentration of 2-10 wt%, the molar ratio of EGCG to APBA is 1:5-5:1, the mass fraction of polyphenol structural units is 0.1-1 wt%, the mass fraction of photoinitiator is 0.02-0.2 wt%, and the concentration of antibacterial agent is 20-200 μg / mL; in step (2), OSM is dissolved in water to prepare an aqueous solution with a concentration of 2-20 wt%, and the mass fraction of photoinitiator is 0.02-0.2 wt%.

6. The method for preparing AB-type bimicrosphere powder capable of in-situ liquid absorption and self-assembly according to claim 1, characterized in that, The continuous phase in step (3) is mineral oil containing 0.5-2.0 wt% Span-80; the particle size of the chitosan polyphenol antibacterial microspheres or sodium alginate microspheres is 50-500 μm.

7. The method for preparing an AB-type double microsphere powder capable of in-situ liquid absorption and self-assembly according to claim 1, characterized in that, The concentration of the FeCl2 aqueous solution in step (4) is 2-20 mM, and the adsorption time is 0.5-12 h.

8. The method for preparing AB-type bimicrosphere powder capable of in-situ liquid absorption and self-assembly according to claim 1, characterized in that, The mass ratio of chitosan polyphenol antibacterial microsphere powder to sodium alginate iron ion microsphere powder in step (5) is 1:9-9:

1.

9. An AB-type bimicrosphere powder capable of in-situ liquid absorption and self-assembly, prepared by the method according to any one of claims 1-8, characterized in that, Composed of chitosan polyphenol antibacterial microspheres and sodium alginate iron ion microspheres, wherein the chitosan polyphenol antibacterial microspheres are chitosan hydrogel microspheres containing carbon-carbon double bonds, amino groups, and polyphenol structural units and loaded with silver nanoparticles; the sodium alginate iron ion microspheres contain carbon-carbon double bonds and aldehyde groups and adsorb Fe 2+ Sodium alginate hydrogel microspheres.

10. The application of the AB-type dual microsphere powder of claim 9, which can self-assemble in situ absorb liquid, in the preparation of antibacterial and anti-inflammatory products for rapid hemostasis and repair of infected wounds.

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