Porous composite hemostatic sponge loaded with hemostatic drug-loading microspheres and preparation method of porous composite hemostatic sponge

Abstract

The invention discloses a porous composite hemostatic sponge loaded with hemostatic drug-loading microspheres and a preparation method of the porous composite hemostatic sponge, and belongs to the technical field of biological medicines. According to the porous composite hemostatic sponge, the liquid absorption, hemostasis and antibacterial effects of a product can be remarkably improved. After a cation solution, hemostatic drug-loaded microspheres and a sponge matrix precursor solution are mixed, the hemostatic drug-loaded microspheres are dispersed and tightly combined with a sponge matrix through the synergistic effect of electrostatic interaction and coordination among microsphere surface groups, matrix components and cations, and the structural stability of the hemostatic drug-loaded microspheres can be maintained in the sponge liquid absorption process; and the continuity of the liquid absorption performance is ensured. Finally, synergistic interaction of hemostasis, bacteriostasis, anti-inflammation and tissue repair is achieved, and the porous composite hemostatic sponge with rapid hemostasis, efficient bacteriostasis and auxiliary tissue healing promoting functions is obtained.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a porous composite hemostatic sponge loaded with hemostatic drug-loaded microspheres and a preparation method thereof. BACKGROUND

[0002] Timely hemostasis and healing intervention for traumatic wounds are of great significance for shortening the treatment cycle, reducing the risk of infection, and reducing scar formation. The current hemostatic methods for traumatic wounds mainly include compression hemostasis and drug hemostasis: compression hemostasis is suitable for superficial and small blood vessel rupture of body surface trauma, and can quickly reduce the amount of bleeding, but the effect of this method on patients with low self-coagulation function is poor. Drug hemostasis is achieved by applying hemostatic powder, hemostatic sponge and hemostatic gel products. However, the existing hemostatic particles and hemostatic sponges generally have the problems of single function and short action time, which are difficult to meet the comprehensive needs of wound healing.

[0003] In order to improve the shortcomings of traditional hemostatic products, Medtrade company launched Celox Rapid, which uses polyurethane sponge as a physical support framework, and embeds micrometer-sized chitosan hemostatic particles into the gap between gauze fibers to form an initial blood clot by promoting the aggregation of red blood cells to play a hemostatic role. However, this product has the problem of uneven pore size distribution, and the combination stability of the particles and the sponge matrix is insufficient, which is easy to cause the phenomenon of particle blocking the sponge pores, which not only weakens the hemostatic effect, but also has an adverse effect on the process of tissue regeneration. QuickClot Combat Gauze developed by Teleflex company is a gauze sponge containing kaolin particles, which mainly plays a hemostatic role through kaolin particles, but this product cannot be absorbed by the human body, and needs to be frequently replaced during use; the replacement is easy to pull the newly formed granulation tissue of the wound, causing repeated mechanical damage.

[0004] Therefore, it is a technical problem to be solved to develop a new type of hemostatic product with good biocompatibility and excellent hemostatic and healing effects. SUMMARY

[0005] In view of the problems of single function, poor healing effect, uneven pore size distribution and unstable combination of microspheres and sponge matrix in the prior art, the application provides a porous composite hemostatic sponge loaded with hemostatic drug-loaded microspheres and a preparation method thereof.

[0006] To achieve the above purpose, the application adopts the following technical solutions: The present application provides a kind of porous composite hemostatic sponge, the porous composite hemostatic sponge composition includes hemostatic drug-loaded microspheres, cation solution and sponge matrix precursor solution, the hemostatic drug-loaded microspheres are antibacterial drugs as core, shell is formed by shell layer solution solidification, wherein shell layer solution composition includes collagen, polyol, mussel mucin and metal ion.

[0007] Preferably, the polyol is selected from at least one of glycerol and propylene glycol.

[0008] Preferably, the preparation method of the hemostatic drug-loaded microspheres is as follows: mixing collagen with polyol to obtain a mixed solution A; mixing mussel mucin with an inorganic salt solution containing metal ions, adjusting the pH to 5.0-6.0 to obtain a mixed solution B; mixing the mixed solution A and the mixed solution B at a volume ratio of 1:1 to obtain a shell layer solution; mixing the shell layer solution with an antibacterial drug solution, spray drying, and then vacuum heat crosslinking to obtain the hemostatic drug-loaded microspheres.

[0009] Preferably, in the hemostatic drug-loaded microspheres, the antibacterial drug is 0.5 wt%-2.5 wt%, the collagen is 2.0 wt%-5.0 wt%, the polyol is 1.0 wt%-5.0 wt%, the mussel mucin is 0.5 wt%-1.0 wt%, and the molar concentration of the metal ion in the hemostatic drug-loaded microspheres is 0.2 mmol / L-2.0 mmol / L.

[0010] Preferably, in the hemostatic drug-loaded microspheres, the metal ion is any one of Ca 2+ , Zn 2+ and Mg 2+ .

[0011] Preferably, in the hemostatic drug-loaded microspheres, when the metal ion is Ca 2+ , the molar concentration is 0.5 mmol / L-1 mmol / L.

[0012] Preferably, in the hemostatic drug-loaded microspheres, when the metal ion is Zn 2+ , the molar concentration is 0.2 mmol / L-0.5 mmol / L.

[0013] Preferably, in the hemostatic drug-loaded microspheres, when the metal ion is Mg 2+ , the molar concentration is 1.0 mmol / L-2.0 mmol / L.

[0014] The coordination crosslinking of the metal ion with collagen and mussel mucin can enhance the stability of the shell structure. In addition, during the preparation of the hemostatic drug-loaded microspheres, Ca 2+The metal ion can form an ion bond with the carboxyl group in the protein, improving the anti-swelling ability of the shell structure in the body fluid environment; when preparing the hemostatic drug-loaded microspheres, Zn 2+ The mussel adhesive protein can synergistically enhance the antibacterial properties of the microspheres; when preparing the hemostatic drug-loaded microspheres, Mg 2+ The mussel adhesive protein can enhance the adhesion performance of the mussel adhesive protein while enhancing the elasticity of the shell structure.

[0015] The hemostatic drug-loaded microspheres provided by the application realize efficient and stable shell structure through the dual regulation of synergistic effect of multiple components and process control. On the one hand, after the collagen protein is mixed with glycerol (propylene glycol or a combination of glycerol and propylene glycol), the glycerol (propylene glycol or the combination of glycerol and propylene glycol) acts as a plasticizer and can form new intermolecular hydrogen bonds with the hydroxyl groups of the collagen protein molecules, weaken the original intermolecular hydrogen bond effect, and increase the flowability of the collagen protein molecular chain, thereby significantly enhancing the flexibility of the shell structure. On the other hand, the ortho-diphenol hydroxyl groups of the dopa groups in the mussel adhesive protein can be tightly combined with the amino groups and hydroxyl groups of the collagen protein through strong hydrogen bond effect, significantly reducing the intermolecular gap and improving the compactness of the shell structure, thereby avoiding the problem of easy deformation caused by loose structure. In addition, the metal ion can form stable coordination bonds with the carboxyl groups and hydroxyl groups of the collagen protein and the mussel adhesive protein in the shell, significantly improving the anti-stress deformation ability of the shell structure, and further strengthening the overall stability of the shell on the basis of the hydrogen bond effect.

[0016] Preferably, the process parameters of the spray drying are as follows: the feeding temperature is 200 ℃-240 ℃, the feeding rate is 15 rpm-20 rpm, and the compressed air volume flow rate is 8 L / min-10 L / min; the temperature of the vacuum thermal crosslinking is 120 ℃-150 ℃, and the crosslinking time is 12 h-24 h.

[0017] After the shell solution is mixed with the antibacterial drug solution, the process parameters in the spray drying step are adjusted to obtain hemostatic drug-loaded microspheres with relatively uniform particle size. The vacuum thermal crosslinking process can promote the formation of covalent crosslinking between collagen protein molecules and strengthen the mussel adhesive protein-metal ion network, and the double solidification mechanism significantly improves the anti-swelling ability and stability of the hemostatic drug-loaded microspheres in the body fluid environment, ensuring the stable release of the antibacterial drug in the microspheres in the later stage.

[0018] Preferably, the antibacterial drug is any one or a combination of the fluoroquinolone antibacterial drugs.

[0019] Further preferably, the antibacterial drug is any one or a combination of ciprofloxacin hydrochloride, levofloxacin, moxifloxacin, and norfloxacin.

[0020] The antibacterial drugs selected in this scheme exist in cationic form under neutral to weakly acidic conditions. They can bind to proteins in the shell matrix through electrostatic interactions, thereby enhancing the stability of the core-shell structure of the hemostatic drug-loaded microspheres.

[0021] This invention also provides a method for preparing a porous composite hemostatic sponge, comprising the following steps: S1, after dispersing hemostatic drug-loaded microspheres in a cationic solution at room temperature, the pH is adjusted to 6.5–7.5 to obtain a hemostatic drug-loaded microsphere-cationic suspension; wherein, the cationic solution is composed of Zn 2+ Ca 2+ Cu 2+ Fe 3+ And Al 3+ At least one of the components; S2, mix collagen solution, elastin solution and alginate solution to obtain sponge matrix precursor solution; S3, mix the hemostatic drug-loaded microsphere-cationic suspension with the sponge matrix precursor solution to obtain the composite precursor solution; S4. After the composite precursor solution is directionally frozen, it is then subjected to vacuum thermal crosslinking to obtain a porous composite hemostatic sponge.

[0022] Preferably, in S1, the mass-to-volume ratio of the hemostatic drug-loaded microspheres to the cationic solution is 1:(5-20) mg / mL.

[0023] By adjusting this ratio, the dispersion concentration of hemostatic drug-loaded microspheres in the cationic solution can be controlled, avoiding the phenomenon that microspheres aggregate and block the sponge pores due to excessive concentration, or that microspheres do not bind sufficiently to the sponge matrix due to insufficient concentration.

[0024] Preferably, the total cation concentration in the cation solution is 20 mmol / L.

[0025] Preferably, the Zn 2+ The molar concentration of the substance in the cation solution is 5 mmol / L to 20 mmol / L; the Fe 3 + Or Al 3+ The molar concentration of the cation in the solution is 0.1 mmol / L to 5 mmol / L; the Cu 2+ or Ca 2+ The molar concentration in the cation solution is 0.1 mmol / L to 8 mmol / L.

[0026] The dopa groups contained in the mussel adhesive protein on the surface of the hemostatic drug-loaded microspheres synergistically interact with the active groups (hydroxyl, carboxyl, etc.) of the matrix components (collagen, elastin, alginate, etc.) in the sponge matrix precursor solution. Through the formation of dynamic reversible coordination bonds with cations in the system, a stable "hemostatic drug-loaded microsphere-cation-sponge matrix" binding system is constructed, thereby dispersing and firmly anchoring the drug-loaded microspheres within the sponge matrix structure. On the one hand, by enhancing the binding stability between the drug-loaded microspheres and the sponge matrix, structural collapse or deformation caused by water absorption and swelling during blood absorption can be effectively avoided, ensuring the continuity of its absorbency and providing structural support for efficient hemostasis. On the other hand, the tight binding between the drug-loaded microspheres and the sponge matrix reduces the shedding of the microspheres during hemostasis, ensuring that the loaded antibacterial drugs can exert their effects precisely on the wound surface.

[0027] In addition, the cations (Ca) in the system 2+ Zn 2+ Cu 2+ Fe 3+ They can also synergistically enhance product efficacy through their respective bioactive mechanisms: Ca 2+ It can activate clotting factors and promote platelet activation, providing core support for the initiation and advancement of the coagulation process; Zn 2+ With Cu 2+ It can exert a potent antibacterial effect by disrupting the integrity of bacterial cell membranes, inhibiting bacterial DNA replication and metabolic enzyme activity; Fe 3+ It can help activate prothrombin and inhibit the release of inflammatory factors by regulating macrophage polarization, while Zn 2+ With Cu 2+ It can further reduce wound inflammation by scavenging reactive oxygen species and blocking inflammatory signaling pathways. Ultimately, through the synergistic effect of these multiple actions, the product combines highly effective hemostasis, long-lasting antibacterial properties, and adjuvant anti-inflammatory effects.

[0028] Preferably, in S1, the hemostatic drug-loaded microspheres are dispersed in the cationic solution at a stirring speed of 100 rpm to 200 rpm.

[0029] Preferably, in S2, the sponge matrix precursor solution is obtained by mixing 0.1 wt% to 1.0 wt% of an elastin solution, 1.0 wt% to 2.5 wt% of a collagen solution, and 0.1 wt% to 0.5 wt% of an alginate solution.

[0030] Preferably, the mass ratio of elastin to collagen is 3:7 to 1:9, and the mass ratio of alginate to collagen is 1:10 to 1:5.

[0031] By limiting the mass ratio of the three components (elastin, collagen, and alginate) in the sponge matrix, it is possible to ensure that the sponge matrix has good support properties while also possessing a certain degree of flexibility. At the same time, this ratio can promote the formation of a three-dimensional cross-linked network in the sponge matrix. This structure not only enhances the hydrophilicity of the matrix to promote blood absorption, but also avoids pore shrinkage caused by excessive gelation, thus laying a good foundation for the subsequent loading of hemostatic microspheres and blood absorption during the hemostasis process.

[0032] Preferably, in S3, the hemostatic drug-loaded microsphere-cationic suspension and the sponge matrix precursor solution are mixed at a volume ratio of 1:1.

[0033] This ratio ensures sufficient contact between the hemostatic drug-loaded microspheres and the sponge matrix precursor solution, maintaining uniform dispersion of the microspheres and preventing the formation of ineffective "dead spaces" due to excessive accumulation. Furthermore, by adjusting the volume ratio, a triple stabilizing effect of "charge-coordination-space" can be achieved. Specifically, under pH conditions of 6.5–7.5 in the mixed system, the hemostatic drug-loaded microspheres carry a positive charge, which causes electrostatic repulsion, inhibiting aggregation. Additionally, the dopa groups can form reversible coordination bonds with metal ions, as well as collagen, elastin, and alginate in the sponge matrix precursor solution. This coordination buffers the interference of external ionic strength changes on the system, further inhibiting microsphere aggregation. Simultaneously, the steric hindrance generated by the microspheres' own structure, synergistically with the above coordination effect, significantly enhances the kinetic stability of the microsphere dispersion system. This effect allows microspheres loaded with antibacterial drugs to be stably dispersed and bound in the sponge matrix precursor. After solidification, the microspheres in the sponge matrix can reduce structural shrinkage during liquid absorption by filling pores. On the other hand, based on the coordination between the mussel adhesive protein on its surface and the matrix components, it can help enhance the compactness and elasticity of the matrix cross-linking network. Thus, it can effectively maintain structural stability, inhibit collapse, and ensure the continuity of liquid absorption performance during sponge liquid absorption.

[0034] Preferably, in S4, the directional freezing includes: placing the composite precursor solution in a mold, completely covering the upper and lower surfaces of the mold with insulating material; first freezing the mold at -80 ℃ for 3 to 5 hours, and then freezing it at -50 ℃ to -40 ℃ for 36 to 48 hours.

[0035] Temperature gradient changes can effectively drive the directional growth of ice crystals, forming an orderly and well-connected pore structure, which is beneficial for subsequent cell proliferation and tissue regeneration, and can also significantly improve the liquid absorption capacity of the sponge matrix.

[0036] Preferably, the vacuum thermal crosslinking temperature is 120 ℃~150 ℃ and the time is 12 h~24 h.

[0037] The porous composite hemostatic sponge prepared in this invention has a pore size of 50 μm to 200 μm. This pore size range is adapted to the passage requirements of red blood cells, which can accelerate blood penetration by relying on the high connectivity of the pores and the hydrophilicity of the matrix, while reducing the risk of pore blockage, thus achieving a balance between absorption rate and liquid retention. Therefore, the product prepared in this invention has strong liquid absorption capacity and can provide sufficient specific surface area to promote platelet adhesion, thereby achieving rapid initiation of the coagulation mechanism.

[0038] Preferably, the collagen, mussel adhesive protein, and elastin can be derived from natural sources or obtained through genetic engineering recombination.

[0039] Compared with the prior art, the present invention has the following beneficial effects: The porous composite hemostatic sponge prepared by this invention significantly improves the product's hemostatic and antibacterial effects. After mixing a cationic solution, hemostatic drug-loaded microspheres, and a sponge matrix precursor solution, the synergistic effects of electrostatic and coordination interactions between microsphere surface groups, matrix components, and cations achieve dispersion of the hemostatic drug-loaded microspheres and tight bonding with the sponge matrix. Furthermore, it maintains structural stability during sponge absorption, ensuring continuous absorption performance. In addition, the collagen, mussel adhesive protein, and metal ions (such as CaO) in the microsphere shell... 2+ Zn 2+ Cu 2+ Fe 3+ Mg 2+ The collagen and elastin in the sponge matrix can synergistically play a role in assisting tissue repair, hemostasis, and antibacterial action; the anti-inflammatory drugs loaded in the microsphere core can significantly enhance the anti-inflammatory effect of the product and further improve its anti-inflammatory and antibacterial properties. Ultimately, this achieves a synergistic effect of hemostasis, antibacterial and anti-inflammatory action, and tissue repair, resulting in a porous composite hemostatic sponge that combines rapid hemostasis, highly efficient antibacterial action, and tissue healing assistance. Attached Figure Description

[0040] Figure 1 This is a diagram showing the reaction phenomenon of flocculent formation during the preparation of microspheres in Comparative Example 4 of this invention; Figure 2 This is a scanning electron microscope image of the hemostatic drug-loaded microspheres prepared in Example 1 of the present invention; Figure 3 The images show cross-sectional scanning electron microscope images of the sponge matrix of the present invention (a is the sponge matrix prepared in Example 1; b is the sponge matrix prepared in Comparative Example 12). Figure 4 The images shown are scanning electron microscope images of the porous composite hemostatic sponge of the present invention (a is the porous composite hemostatic sponge prepared in Example 1; b is the porous composite hemostatic sponge prepared in Comparative Example 11). Figure 5The porous composite hemostatic sponge prepared in Example 1 and Comparative Example 13 of this invention S. aureus and E. coli Plate antibacterial graph (A is) S. aureus Plate antibacterial graph; B represents... E. coli (Platelet antibacterial diagram). Detailed Implementation

[0041] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0042] Unless otherwise specified, wt% in this invention refers to mass percentage; "room temperature" refers to 10-30℃.

[0043] The present invention will now be described in further detail with reference to the accompanying drawings: Example 1 This embodiment provides a porous composite hemostatic sponge, and the specific preparation steps are as follows: S1: Hemostatic drug-loaded microspheres were dispersed in a cationic solution at a mass-to-volume ratio of 1:8 mg / mL. The pH of the system was adjusted to 7.0, and the mixture was stirred at 100 rpm at room temperature until the microspheres were uniformly dispersed, resulting in a hemostatic drug-loaded microsphere-cationic suspension. The cationic solution was a 1:1 volume mixture of 15 mmol / L ZnSO4•7H2O and 5 mmol / L AlCl3•6H2O solutions.

[0044] S2: Mix 1.5 wt% collagen solution, 0.3 wt% elastin solution and 0.24 wt% sodium alginate solution evenly to obtain a sponge matrix precursor solution, wherein the mass ratio of elastin to collagen is 1:5 and the mass ratio of sodium alginate to collagen is 4:25.

[0045] S3: The hemostatic drug-loaded microsphere-cationic suspension and the sponge matrix precursor solution were mixed evenly at a volume ratio of 1:1 to obtain a composite precursor solution. The solution was then filled into a mold, and the mold was completely covered with insulation material. The mold was frozen at -80 ℃ for 4 h, then transferred to -45 ℃ for 42 h, and then subjected to vacuum thermal crosslinking treatment at 150 ℃ for 16 h to obtain a porous composite hemostatic sponge. The pore size of the prepared porous composite hemostatic sponge was 150 μm, as shown by scanning electron microscopy.

[0046] The hemostatic drug-loaded microspheres in this embodiment consist of: 2.0 wt% glycerol, 3.5 wt% collagen, 0.7 wt% mussel adhesive protein, 0.6 mmol / L CaCl2, and 2.5 wt% antibacterial drug.

[0047] The method for preparing hemostatic drug-loaded microspheres in this embodiment is as follows: (1) Add glycerol to the collagen solution and mix well to form solution A.

[0048] (2) Add mussel adhesive protein to calcium chloride solution and adjust the pH of the system to 5.5 to obtain mixed solution B.

[0049] (3) Mix solution A and solution B at a volume ratio of 1:1 to obtain a shell solution.

[0050] (4) The shell solution and antibacterial drugs (ciprofloxacin hydrochloride, levofloxacin, moxifloxacin, norfloxacin, or any combination thereof) are dispersed evenly at room temperature, spray-dried, and then transferred to a vacuum drying oven for vacuum thermal crosslinking to obtain hemostatic drug-loaded microspheres. The spray drying process parameters are as follows: a spray drying device with a nozzle diameter of 0.5 mm is used, the feed temperature is set to 220 ℃, the feed rate is 18 rpm, and the compressed air speed is 8 L / min; the vacuum thermal crosslinking process parameters are as follows: the temperature is set to 150 ℃ and the time is 16 h.

[0051] Example 2 This embodiment provides a porous composite hemostatic sponge, and the specific preparation steps are as follows: S1: Hemostatic drug-loaded microspheres were dispersed in a cationic solution at a mass-to-volume ratio of 1:20 mg / mL. The pH of the system was adjusted to 7.5, and the mixture was stirred at 200 rpm at room temperature until the microspheres were uniformly dispersed, resulting in a hemostatic drug-loaded microsphere-cationic suspension. The cationic solution was a 20 mmol / L ZnSO4•7H2O solution.

[0052] S2: Mix 1.0 wt% collagen solution, 0.1 wt% elastin solution and 0.1 wt% potassium alginate solution evenly to obtain a sponge matrix precursor solution, wherein the mass ratio of elastin to collagen is 1:9 and the mass ratio of sodium alginate to collagen is 1:10.

[0053] S3: The hemostatic drug-loaded microsphere-cationic suspension and the sponge matrix precursor solution were mixed evenly at a volume ratio of 1:1 to obtain a composite precursor solution. The solution was then filled into a mold, and the mold was completely covered with thermal insulation material. The mold was then frozen at -80 ℃ for 3 h and then transferred to -45 ℃ for 36 h. Finally, it was subjected to vacuum thermal crosslinking treatment at 120 ℃ for 12 h to obtain a porous composite hemostatic sponge. The pore size of the prepared porous composite hemostatic sponge was 200 μm, as shown by scanning electron microscopy.

[0054] The hemostatic drug-loaded microspheres in this embodiment consist of: 1.0 wt% propylene glycol, 2.0 wt% collagen, 0.5 wt% mussel adhesive protein, 0.5 mmol / L CaCl2 (or 0.2 mmol / L zinc sulfate or 1.0 mmol / L magnesium chloride), and 0.5 wt% antibacterial drug.

[0055] The method for preparing hemostatic drug-loaded microspheres in this embodiment is as follows: (1) Add propylene glycol to the collagen solution and mix well to form solution A.

[0056] (2) Add mussel adhesive protein to calcium chloride solution (zinc sulfate or magnesium chloride), adjust the pH of the system to 5.0, and obtain mixed solution B.

[0057] (3) Mix solution A and solution B at a volume ratio of 1:1 to obtain a shell solution.

[0058] (4) The shell solution and antibacterial drugs (ciprofloxacin hydrochloride, levofloxacin, moxifloxacin, norfloxacin, or any combination thereof) are dispersed evenly at room temperature, spray-dried, and then transferred to a vacuum drying oven for vacuum thermal crosslinking to obtain hemostatic drug-loaded microspheres. The spray drying process parameters are as follows: a spray drying device with a nozzle diameter of 0.5 mm is used, the feed temperature is set to 200 ℃, the feed rate is 15 rpm, and the compressed air speed is 8 L / min; the vacuum thermal crosslinking process parameters are as follows: the temperature is set to 120 ℃ and the time is 12 h.

[0059] Example 3 This embodiment provides a porous composite hemostatic sponge, and the specific preparation steps are as follows: S1: Hemostatic drug-loaded microspheres were dispersed in a cationic solution at a mass-to-volume ratio of 1:5 mg / mL. The pH of the system was adjusted to 7.5, and the mixture was stirred at 200 rpm at room temperature until the microspheres were uniformly dispersed, resulting in a hemostatic drug-loaded microsphere-cationic suspension. The cationic solution was prepared by mixing 7 mmol / L ZnSO4•7H2O, 8 mmol / L CuSO4, and 5 mmol / L AlCl3·6H2O solutions in a volume ratio of 1:1:1.

[0060] S2: Mix 2.5 wt% collagen solution, 1.0 wt% elastin solution and 0.5 wt% sodium alginate solution evenly to obtain a sponge matrix precursor solution, wherein the mass ratio of elastin to collagen is 3:7 and the mass ratio of sodium alginate to collagen is 1:5.

[0061] S3: The hemostatic drug-loaded microsphere-cationic suspension and the sponge matrix precursor solution were mixed evenly at a volume ratio of 1:1 to obtain a composite precursor solution. The solution was then filled into a mold, and the mold was completely covered with insulation material. The mold was frozen at -80 ℃ for 5 h, then transferred to -45 ℃ for 48 h, and then transferred to a vacuum drying oven for vacuum thermal crosslinking at 150 ℃ for 24 h. Finally, a porous composite hemostatic sponge was obtained. The pore size of the prepared porous composite hemostatic sponge was 80 μm, as shown by scanning electron microscopy.

[0062] The hemostatic drug-loaded microspheres in this embodiment consist of: 2.0 wt% glycerol, 3.0 wt% propylene glycol, 5.0 wt% collagen, 1.0 wt% mussel adhesive protein, 1 mmol / L CaCl2 (or 0.5 mmol / L zinc sulfate or 2.0 mmol / L magnesium chloride), and 2.5 wt% antibacterial drug.

[0063] The method for preparing hemostatic drug-loaded microspheres in this embodiment is as follows: (1) Add glycerol and propylene glycol to the collagen solution and mix well to form solution A.

[0064] (2) Add mussel adhesive protein to calcium chloride solution and adjust the pH of the system to 6.0 to obtain mixed solution B.

[0065] (3) Mix solution A and solution B at a volume ratio of 1:1 to obtain a shell solution.

[0066] (4) The shell solution and antibacterial drugs (ciprofloxacin hydrochloride, levofloxacin, moxifloxacin, norfloxacin, or any combination thereof) are dispersed evenly at room temperature, spray-dried, and then transferred to a vacuum drying oven for vacuum thermal crosslinking to obtain hemostatic drug-loaded microspheres. The spray drying process parameters are as follows: a spray drying device with a nozzle diameter of 0.5 mm is used, the feed temperature is set at 240 ℃, the feed rate is 20 rpm, and the compressed air speed is 10 L / min; the vacuum thermal crosslinking process parameters are as follows: the temperature is set at 150 ℃ and the time is 24 h.

[0067] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that the hemostatic drug-loaded microspheres in Comparative Example 1 contain 0.5 wt% glycerol, while the content of the remaining components and the preparation method are the same as in Example 1.

[0068] Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that the hemostatic drug-loaded microspheres in Comparative Example 2 contain 7 wt% glycerol, while the content of the remaining components and the preparation method are the same as in Example 1.

[0069] Comparative Example 3 The difference between Comparative Example 3 and Example 1 is that the CaCl2 content in the hemostatic drug-loaded microspheres in Comparative Example 3 is 0.2 mmol / L, while the content of the remaining components and the preparation method are the same as in Example 1.

[0070] Comparative Example 4 The difference between Comparative Example 4 and Example 1 is that the CaCl2 content in the hemostatic drug-loaded microspheres of Comparative Example 4 is 1.5 mmol / L, while the content of other components and the preparation method are the same as in Example 1. During the preparation process, it was found that the excessively high CaCl2 content led to the formation of flocculent complexes in the system. Figure 1 Therefore, Comparative Example 4 could not prepare hemostatic drug-loaded microspheres.

[0071] Comparative Example 5 The difference between Comparative Example 5 and Example 1 is that in step S2 of preparing the porous composite hemostatic sponge in Comparative Example 5, the elastin content is 0.05 wt%, and the ratio of elastin to collagen is 1:30. The content of the remaining components and the preparation method are the same as in Example 1.

[0072] Comparative Example 6 The difference between Comparative Example 6 and Example 1 is that in step S2 of preparing the porous composite hemostatic sponge loaded with hemostatic drug-loaded microspheres in Comparative Example 6, the elastin content is 1.5 wt%, wherein the mass ratio of elastin to collagen is 1:1, and the content and preparation method of the remaining components are the same as those in Example 1.

[0073] Comparative Example 7 The difference between Comparative Example 7 and Example 1 is that in step S2 of preparing the porous composite hemostatic sponge loaded with hemostatic drug-loaded microspheres in Comparative Example 7, sodium alginate is 0.05 wt%, and the mass ratio of sodium alginate to collagen protein is 1:30. The content of the remaining components and the preparation method are the same as in Example 1.

[0074] Comparative Example 8 The difference between Comparative Example 8 and Example 1 is that in step S2 of preparing the porous composite hemostatic sponge loaded with hemostatic drug-loaded microspheres in Comparative Example 8, sodium alginate is 1.0 wt%, wherein the mass ratio of sodium alginate to collagen protein is 2:3, and the content and preparation method of the remaining components are the same as those in Example 1.

[0075] Comparative Example 9 The difference between Comparative Example 9 and Example 1 is that in step S1 of preparing the porous composite hemostatic sponge, the hemostatic drug-loaded microspheres are dispersed in a cationic solution at a mass-to-volume ratio of 1:25 mg / mL, while the content of the remaining components and the preparation method are the same as in Example 1.

[0076] Comparative Example 10 The difference between Comparative Example 10 and Example 1 is that in step S1 of preparing the porous composite hemostatic sponge in Comparative Example 10, the hemostatic drug-loaded microspheres are dispersed in a cationic solution at a mass-to-volume ratio of 1:3 mg / mL, while the content of the remaining components and the preparation method are the same as in Example 1.

[0077] Comparative Example 11 The difference between Comparative Example 11 and Example 1 is that in Comparative Example 11, hemostatic drug-loaded microspheres are directly loaded onto a porous composite hemostatic sponge. The specific operation steps are as follows: S1: Mix 1.5 wt% collagen solution, 0.3 wt% elastin solution and 0.24 wt% sodium alginate solution evenly to obtain a sponge matrix precursor solution, wherein the mass ratio of elastin to collagen is 1:5 and the mass ratio of sodium alginate to collagen is 4:25.

[0078] S2: Disperse the hemostatic drug-loaded microspheres in the sponge matrix precursor solution at a mass-to-volume ratio of 1:8 mg / mL, and stir at 100 rpm at room temperature until the microspheres are uniformly dispersed to obtain the composite precursor solution.

[0079] S3: The composite precursor solution is then filled into the mold, and the mold is completely covered with insulation material. After freezing in a -80℃ freezer for 4 hours, it is transferred to a -45℃ freezer for 42 hours. Then it is transferred to a vacuum drying oven for vacuum thermal crosslinking at 150℃ for 16 hours, finally obtaining a porous composite hemostatic sponge loaded with hemostatic drug-loaded microspheres.

[0080] The composition, component ratio, and preparation method of the hemostatic drug-loaded microspheres in Comparative Example 11 were the same as those in Example 1.

[0081] Comparative Example 12 The difference between Comparative Example 11 and Example 1 is that Comparative Example 12 uses direct freeze-drying to prepare the porous composite hemostatic sponge. The specific operation steps are as follows: S1: Hemostatic drug-loaded microspheres were dispersed in a cationic solution at a mass-to-volume ratio of 1:8 mg / mL. The pH of the system was adjusted to 7.0, and the mixture was stirred at 100 rpm at room temperature until the microspheres were uniformly dispersed, resulting in a hemostatic drug-loaded microsphere-cationic suspension. The cationic solution was a 1:1 volume mixture of 15 mmol / L ZnSO4•7H2O and 5 mmol / L AlCl3•6H2O solutions.

[0082] S2: Mix 1.5 wt% collagen solution, 0.3 wt% elastin solution and 0.24 wt% sodium alginate solution evenly to obtain a sponge matrix precursor solution, wherein the mass ratio of elastin to collagen is 1:5 and the mass ratio of sodium alginate to collagen is 4:25.

[0083] S3: The hemostatic drug-loaded microsphere-cationic suspension and the sponge matrix precursor solution were mixed uniformly at a volume ratio of 1:1 to obtain a composite precursor solution. This solution was then filled into a mold and frozen at -45℃ for 46 hours. Afterward, it was transferred to a vacuum drying oven for vacuum thermal crosslinking at 150℃ for 16 hours, finally obtaining a porous composite hemostatic sponge. Testing showed that the pore size of the prepared porous composite hemostatic sponge loaded with hemostatic drug-loaded microspheres was 60 μm.

[0084] The composition, component ratio, and preparation method of the hemostatic drug-loaded microspheres in Comparative Example 12 were the same as those in Example 1.

[0085] Comparative Example 13 The difference between Comparative Example 13 and Example 1 is that the hemostatic drug-loaded microspheres prepared in Comparative Example 13 do not contain antibacterial drugs, while the content of the remaining components and the preparation method are the same as those in Example 1.

[0086] Comparative Example 14 The difference between Comparative Example 14 and Example 1 is that in Comparative Example 14, the antibacterial drug was directly added to the sponge matrix precursor solution. The specific operation steps are as follows: S1: Hemostatic drug-loaded microspheres were dispersed in a cationic solution at a mass-to-volume ratio of 1:8 mg / mL. The pH of the system was adjusted to 7.0, and the mixture was stirred at 100 rpm at room temperature until the microspheres were uniformly dispersed, resulting in a hemostatic drug-loaded microsphere-cationic suspension. The cationic solution was obtained by mixing 15 mmol / L ZnSO4•7H2O and 5 mmol / L AlCl3•6H2O solutions at a volume ratio of 1:1.

[0087] S2: Mix 1.5 wt% collagen solution, 0.3 wt% elastin solution and 0.24 wt% sodium alginate solution evenly to obtain a sponge matrix precursor solution, wherein the mass ratio of elastin to collagen is 1:5 and the mass ratio of sodium alginate to collagen is 4:25.

[0088] S3: Mix the antibacterial drug (ciprofloxacin hydrochloride, levofloxacin, moxifloxacin, norfloxacin or any combination thereof) with the sponge matrix precursor solution to obtain a sponge matrix precursor solution containing the antibacterial drug, wherein the antibacterial drug in the solution system is 2.5 wt%.

[0089] S4: The hemostatic drug-loaded microsphere-cationic suspension and the sponge matrix precursor solution containing antibacterial drugs were mixed evenly at a volume ratio of 1:1 to obtain a composite precursor solution. The solution was then filled into a mold, and the mold was completely covered with heat-insulating material. The mold was then frozen in a -80℃ freezer for 4 hours and then transferred to a -45℃ freezer for 42 hours. Finally, it was transferred to a vacuum drying oven for vacuum thermal crosslinking at 150℃ for 16 hours to obtain a porous composite hemostatic sponge.

[0090] The composition of the hemostatic drug-loaded microspheres in Comparative Example 14 included: 2.0 wt% glycerol, 3.5 wt% collagen, 0.7 wt% mussel adhesive protein, and 0.6 mmol / L CaCl2.

[0091] The preparation method of the hemostatic drug-loaded microspheres in Comparative Example 14 is as follows: (1) Add glycerol to the collagen solution and mix well to form solution A.

[0092] (2) Add mussel adhesive protein to calcium chloride solution and adjust the pH of the system to 5.5 to obtain mixed solution B.

[0093] (3) Mix solution A and solution B at a volume ratio of 1:1 to obtain a shell solution.

[0094] (4) The shell solution was spray-dried and then transferred to a vacuum drying oven for vacuum thermal crosslinking to obtain hemostatic drug-loaded microspheres. The spray drying process parameters were as follows: a spray drying device with a nozzle diameter of 0.5 mm was used, the feed temperature was set to 220 ℃, the feed rate was 18 rpm, and the compressed air speed was 8 L / min; the vacuum thermal crosslinking process parameters were as follows: the temperature was set to 150 ℃ and the time was 16 h.

[0095] Experimental Example 1. Microsphere encapsulation rate test: The encapsulation efficiency (EE) of the hemostatic drug-loaded microspheres prepared in Examples 1-3 and Comparative Examples 1-3 was determined, and the specific steps are as follows: Take the hemostatic drug-loaded microsphere samples from Examples 1-3 and Comparative Examples 1-3, and add physiological saline to prepare a hemostatic drug-loaded microsphere suspension with a mass concentration of 10 mg / mL. Accurately transfer 5 mL of the above hemostatic drug-loaded microsphere suspension into a centrifuge tube, centrifuge at 6000 rpm for 5 min, and collect the supernatant (i.e., the free drug solution). Use a UV-Vis spectrophotometer to perform a full-wavelength scan of the supernatant to determine the maximum absorption wavelength (λ) of the target component. max ); then at λ max The absorbance of the supernatant was measured, and the concentration and mass of the free drug in the supernatant were calculated based on the pre-established standard curve of the target component.

[0096] Simultaneously, an equal amount of hemostatic drug-loaded microsphere samples were taken for determination of the total drug content encapsulated within the microspheres: the microspheres were broken up by ultrasound to completely release the encapsulated drug. After removing carrier residue by centrifugation or filtration, the total drug mass was determined using the same method. The encapsulation rate was calculated using the following formula:

[0097] In the formula, EE: encapsulation rate (%); A: dosage (mg); B: mass of antibacterial drug in the combined supernatant (mg). The test results are shown in Table 1.

[0098] Table 1: Encapsulation efficiency results of Examples 1-3 and Comparative Examples 1-3

[0099] (Note: * indicates p ≤ 0.05 compared to Example 1, ** indicates p ≤ 0.01 compared to Example 1) As shown in Table 1, the hemostatic drug-loaded microspheres of Examples 1-3 exhibit excellent performance, with an average encapsulation efficiency of over 98%, and the measured values ​​of each example show extremely low dispersion (e.g., the standard deviation of Example 1 is ±0.0011). This result indicates that the measurement data of the encapsulation efficiency of the hemostatic drug-loaded microspheres prepared in Examples 1-3 are highly consistent and have excellent experimental repeatability. Combined with their high encapsulation efficiency, this further confirms that the preparation process has good stability and reliability, and can achieve efficient and stable encapsulation of the target component.

[0100] In the preparation of hemostatic drug-loaded microspheres, glycerol (propylene glycol or a combination of glycerol and propylene glycol) plays a key role as a functional adjuvant, mainly in two aspects: first, it significantly improves the extensibility of the microsphere shell (mainly composed of collagen) for film formation; second, it protects the protein. Its specific mechanism of action is as follows: The multiple hydroxyl groups in glycerol molecules (propylene glycol or a combination of glycerol and propylene glycol) can undergo solvation interactions with collagen molecular chains, covering the nonpolar regions of collagen through a "wrap-up effect" and reducing intermolecular aggregation caused by van der Waals forces in these regions. At the same time, this effect can effectively prevent collagen molecular chains from forming rigid aggregates through hydrophobic interactions, allowing the shell material to maintain appropriate flexibility and providing the necessary extensibility support for uniformly wrapping the core material.

[0101] The results of the comparative experiments show that the amount of glycerol added must be strictly controlled within the set range; otherwise, the encapsulation efficiency of the microspheres will be significantly reduced. In Comparative Example 1, the amount of glycerol added was lower than the set range, and the microsphere encapsulation rate was significantly lower than that of the Example Group. The main reason was that the insufficient amount of glycerol resulted in insufficient extensibility of the shell film, which led to a decrease in the integrity of the shell's encapsulation of the core material, and uneven encapsulation thickness. Some core materials could not be effectively encapsulated and were exposed, ultimately resulting in a reduced encapsulation rate.

[0102] In Comparative Example 2, the amount of glycerol added exceeded the set range, which also led to a decrease in the microsphere encapsulation efficiency. This is because excessive glycerol has a destructive effect on the intermolecular interactions and network structure of collagen: on the one hand, excessive glycerol competitively disrupts the hydrogen bonds and cross-linking structures between collagen molecules, resulting in a significant decrease in the mechanical strength of the shell material; on the other hand, excessive glycerol interferes with the normal assembly process of the collagen network, causing uneven distribution of the shell's pore structure. These structural defects can lead to shell damage during preparation or subsequent processing, causing leakage of the encapsulated core material and ultimately resulting in a decrease in encapsulation efficiency.

[0103] The key difference between Comparative Example 3 and the Examples lies in the Ca content during the microsphere shell preparation stage. 2+ Based on the encapsulation efficiency data, the encapsulation efficiency of the microspheres in Comparative Example 3 was significantly lower than that in the other examples. The fundamental reason for this phenomenon lies in the fact that Ca in Comparative Example 3...2+ Insufficient addition prevented the full realization of its effect of enhancing the adhesion performance of mussel adhesive proteins through metal coordination, resulting in a decrease in the stability of the microsphere core-shell structure and thus adversely affecting the encapsulation effect of the microspheres. 2. Microscopic morphology characterization The surface morphology of the hemostatic drug-loaded microspheres prepared in Example 1 was observed using a scanning electron microscope (SEM), and the results are as follows: Figure 2 As shown, the hemostatic drug-loaded microspheres have a complete spherical structure without obvious damage or collapse. Statistical analysis of the SEM images shows that the particle size distribution ranges from 20 μm to 80 μm, with relatively uniform particle size and regular surface morphology, indicating that the preparation process can stably obtain microsphere products with complete structures.

[0104] The microstructure of the sponge matrix prepared in Example 1 and Comparative Example 12 was characterized and analyzed using scanning electron microscopy (SEM). Typical morphologies are shown below. Figure 3 As shown: Among them Figure 3 (a) is a SEM image of the sponge matrix prepared by the directional freezing method of this scheme in Example 1. Figure 3 (b) is a SEM image of the sponge matrix prepared by direct freeze-drying method in Comparative Example 12.

[0105] SEM results showed that the sponge matrix of Example 1 exhibited a loose and porous microstructure with orderly and regular pore arrangement and good interconnection between pores; while the sponge matrix of Comparative Example 12 also showed a loose and porous structure, but the pore arrangement was disordered, the interconnection was significantly reduced, and some closed pores existed.

[0106] The structural differences between Comparative Example 12 and Example 1 affect the performance as follows: the disordered channels and closed pores in Comparative Example 12 can hinder the blood permeation process, thereby significantly reducing its liquid absorption performance; at the same time, the disordered channel structure is not conducive to cell migration and adhesion inside the sponge, resulting in a prolonged wound healing period and an increased risk of infection.

[0107] The microstructure of the porous composite hemostatic sponges prepared in Example 1 and Comparative Example 11 was characterized and analyzed using scanning electron microscopy (SEM). Their typical morphologies are shown in the figure below. Figure 4 As shown: Among them Figure 4 (a) is a SEM image of the porous composite hemostatic sponge prepared in Example 1. Figure 4 (b) is a SEM image of the porous composite hemostatic sponge prepared in Comparative Example 11.

[0108] SEM characterization results showed that the product prepared in Example 1 had hemostatic drug-loaded microspheres dispersed in a lamellar structure within the pores of the sponge, forming a stable physical attachment with the sponge scaffold. This uniformly distributed and well-attached microstructure has significant advantages: on the one hand, it facilitates the continuous and stable release of antibacterial drugs by the microspheres during the hemostatic sponge's function, thereby exerting a uniform and long-lasting antibacterial effect in wound treatment and providing reliable antibacterial protection for the wound; on the other hand, good physical attachment can effectively reduce the risk of microsphere detachment during use, ensuring the integrity of the overall structure and function of the hemostatic sponge.

[0109] In contrast, the porous composite hemostatic sponge prepared in Comparative Example 11 has obvious defects in its microstructure. In this product, the hemostatic drug-loaded microspheres aggregate and accumulate in the lamellar structure within the sponge pores. This aggregation can seriously affect the continuous and stable release of antibacterial drugs in the microspheres, resulting in unstable antibacterial effects and failing to provide effective antibacterial protection for the wound. In addition, because the microspheres are aggregated in the lamellar structure within the sponge pores, they are prone to falling off during subsequent use, which in turn affects the overall performance and service life of the hemostatic sponge.

[0110] Further analysis revealed that the main reason for the aforementioned microstructural differences stemmed from the surface energy mismatch between the hemostatic drug-loaded microspheres and the sponge matrix. In Comparative Example 11, the hemostatic drug-loaded microspheres were directly mixed with the sponge matrix precursor solution during the preparation process. The surface of the hemostatic drug-loaded microspheres was mainly composed of protein components, while the sponge matrix precursor solution was a collagen-elastin-sodium alginate complex system. The surface energy difference between the two was significant. This surface energy difference caused the hemostatic drug-loaded microspheres to preferentially self-aggregate rather than bind to the sponge matrix after being directly mixed with the sponge matrix precursor solution in Comparative Example 11. Ultimately, this resulted in microsphere aggregation, affecting the performance and quality of the product.

[0111] 3. Water absorption ratio test To evaluate the liquid absorption capacity of the porous composite hemostatic sponge products prepared in Examples 1-3 and Comparative Examples 7-12, a water absorption ratio test was conducted. The specific test method is as follows: The water absorption ratio test method is as follows: Accurately weigh 20 mg of each of the porous composite hemostatic sponge products prepared in Examples 1-3 and Comparative Examples 7-12, and record it as the initial mass m1. Before the test, gently rub the sponge sample with your fingers until all the air inside is completely expelled; then immerse all the above samples in beakers containing distilled water and let them stand for 30 minutes; after soaking, remove the test sample with small tweezers and hang it to drain the water until no more water droplets drip from the surface of the sponge. At this time, accurately weigh the sample mass and record it as the mass after water absorption m2.

[0112] Meanwhile, a commercially available medical collagen sponge (50×25×5 mm thick) produced by Wuxi Bedy Biotechnology Co., Ltd. was selected as a control group. Five parallel samples were set up for each experimental group and control group. After the experiment, the average water absorption ratio A of each group was calculated to ensure the reliability and repeatability of the experimental results. The results are shown in Table 2. The water absorption ratio A was calculated using the following formula: A=(m2-m1) / m1, where: A: water absorption ratio of the sample; m1: mass of the sample before wetting, in grams (g); m2: mass of the sample after wetting, in grams (g).

[0113] Table 2: Statistical table of water absorption ratio results for Examples 1-3, Comparative Examples 7-12, and commercially available control products.

[0114] (Note: * indicates p ≤ 0.05 compared to Example 1, ** indicates p ≤ 0.01 compared to Example 1) According to the industry standard YY / T 1511-2017 "Collagen Sponge", the liquid absorption capacity of the collagen sponge sample should not be less than 20 times its own weight. As can be seen from the experimental results in Table 2, the water absorption rate of Examples 1-3 and the commercially available control samples all meet the requirements of this standard. Its performance advantage stems from the synergistic effect of multiple factors: (1) The introduction of elastin effectively enhances the elastic deformation ability of the sponge pore structure, which can maintain the stability of the pore morphology during water absorption, reduce the phenomenon of pore collapse, and provide structural guarantee for rapid liquid penetration; (2) Sodium alginate molecules are rich in hydrophilic groups such as carboxyl (-COOH) and hydroxyl (-OH), which are extremely hydrophilic and can form hydrogen bond interactions with collagen molecules to build a dense three-dimensional hydrophilic network structure, which significantly enhances the sponge's adsorption capacity for water molecules; (3) Sponges are prepared by directional freezing technology, and by precisely controlling the direction of ice crystal growth, layered or tubular interconnected pores (similar to "pipe" structures) perpendicular to the freezing direction are formed. This ordered pore structure significantly improves pore connectivity and optimizes the liquid transport path, thereby significantly increasing the liquid absorption capacity of the sponge.

[0115] The mass ratio of collagen to sodium alginate is a key parameter for regulating the liquid absorption performance of a sponge; both excessively high and low ratios will adversely affect absorption performance. The difference between Comparative Example 7 and Example 1 lies in the fact that the mass ratio of collagen to sodium alginate during the sponge matrix preparation process is greater than 10:1 (lower sodium alginate concentration). Table 2 shows that its liquid absorption is significantly lower than that of Example 1. This is because the low concentration of sodium alginate provides insufficient hydrophilic groups, leading to a weakened osmotic pressure effect, reduced water molecule adsorption kinetics, and consequently, a decrease in water absorption rate and volume. Simultaneously, the low concentration of sodium alginate and collagen results in insufficient hydrogen bond cross-linking density, failing to construct a dense hydrogen bond network, reducing the spreading efficiency of the liquid within the sponge, and thus affecting the water absorption effect.

[0116] Conversely, the difference between Comparative Example 8 and Example 1 lies in the mass ratio of collagen to sodium alginate being less than 5:1 (the sodium alginate concentration was too high). Experimental results show that its liquid absorption performance also decreased. Specifically, high concentrations of sodium alginate significantly increase the system viscosity, leading to reduced pore size and porosity during sponge molding, thus hindering liquid penetration and diffusion. Furthermore, high concentrations of sodium alginate undergo excessive cross-linking with collagen, densifying the sponge structure and further inhibiting liquid absorption. These results indicate that the mass ratio of collagen to sodium alginate needs to be controlled within a reasonable range to maximize the liquid absorption performance of the sponge by optimizing the number of hydrophilic groups, hydrogen bond network density, and pore structure characteristics.

[0117] The loading capacity of hemostatic drug-loaded microspheres (characterized by the solid-liquid ratio of the hemostatic drug-loaded microspheres to the sponge matrix solution) is a crucial factor affecting the liquid absorption performance of the sponge; both excessively high and low loading capacities lead to performance degradation. The difference between Comparative Example 9 and Example 1 lies in the solid-liquid ratio of the loaded microspheres being less than 1:10 (lower microsphere content). Table 2 shows that the liquid absorption performance of Example 1 is significantly better than that of Comparative Example 9. This is because the collagen microspheres prepared in this study contain mussel adhesive protein, whose hydrophilicity and interfacial cross-linking enhance the sponge's water absorption and retention properties, forming a synergistic effect of "structure retention-functional enhancement." In contrast, the microsphere content in Comparative Example 9 is insufficient, only filling part of the pore space, failing to fully utilize this synergistic effect, thus limiting absorption performance.

[0118] Conversely, the difference between Comparative Example 10 and Example 1 lies in the solid-liquid ratio of the loaded microspheres being greater than 1:5 (excessive microsphere content). Experimental results show that its liquid absorption performance also decreases. Specifically, high-load microspheres easily clog sponge pores, leading to a decrease in effective porosity, hindering liquid penetration and slowing down the absorption rate. Simultaneously, a large number of microspheres may damage the structural integrity of the collagen sponge, reducing its mechanical properties and causing the sponge to easily deform and collapse during water absorption, ultimately affecting the liquid absorption effect and capacity. These results indicate that the solid-liquid ratio of the hemostatic drug-loaded microspheres needs to be controlled within a reasonable range to fully utilize the synergistic enhancing effect of the microspheres and avoid pore blockage or structural damage, thereby optimizing the sponge's liquid absorption performance.

[0119] The core difference between Comparative Example 11 and Example 1 lies in the loading method of the microspheres: Comparative Example 11 uses a direct loading method, that is, directly mixing positively charged microspheres with a collagen sponge matrix solution, and then preparing sponge materials through subsequent freeze-drying and other processes. However, microspheres are prone to aggregation under this loading method. The main reason is that the elastin and sodium alginate in the sponge matrix solution are negatively charged as a whole in an environment of pH 6.5~7.5. When positively charged microspheres are mixed with the overall weakly negatively charged matrix solution, the positive charge on the surface of the microspheres will electrostatically attract the negative charge of the elastin and sodium alginate in the matrix. On the one hand, the microspheres will preferentially adsorb onto the surface of the negatively charged matrix components; on the other hand, adjacent microspheres may aggregate through "negatively charged matrix components bridging" (i.e., multiple microspheres simultaneously bind to the same negatively charged molecular chain of the matrix), ultimately leading to microsphere aggregation. The aggregated microspheres fill or block the porous structure formed during the freeze-drying process of the sponge, resulting in a significant reduction in the effective porosity of the sponge matrix. Secondly, the penetration path of liquid inside the sponge is blocked, increasing the osmotic resistance. Under the combined effect of these two factors, the liquid absorption rate of the sponge slows down significantly, and the saturated liquid absorption capacity per unit mass of sponge also decreases significantly, ultimately affecting its blood absorption effect during hemostasis.

[0120] The difference between Comparative Example 12 and Example 1 lies in the use of direct freezing to prepare the sponge. Table 2 shows that the sponge prepared using the directional freezing method in Example 1 exhibits significantly better liquid absorption performance than the directly frozen sample. Directional freezing, by controlling the direction of ice crystal growth, forms an ordered, interconnected pore structure, characterized by high porosity and a regular arrangement of collagen fibers. Figure 3 a) This method fundamentally optimizes the liquid transport path and surface wettability, and significantly improves liquid absorption performance by controlling ice crystal growth to form an ordered microstructure. In contrast, the direct freezing process in Comparative Example 12 resulted in a non-directional temperature distribution, leading to random ice crystal growth. This resulted in an irregular pore structure, uneven pore size, and poor connectivity within the sponge (Figure 3b), significantly increasing liquid permeation resistance and reducing absorption efficiency.

[0121] 4. In vitro coagulation test Hemostatic function is a core indicator for evaluating the efficacy of hemostatic products. The hemostatic efficacy of porous composite hemostatic sponges loaded with drug-eluting hemostatic microspheres was compared and evaluated using in vitro coagulation experiments. The specific testing methods are as follows: (1) In vitro whole blood clotting time test Take 5 mg of each of the porous composite hemostatic sponges loaded with drug-eluting hemostatic microspheres prepared in Examples 1-3 and Comparative Examples 5-12, and place them at the bottom of test tubes, ensuring that the sponges are flat and wrinkle-free. Quickly add 1 mL of recalcified rabbit whole blood (whole blood recalcification treatment is performed according to the "National Clinical Laboratory Operation Procedures (4th Edition)") to each test tube containing the sponge sample, and start the timer immediately upon addition. Gently tilt the test tube every 20 seconds (tilt angle of about 45° to avoid external force interfering with the coagulation process) and observe the blood flow. When the blood stops flowing for the first time and no liquid drips when the test tube is inverted (defined as "initial coagulation state"), stop the timer immediately and record this time as the single coagulation time. Repeat the test 3 times in parallel for each group of samples, and take the average of the 3 test results as the final in vitro whole blood coagulation time of the sample.

[0122] (2) Blood Clotting Index (BCI) Detection Fresh whole blood was collected from rats and mixed with 3.8% sodium citrate anticoagulant (blood to anticoagulant volume ratio 9:1). The mixture was then gently inverted and stored temporarily at 4°C. Before use, the blood was brought to room temperature and recalcified. 5 mg of each of the porous composite hemostatic sponges prepared in Examples 1-3 and Comparative Examples 5-12 were placed at the bottom of test tubes. The test tubes were preheated in a 37°C water bath for 10 min (simulating physiological temperature in vivo to ensure consistent experimental conditions). 50 μL of recalcified whole blood containing 10 mmol / L CaCl2 was added to each preheated test tube containing the sponge. The mixture was quickly mixed and incubated in a 37°C water bath for 60 s. After incubation, 3 mL of deionized water was immediately added to each test tube, and the mixture was gently shaken to dissolve the uncoagulated blood. The absorbance (OD value, denoted as OD) of each solution was measured at 540 nm using an ELISA reader. 试验组 The absorbance measured at the same wavelength (540 nm) after adding 50 μL of recalcified whole blood directly to 3 mL of deionized water was used as a reference value (denoted as OD). 参考值 ).

[0123] Calculate the coagulation index (BCI) for each group of samples using the following formula: BCI = (OD) 试验组 / OD 参考值 ) × 100% A lower BCI value indicates a stronger promoting effect of the material on blood clotting, meaning better hemostatic efficacy; conversely, a higher BCI value indicates poorer hemostatic efficacy. Specific evaluation results are shown in Table 3.

[0124] Table 3: In vitro whole blood clotting time and coagulation index of Examples 1-3, Comparative Examples 5-12, and commercially available control products.

[0125] (Note: * indicates p ≤ 0.05 compared to Example 1, ** indicates p ≤ 0.01 compared to Example 1, *** indicates p ≤ 0.001 compared to Example 1) The results of in vitro whole blood clotting time (s) and coagulation index tests showed that, compared with the commercially available control, the porous composite hemostatic sponge prepared in Example 1 of this invention had a 38.4%–36.2% shorter in vitro whole blood clotting time and a 46.1%–52.53% higher coagulation index, demonstrating significant hemostatic efficacy advantages and achieving more efficient hemostasis. This is mainly because the mass ratio of elastin to collagen in the porous composite hemostatic sponge prepared in this example is closer to the composition ratio of the ECM of skin wounds, which can better adapt to the wound repair microenvironment. Specifically, the hydrophobic amino acids (such as proline and valine) of elastin form a balance with the hydrophilic groups of collagen, thereby achieving rapid liquid adsorption through hydrophilic groups while reducing blood retention and adhesion on the material surface through hydrophobic interactions. This avoids the "surface liquid film barrier" problem caused by excessive hydrophilicity and significantly improves the permeation rate of blood into the porous structure inside the material.

[0126] The product of this invention enhances the efficiency of coagulation initiation through charge synergy. The RGD (Arg-Gly-Asp) sequence in collagen molecules can specifically bind to platelet surface integrin αⅡbβ3, activating platelet adhesion and aggregation. At the same time, the carboxyl group in sodium alginate molecules adsorbs positively charged coagulation factors (such as prothrombin and factor IX) in the blood through electrostatic interaction, thereby increasing the local concentration of coagulation factors. The charge balance formed by the two can simultaneously initiate the dual pathways of "platelet activation" and "coagulation factor enrichment", laying the foundation for the coagulation cascade reaction.

[0127] In this invention, the surface active proteins and ionic components of the microspheres regulate and accelerate the terminal coagulation process. When hemostatic drug-loaded microspheres with a mass-to-volume ratio of 1:5 to 20 mg / mL to a cationic solution are attached to the pores of a sponge, the densely distributed polyphenolic groups and amino groups on the surface of the hemostatic drug-loaded microspheres can exert a synergistic effect: polyphenols rapidly capture platelets through hydrogen bonding and hydrophobic interactions, while amino groups cross-link with the carboxyl groups of fibrinogen through Schiff base reactions, accelerating the conversion of fibrinogen into insoluble fibrin; in addition, the hemostatic drug-loaded microspheres chelate Ca on their surface carboxyl groups. 2+It is rapidly released within 10-15 seconds after contact with blood, providing the necessary coenzyme for the formation of prothrombin activator, further triggering the conversion of prothrombin to thrombin, and ultimately achieving a highly efficient hemostasis chain of "rapid blood absorption - platelet activation - fibrin clot formation", significantly shortening the clotting time.

[0128] In the preparation of the porous composite hemostatic sponges of Comparative Examples 5 and 6, both excessively low or high mass ratios of elastin to collagen significantly affected the in vitro clotting time and clotting index of the products. When the mass ratio of collagen to elastin is too small, the proportion of hydrophilic groups (hydroxyl and carboxyl groups) on the material surface is high. Excessive hydrophilicity causes liquid to only be adsorbed on the surface and difficult to diffuse into the interior. In addition, the material surface is relatively smooth, which reduces the initial adhesion of platelets and slows down the aggregation rate. Ultimately, the blood clotting time is prolonged, and the coagulation cascade reaction is inhibited. The clotting time of Comparative Example 5 decreased by 32.4% compared with Example 1, and the BCI index decreased by 35.25% to 21.01%. When the mass ratio of collagen to elastin is too large, the hemostatic sponge structure is too dense and the pores are smaller, which hinders the diffusion and transport of coagulation factors in the blood and affects the interaction between coagulation factors, thus prolonging the clotting time. The clotting time of Comparative Example 6 decreased by 62.3% to 59.1% compared with Example 1, and the BCI index decreased by 50% to 47.38%.

[0129] In the preparation of the porous composite hemostatic sponges of Comparative Examples 7 and 8, both excessively high and low mass ratios of collagen to sodium alginate significantly affected the in vitro clotting time and coagulation index of the products. When the collagen-to-sodium alginate mass ratio was too high, the negative charge on the collagen surface was insufficient, failing to fully utilize the hydrophilicity and network synergistic effect of sodium alginate, and reducing ion exchange sites. This slowed the conversion rate of prothrombin to thrombin, delayed the initiation of intrinsic coagulation, and ultimately hindered rapid blood coagulation. The clotting time of Comparative Example 7 decreased by 37.8%–48.2% compared to Example 1, and the BCI index decreased by 46.95%–33.97%. When the collagen-to-sodium alginate mass ratio was too low, on the one hand, excessively high concentrations of sodium alginate easily formed a rigid gel network, making the hemostatic sponge gel too strong and the pores too small, increasing blood flow resistance and hindering the diffusion and interaction of coagulation factors; on the other hand, the high proportion of sodium alginate and the Ca on the surface of the microspheres... 2+ Excessive chelation leads to the formation of an insoluble calcium alginate gel layer covering the material surface, blocking the contact between coagulation factors and active sites, while simultaneously reducing platelet adhesion, spreading, and activity. These two factors combined prolong clotting time, negatively impacting in vitro coagulation performance. In Comparative Example 8, clotting time decreased by 71.2%–74.5% compared to Example 1, and the BCI index decreased by 69.9%–55.2%.

[0130] The porous composite hemostatic sponges prepared in Comparative Examples 9 and 10 were used to control the microsphere loading within the pores of the sponge by adjusting the mass-volume ratio of drug-loaded hemostatic microspheres to cationic solution. The experimental results showed that when the number of microspheres loaded within the pores of the porous composite hemostatic sponge was too small, the platelet adhesion sites and coagulation factor activation sites provided by the microspheres were insufficient. Although the porous structure of collagen sponges facilitates blood penetration, the lack of sufficient microsphere surface active groups prevents the rapid initiation of the coagulation cascade reaction. In Comparative Example 9, the coagulation time decreased by 41.6%–37.0% compared to Example 1, and the BCI index decreased by 44.5%–32.88%. Furthermore, when the number of microspheres loaded within the pores of the porous composite hemostatic sponge is excessive, microsphere aggregation easily occurs, clogging the sponge pores. Simultaneously, some microspheres cannot be fully exposed to the blood, reducing the contact area with coagulation factors and platelets, ultimately affecting the coagulation effect. In Comparative Example 10, the coagulation time decreased by 78.8%–85.7% compared to Example 1, and the BCI index decreased by 76.11%–69.65%.

[0131] Comparative Example 11: Microspheres were directly added to the sponge matrix solution, resulting in the microspheres being concentrated on the sponge surface and agglomerated. Furthermore, some sponge pores were blocked due to microsphere agglomeration, affecting blood absorption (reference). Figure 4 (b) In Comparative Example 11, the clotting time decreased by 55.1% to 68.7% compared to Example 1, and the BCI index decreased by 51.35% to 37.31%.

[0132] Comparative Example 12 used direct freezing to prepare the sponge, which resulted in large temperature fluctuations and uneven ice crystal nucleation and growth, leading to uneven sponge pore size and ultimately affecting the coagulation effect. The coagulation time of Comparative Example 12 decreased by 72.4% to 59.5% compared to Example 1, and the BCI index decreased by 63.85% to 52.81%.

[0133] 5. Tensile property test The porous composite hemostatic sponges prepared in Examples 1-3 and Comparative Examples 5-6 were cut into 1cm × 2cm specimen strips, ensuring that the specimens were free of obvious damage or defects. These porous composite hemostatic sponges were symmetrically clamped in the fixture of a high-low temperature controlled tensile testing machine, with the longitudinal axis of the specimen coinciding with the tensile direction. The tensile speed was set to 100 mm / min, and the testing machine was started to perform tensile tests on the specimens. The force-displacement curve of the specimens during the tensile process was recorded in real time. The test continued until the specimens fractured, and the maximum tensile force at fracture was recorded, as shown in Table 4. The tensile strength calculation formula is: ∂... In the formula: F: the maximum load (N) that the specimen can withstand before fracture; S: the cross-sectional area of ​​the specimen (m²). 2 ); Table 4: Hemostatic Tensile Strength of Examples 1-3 and Comparative Examples 5-6

[0134] (Note: * indicates p≤0.05 compared to Example 1) As can be seen from the tensile strength test results in Table 4, compared with the commercially available control product, the tensile strength of the porous composite hemostatic sponge prepared in Example 1 was increased by 7.1% to 12.7%. The main reason is that after the addition of elastin, the microfibrils of elastin form covalent bonds with the carboxyl groups in the RGD sequence on the surface of collagen fibers through the amino groups of lysine residues. This unique combination mode enables the material to simultaneously possess the "rigid load-bearing framework" of collagen and the "elastic buffer network" of elastin. The two work together to resist mechanical external forces, thereby improving the flexibility and tensile strength of the sponge structure.

[0135] In both Comparative Examples 5 and 6, excessively low or high levels of elastin addition significantly impacted the tensile strength of the sponge structure. In Comparative Example 5, the elastin addition was too low, preventing the formation of a continuous elastic network within the material. Furthermore, the lack of external force buffering from the amorphous regions of elastin (rich in glycine and proline) meant that the material's tensile strength primarily relied on collagen fibers. While collagen could form a dense, rigid network through hydrogen bonds, van der Waals forces, and β-sheet structures, its insufficient toughness resulted in relatively weak tensile strength in Comparative Example 5. Compared to Example 1, the tensile strength of the sponge structure prepared in Comparative Example 5 decreased by 10.5%–13.7%. In Comparative Example 6, the elastin concentration was high, preventing the formation of a continuous load-bearing phase in the collagen fiber network, significantly reducing the overall load-bearing capacity of the material. Although elastin could form a highly elastic network through disulfide bonds and lysine crosslinking, its tensile strength was far lower than that of collagen, ultimately leading to a 19.9%–22.1% decrease in tensile strength in Comparative Example 6 compared to Example 1. 6. Cell migration experiment Take target cells in the logarithmic growth phase, and use 5 × 10 5 Cells were seeded at a density of 1 cell / well in six-well cell culture plates, with each well containing 2 mL of culture medium. The plates were then incubated at 37°C with 5% CO2 for 24 hours until cells adhered and formed a confluent monolayer of over 90%. Once the cells had reached confluence, five equally spaced parallel horizontal lines were drawn on the bottom of each well using a marker, using a sterile steel ruler as a reference. Then, using a 200 μL sterile pipette tip, vertical scratches were made along the horizontal lines to create a crisscrossing grid pattern. After scratch preparation, each well was gently rinsed three times with 3 mL of sterile PBS buffer, discarding the PBS buffer after each rinse to thoroughly remove any detached cells from the scratched areas and prevent interference with subsequent migration observation.

[0136] Add 2 mL of H-DMEM sample culture medium containing 1% fetal bovine serum (FBS) to the wells of each experimental group (Examples 1-3 and Comparative Examples 1-14); add only 2 mL of H-DMEM basal culture medium containing 1% FBS to the wells of the blank control group. Place the treated culture plates back into a 37℃, 5% CO2 incubator and continue incubation for 48 h. After 48 h of incubation, remove the culture plates and observe and photograph the scratched areas of each well under an inverted phase-contrast microscope. Use ImageJ image analysis software to process the images, and measure and record the initial scratched area (A0, i.e., the scratched area immediately after scratch preparation) and the fixed scratched area (A1, i.e., the remaining scratched area after 48 h) for each group of samples. The specific results are shown in Table 5 below.

[0137] Cell migration rate (A) = migration area of ​​fixed scratch zone (A1) / area of ​​initial scratch zone (A0) × 100%; (Note: The area of ​​the transitional cells in the fixed scratch zone = the area of ​​the initial scratch zone (A0) - the area of ​​the fixed scratch zone (A1)) Table 5: Cell migration rate (%) at 48 hours for Examples 1-3 and Comparative Examples 1-12

[0138] (Note: * indicates p ≤ 0.05 compared to Example 1, ** indicates p ≤ 0.01 compared to Example 1) As shown in Table 5, the cell migration rate of Examples 1-3 and Comparative Examples 1-12 was significantly improved compared with the blank control group (among which, Example 1 improved by 354.8% to 359.0% compared with the blank control group). This indicates that the materials used in this scheme not only have good biocompatibility, but also have the ability to significantly promote the migration of target cells, providing strong support for wound repair. Specifically, Examples 1-3 exhibited the best cell migration rates. This is because, on the one hand, Examples 1-3 incorporated 1-5 wt% glycerol (propylene glycol or a combination of glycerol and propylene glycol) during the preparation process. This glycerol, through appropriate hydration, regulates the extracellular microenvironment, maintaining the dynamic interaction between cells and the matrix (such as collagen fibers) and preventing a decline in cell adhesion due to excessive dryness. It also promotes the enzymatic degradation of the collagen matrix (such as activating the activity of matrix metalloproteinases MMP-2 / MMP-9) and structural remodeling, enabling the collagen fiber network to form looser channels that facilitate cell migration. This creates a positive feedback loop of "cell migration-matrix degradation," accelerating cell migration to the scratched area. On the other hand, during the preparation of collagen microspheres, the addition of metal ions and mussel adhesive proteins optimizes the material structure and biological function through a synergistic effect of "ionic cross-linking-biomimetic adhesion." Metal ions form ionic bonds with the carboxyl groups of collagen molecules and the phenolic hydroxyl groups of mussel adhesive protein, enabling rapid microsphere molding while enhancing the mechanical stability of the microspheres (such as compressive strength and anti-swelling properties), preventing microsphere disintegration in cell culture environments. The adhesive properties of mussel adhesive protein strengthen the binding force between the microspheres and the sponge matrix, reducing microsphere detachment. Simultaneously, its surface-active groups (such as amino and phenolic hydroxyl groups) can mimic the adhesion sites of the natural extracellular matrix, enhancing the adhesion between cells and the material surface. The synergistic construction of this "high-strength, high-biocompatibility" porous network retains the porous structural advantages of the sponge (providing physical channels for cell migration) while endowing the material with dual functions of "hemostasis and repair." Furthermore, the bioactivity of mussel adhesive protein can further activate intracellular migration-related signaling pathways (such as the FAK-RhoA pathway), synergistically enhancing the matrix-regulating effects of glycerol (propylene glycol or a combination of glycerol and propylene glycol), significantly improving cell migration efficiency.

[0139] The results in Table 5 also show that although the cell migration rates of the porous composite hemostatic sponges prepared in Comparative Examples 5-12 were significantly higher than those in the blank control group, demonstrating their basic biocompatibility and certain promoting effect on cell migration, there was still a significant difference compared to Examples 1-3. The core reason for this difference lies in the changes in the preparation process (component ratio, preparation method) of Comparative Examples 5-12, which caused the key structural features of the material (pore size, pore connectivity) and microsphere dispersibility to deviate from the optimal range, thus affecting the cell migration efficiency. Comparative Examples 5 and 6 (elasticin addition), Comparative Examples 7 and 8 (sodium alginate addition), and Comparative Examples 9 and 10 (hemostatic drug-loaded microsphere loading) all affect the uniformity of sponge pore size and pore connectivity. Pore channels are the core physical channels for cell migration. Uneven pore size or blockage increases the physical resistance to cell migration and reduces the efficiency of cell migration into the material interior or scratched areas. In addition, imbalanced component ratios can lead to insufficient optimization of pore structure due to differences in microspheres on the sponge surface / preparation method, weakening the adaptability to the migration environment. Comparative Examples 11 (direct loading of hemostatic drug-loaded microspheres onto the sponge surface) and 12 (direct freezing preparation of porous composite hemostatic sponge) and Examples 1-3 (directional loading) are also affected. The differences in the preparation methods of cryogenic preparation directly affect the orientation and connectivity of sponge pores. During direct freezing, the temperature fluctuates greatly, and the nucleation and growth of ice crystals are disordered, resulting in pores with no obvious orientation and poor connectivity (some pores are closed pores), which cannot provide continuous channels for directional cell migration. In contrast, the directional freezing process of Examples 1-3 can construct oriented pores (uniform pore size, connectivity >90%), which can reduce the physical obstacles to cell migration and guide the directional migration of cells (such as migration towards the center of the wound) through pore orientation. This structural difference directly leads to the cell migration efficiency of Comparative Example 12 being significantly lower than that of Examples 1-3, and also further confirms the strong correlation between material structure and cell migration efficiency.

[0140] 6. Antibacterial properties Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922) were selected as indicator strains. The porous composite hemostatic sponges loaded with drug-eluting hemostatic microspheres prepared in Examples 1 and 13 were cut into cylindrical samples with a diameter of 0.5 cm and a height of 0.5 cm. The samples were sterilized using ultraviolet light in a clean bench and then stored in sterile petri dishes. Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922) were inoculated separately into LB liquid medium and cultured in a shaker at 37°C and 180 r / min for 12 h to prepare a concentration of 1×10⁻⁶. 6 ~1×10 7 CFU / mL bacterial suspension. Take 100 μL of each of the above bacterial suspensions and evenly spread them on the surface of LB solid culture plates to form bacterial culture plates. Let them stand for 5 minutes to allow the bacterial suspension to be completely absorbed. Place sterilized “Example 1 porous composite hemostatic sponge sample” and “Comparative Example 13 porous composite hemostatic sponge sample” in different areas of each bacterial culture plate. Slowly drop 20 μL of sterile simulated body fluid onto the surface of each porous composite hemostatic sponge sample, ensuring that SBF evenly wets the sponge without overflowing. After sealing all the culture plates containing samples, place them in a 37℃ constant temperature incubator for static incubation for 24 hours. Remove all the culture plates and place them in a laminar flow hood. Observe and record the diameter of the inhibition zone around each group of samples. See details. Figure 5 .

[0141] The difference between Comparative Example 13 and Example 1 is that the hemostatic drug-loaded microspheres prepared in Comparative Example 13 did not encapsulate antibacterial drugs. The antibacterial results of the porous composite hemostatic sponges prepared in Comparative Example 13 and Example 1 were evaluated according to the result judgment method in WST 650-2019 antibacterial and bacteriostatic effect evaluation criteria (an inhibition ring diameter > 7 mm is judged as having bacteriostatic effect; an inhibition ring diameter ≤ 7 mm is judged as having no bacteriostatic effect). Figure 5 It can be seen that the antibacterial effect of Comparative Example 13 is weak, while the porous composite hemostatic sponge obtained by the preparation method of Example 1 has a significant antibacterial effect. Example 1, in particular, shows a strong antibacterial effect. S. aureus The diameter of the inhibition zone can reach (25.09±1.97) mm, which is effective against... E. coli The diameter of the inhibition zone can reach (13.24±1.02) mm, which is greater than the 7 mm determined by the above standard, indicating that the porous composite hemostatic sponge prepared in Example 1 exhibits good antibacterial properties.

[0142] 7. Long-lasting antibacterial properties Take sterile centrifuge tubes (50 mL), and add 10 mL of sterile simulated body fluid and 1 mL of a 1×10⁻⁶ solution to each tube. 6 CFU / mL bacterial suspension was used to place the sterilized porous composite hemostatic sponge products prepared in Example 1, Comparative Example 13, and Comparative Example 14 into corresponding centrifuge tubes, which were then sealed and placed in a 37°C incubator for static culture for 21 days. Samples were taken at 0 h, 12 h, 24 h, 48 h, 72 h, 7 d, 14 d, and 21 d: the porous composite hemostatic sponge samples from each centrifuge tube were removed and gently rinsed three times with 5 mL of sterile PBS buffer (each rinse lasting 10 s) to remove unadsorbed free bacteria from the sample surface. After rinsing, the porous composite hemostatic sponge sample was transferred to a new sterile centrifuge tube, and 10 mL of sterile PBS buffer was added. The sample was then ultrasonically treated with an ultrasonic cleaner to ensure that the bacteria attached to the pores of the porous composite hemostatic sponge were completely detached into the PBS buffer to form a bacterial eluent.

[0143] Take the bacterial eluent and perform 10-fold serial dilutions with sterile PBS. Take 0.1 mL of each dilution and spread it evenly on nutrient agar plates (90 mm in diameter). Set up 3 parallel plates for each dilution. Invert the plates after spreading and incubate them in a 37°C incubator for 24 h. Observe and count the number of colonies on the plates and calculate the average number of colonies for each parallel sample.

[0144] The antibacterial rate of each group of experimental materials was calculated according to the formula: Antibacterial rate (%) = (number of colonies in blank control group - number of colonies in experimental group) / number of colonies in blank control group × 100%. The specific results are shown in Table 6.

[0145] Table 6: Comparison of bactericidal rate and antibacterial duration between Example 1 and Comparative Examples 13-14

[0146] The difference between Comparative Example 13 and the porous composite hemostatic sponge of Example 1 is that Comparative Example 13 did not contain any antibacterial drugs. Therefore, the antibacterial rate of Comparative Example 13 was very low. Its antibacterial effect was solely due to the phenolic hydroxyl groups of mussel adhesive protein, the polar groups (hydroxyl and amino groups) of collagen, and metal ions. However, this mechanism of action is achieved by disrupting the integrity of bacterial cell membranes and inhibiting bacterial adhesion. This effect is not concentration-dependent or has a sustained release characteristic, and therefore cannot form an effective antibacterial concentration gradient. By day 7, Comparative Example 13 had no antibacterial effect. Comparative Example 14 involved directly mixing antibacterial drugs into the sponge matrix. As shown in Table 6, the antibacterial rate data for Comparative Example 14 indicates that it exhibited highly efficient antibacterial performance in the early stages of the experiment, but by day 21, it had no antibacterial effect. This is because simply mixing antibacterial drugs into the sponge matrix cannot achieve sustained release of the antibacterial drugs; the drugs dissolve rapidly, resulting in a lack of sustained antibacterial effect. Example 1 achieves gradual release of antibacterial drugs by encapsulating them in microspheres, avoiding rapid dissolution of the antibacterial drugs and demonstrating the advantage of long-lasting and stable antibacterial effects.

[0147] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A porous composite hemostatic sponge, characterized in that, The porous composite hemostatic sponge comprises hemostatic drug-loaded microspheres, a cationic solution, and a sponge matrix precursor solution. The hemostatic drug-loaded microspheres have an antibacterial drug as the core and the outer shell is formed by solidifying a shell solution, wherein the shell solution comprises collagen, polyol, mussel adhesive protein, and metal ions.

2. The porous composite hemostatic sponge according to claim 1, characterized in that, The sponge matrix precursor solution is obtained by mixing 0.1 wt% to 1.0 wt% of elastin solution, 1.0 wt% to 3.0 wt% of collagen solution and 0.1 wt% to 0.5 wt% of alginate solution.

3. The porous composite hemostatic sponge according to claim 1, characterized in that, In the hemostatic drug-loaded microspheres, the metal ion is Ca. 2+ Zn 2+ and Mg 2+ Any one of the following; the polyol is selected from at least one of glycerol and propylene glycol.

4. The porous composite hemostatic sponge according to claim 1, characterized in that, The hemostatic drug-loaded microspheres contain 0.5 wt%–2.5 wt% antibacterial drugs, 2.0 wt%–5.0 wt% collagen, 1.0 wt%–5.0 wt% polyol, 0.5 wt%–1.0 wt% mussel adhesive protein, and the molar concentration of metal ions in the hemostatic drug-loaded microspheres is 0.2 mmol / L–2.0 mmol / L.

5. A method for preparing a porous composite hemostatic sponge according to any one of claims 1 to 4, characterized in that, include: S1, at room temperature, hemostatic drug-loaded microspheres are dispersed in a cationic solution, and the pH is adjusted to 6.5–7.5 to obtain a hemostatic drug-loaded microsphere-cationic suspension; wherein, the cationic solution is composed of Zn 2+ Ca 2+ Cu 2+ Fe 3+ And Al 3+ At least one of the components; S2, mix collagen solution, elastin solution and alginate solution to obtain sponge matrix precursor solution; S3, mix the hemostatic drug-loaded microsphere-cationic suspension with the sponge matrix precursor solution to obtain a composite precursor solution; S4. After the composite precursor solution is directionally frozen, it is then subjected to vacuum thermal crosslinking to obtain a porous composite hemostatic sponge.

6. The method for preparing a porous composite hemostatic sponge according to claim 5, characterized in that, In S1, the mass-to-volume ratio of the hemostatic drug-loaded microspheres to the cationic solution is 1:(5-20) mg / mL; the total cation concentration in the cationic solution is 20 mmol / L.

7. The method for preparing a porous composite hemostatic sponge according to claim 5, characterized in that, In S2, the mass ratio of elastin to collagen is 3:7 to 1:9, and the mass ratio of sodium alginate to collagen is 1:10 to 1:

5.

8. The method for preparing a porous composite hemostatic sponge according to claim 5, characterized in that, In S3, the volume ratio of the hemostatic drug-loaded microsphere-cationic suspension to the sponge matrix precursor solution is 1:

1.

9. The method for preparing a porous composite hemostatic sponge according to claim 5, characterized in that, In S4, the directional freezing includes: placing the composite precursor solution in a mold and completely covering the upper and lower surfaces of the mold with insulating material; freezing the mold at -80 ℃ for 3 h to 5 h, and then freezing it at -50 ℃ to -40 ℃ for 36 h to 48 h; the vacuum thermal crosslinking temperature is 120 ℃ to 150 ℃, and the time is 12 h to 24 h.

10. A method for preparing hemostatic drug-loaded microspheres as described in claim 1, characterized in that, Collagen and polyol were mixed to obtain mixed solution A; mussel adhesive protein solution was mixed with inorganic salt solution containing metal ions, and the pH was adjusted to 5.0-6.0 to obtain mixed solution B; mixed solution A and mixed solution B were mixed at a volume ratio of 1:1 to obtain shell solution. The shell solution was mixed with the antibacterial drug solution, spray-dried, and then vacuum thermal cross-linked to obtain hemostatic drug-loaded microspheres.

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