Heterogeneous wettability hemostatic sponge and method of making same

Abstract

A heterogeneous wetting hemostatic sponge and its preparation method are disclosed. The method includes the following steps: dissolving chitosan, cerium oxide stabilized by Panax notoginseng polysaccharide, and silver nanoparticles stabilized by bacterial cellulose in an acidic aqueous solution to obtain a first solution; adding alkali to the first solution to obtain a first suspension; dissolving chitosan and hexadecyltrimethoxysilane in an acidic aqueous solution to obtain a second solution; adding alkali to the second solution to obtain a second suspension; freezing the second suspension to obtain a frozen solid; subsequently adding the first suspension, the frozen solid, and the first suspension to a mold in sequence, and directionally freezing the first suspension on both sides of the frozen solid. The heterogeneous wetting hemostatic sponge of this invention can rapidly absorb blood from the wound site using a hydrophilic oriented layer; the synergistic activation of coagulation factors by chitosan and Panax notoginseng polysaccharide can also accelerate coagulation; the hydrophobic random layer can apply pressure to inhibit the continued longitudinal diffusion of blood, concentrate blood components, and improve hemostatic efficiency.

Description

Technical Field

[0001] This invention relates to the field of hemostatic materials, specifically to a heterogeneous wettable hemostatic sponge and its preparation method. Background Technology

[0002] Uncompressible bleeding is a major contributing factor to the high mortality rate in trauma patients. Timely and effective hemostasis is crucial for maintaining vital signs and reducing blood loss-related complications. Ideal hemostatic materials should not only possess rapid hemostasis capabilities but also have antibacterial, anti-inflammatory, and reparative properties to prevent bacterial infection and inflammatory responses during hemostasis from adversely affecting subsequent tissue repair.

[0003] Currently, commonly used hemostatic methods such as surgical sutures and gauze pressure still have limitations in hemostatic efficiency, are complex to operate, and are prone to inducing inflammatory reactions, especially in managing bleeding in non-compressible areas. Therefore, novel multifunctional hemostatic materials such as sponges, cryogels, hemostatic powders, adhesives, and hydrogels have attracted widespread attention. Among them, chitosan-based hemostatic materials have become a research hotspot due to their unique coagulation mechanism and cost advantages.

[0004] However, sponges prepared by traditional methods still have significant limitations in terms of pore connectivity and liquid absorption rate. Anisotropically oriented porous structures constructed by directional freezing can significantly improve the internal connectivity and pore structure of the material, thereby further enhancing the liquid absorption rate and hemostatic performance. However, due to the material's high hydrophilicity, porous structure, and capillary action between pores, a large amount of blood can be absorbed before the bleeding has stopped. Summary of the Invention

[0005] One objective of this invention is to provide a method for preparing a heterogeneous wettable hemostatic sponge. This method utilizes a directional freezing method to form a first and a second hydrophilic oriented layer on either side of a hydrophobic random layer. The hydrophilic oriented layer absorbs blood, accelerating the coagulation process, while the hydrophobic layer applies pressure to the blood to slow its diffusion. This achieves rapid blood absorption in the early stages of bleeding while avoiding the problem of large amounts of blood being absorbed before the bleeding stops. Simultaneously, loading Panax notoginseng polysaccharide to stabilize cerium oxide and bacterial cellulose to stabilize silver nanoparticles in the hydrophilic oriented layer effectively promotes wound tissue repair, showing broad application prospects in the fields of acute bleeding and tissue repair.

[0006] This invention is achieved through the following technical solution:

[0007] A method for preparing a heterogeneous wetting hemostatic sponge includes the following steps:

[0008] Chitosan, Panax notoginseng polysaccharide-stabilized cerium oxide, and bacterial cellulose-stabilized silver nanoparticles were dissolved in an acidic aqueous solution to obtain a first solution. An alkali was then added to the first solution to obtain a first suspension.

[0009] Chitosan and hexadecyltrimethoxysilane were dissolved in an acidic aqueous solution to obtain a second solution. An alkali was then added to the second solution to obtain a second suspension.

[0010] The second suspension is frozen to obtain a frozen solid. Then, the first suspension, the frozen solid, and the first suspension are added to the mold in sequence, and the first suspension on both sides of the frozen solid is frozen in a directional manner to obtain the heterogeneous wettable hemostatic sponge.

[0011] In this technical solution, chitosan, Panax notoginseng polysaccharide-stabilized cerium oxide, and bacterial cellulose-stabilized silver nanoparticles are dissolved together in deionized water. An acid, such as hydrochloric acid, is then added and stirred to obtain a first solution. An alkali, such as sodium hydroxide, is then added to the first solution to adjust the pH value, resulting in a first suspension. This first suspension is used to subsequently form the superhydrophilic portion of the hemostatic sponge.

[0012] In this technical solution, chitosan and hexadecyltrimethoxysilane are dissolved in deionized water. Similarly, acid is added and stirred to obtain a second solution. Then, alkali is added to the second solution to prepare a second suspension. This second suspension is used to subsequently form the hydrophobic portion of the hemostatic sponge.

[0013] In some preferred embodiments, the first and second suspensions are dispersed using a high-speed disperser, then centrifuged, and then epichlorohydrin (ECH) is added and mixed evenly before crosslinking at 37 °C.

[0014] In this technical solution, to achieve different orientations for the first and second suspensions, the second suspension is first frozen separately, for example, by freezing it in a refrigerator to obtain a frozen solid. Since the cold source for the second suspension comes from any direction, the formation of ice crystals is disordered, resulting in a randomly distributed porous structure during the freezing process. Subsequently, the first suspension, the frozen solid, and the first suspension are added to a mold, ensuring that both sides of the frozen solid have the first suspension. Then, the first suspensions on both sides of the frozen solid are subjected to directional freezing. For example, the mold is placed with one end facing down in a cold trap, so that the end of the mold is in contact with a low-temperature copper block. The copper block acts as a cold source, freezing the first suspension inside the mold from bottom to top, causing ice crystals to form from bottom to top, thus creating an oriented porous structure. After completing the directional freezing of the first suspension on one side, the mold is inverted so that the end of the first suspension on the other side of the frozen solid is close to the low-temperature copper block, thus completing the directional freezing of the first suspension on the other side.

[0015] After freezing, the resulting heterogeneous wetting hemostatic sponge has a three-layer structure. The addition of hexadecyltrimethoxysilane to the middle layer of the heterogeneous wetting hemostatic sponge, along with its own disordered pore structure, determines its hydrophobicity, i.e., a hydrophobic random layer. The sponge layers on both sides of the hydrophobic random layer do not contain hexadecyltrimethoxysilane, and an ordered oriented pore structure is formed by directional freezing, which greatly enhances the hydrophilicity of the sponge layers on both sides. The first and second hydrophilic oriented layers formed on both sides of the hydrophobic random layer are superhydrophilic.

[0016] The heterogeneous wetting hemostatic sponge features a superhydrophilic-hydrophobic-superhydrophilic three-layer structure. During hemostasis, the hydrophilic oriented layer contacts the wound, and its oriented porous structure significantly increases the blood absorption rate, rapidly absorbing blood from the wound site. Furthermore, chitosan and Panax notoginseng polysaccharide synergistically activate coagulation factors, accelerating clotting. Subsequently, regardless of which side of the hydrophilic oriented layer absorbs blood, the hydrophobic random layer applies pressure to inhibit the continued longitudinal diffusion of blood, further concentrating blood components, promoting clot formation, improving hemostasis efficiency, and preventing the problem of large amounts of blood being absorbed before bleeding stops.

[0017] Meanwhile, in the three-layer structure, although the middle hydrophobic random layer is hydrophobic, it still has the ability to absorb blood. Therefore, when the amount of bleeding in the hydrophilic orientation layer that is attached to the wound is too large, the excess blood can still move through the hydrophobic random layer to the hydrophilic orientation layer that is not attached to the wound for coagulation, thus avoiding excessive bleeding at the wound site due to the pressure applied by the hydrophobic random layer.

[0018] In addition, this three-layer structure of heterogeneous wettable hemostatic sponge is particularly suitable for hemostasis of wounds in non-compressible areas. For example, pressure hemostasis is often not suitable for internal wounds. In this case, the three-layer sponge can be compressed and placed on the wound. Both hydrophilic oriented layers can play a hemostatic role. By absorbing blood and expanding, it generates pressure to achieve the purpose of hemostasis.

[0019] In this technical solution, the first and second hydrophilic orientation layers also contain bacterial cellulose-stabilized silver nanoparticles and Panax notoginseng polysaccharide-stabilized cerium oxide. During wound repair and regeneration, bacterial cellulose-stabilized silver nanoparticles can effectively prevent bacterial growth in wounds due to their excellent antibacterial ability, while Panax notoginseng polysaccharide-stabilized cerium oxide can effectively regulate the immune microenvironment to promote the repair process and promote cell migration and angiogenesis due to its excellent intracellular ROS scavenging ability and regeneration-promoting properties.

[0020] Specifically, the antibacterial mechanism of bacterial cellulose-stabilized silver nanoparticles lies in the synergistic effect of the nanoparticles themselves and the continuously released silver ions, resulting in multi-target synergy. On the one hand, the nanoparticles can act as a reservoir to continuously release silver ions, which attack bacteria through multiple pathways, including disrupting cell membranes, binding to key groups of enzymes and DNA to inactivate them, and inducing oxidative stress. On the other hand, the tiny nanoparticles themselves can also directly penetrate or adsorb onto the bacterial surface, physically disrupting the membrane structure and interfering with its normal function. This dual-action mode gives silver nanoparticles broad-spectrum, high-efficiency, and low-prone-to-inducing bacterial resistance characteristics.

[0021] The inflammatory regulation and tissue repair mechanism of cerium oxide stabilized by Panax notoginseng polysaccharide lies in the fact that both regulate intracellular ROS homeostasis and inhibit the activation of the IL-17 signaling pathway, thereby reducing the expression levels of various inflammatory cytokines such as IL-6 and CXCL2. This process blocks the NF-κB, TNF, and HIF-1 signaling pathways, downregulates the expression of metalloproteinases and inducible iNOS, effectively alleviates the inflammatory response, improves the hypoxic microenvironment, and inhibits the polarization of M1 macrophages. Experiments have shown that the introduction of cerium oxide stabilized by Panax notoginseng polysaccharide can effectively regulate the immune microenvironment to promote the repair process, reduce the expression of the M1 macrophage marker CD86, and promote cell migration and angiogenesis. In addition, cerium oxide stabilized by Panax notoginseng polysaccharide can also promote the secretion of anti-inflammatory cytokines by activating the PI3K-Akt and TGF-β signaling pathways, thereby activating the VEGF and RAP1 signaling pathways, ultimately enhancing cell migration, promoting angiogenesis, and inducing M2 macrophage expression.

[0022] In this technical solution, the hydrophobicity of the hydrophobic random layer effectively prevents the longitudinal diffusion of absorbed blood, while simultaneously creating a high-concentration local hemostatic environment within the hydrophilic orientation layer. In this environment, Panax notoginseng polysaccharides differ from other polysaccharides; they not only improve microcirculation and prevent local necrosis due to excessively high local blood concentration, but also promote macrophage polarization from M1 to M2, actively activate platelets and initiate immune repair processes, regulate the immune microenvironment to accelerate healing, and significantly improve wound healing and repair outcomes.

[0023] In a preferred embodiment of the present invention, the mass ratio of hexadecyltrimethoxysilane to chitosan in the second suspension is 1:1 to 1:3.

[0024] The amount of hexadecyltrimethoxysilane used determines the hydrophobicity of the hydrophobic random layer. If its content is too low, the hydrophobicity of the hydrophobic random layer is weak, making it difficult to apply the desired pressure to the blood. Conversely, if the content of hexadecyltrimethoxysilane is too high, the greater hydrophobicity of the hydrophobic random layer causes it to act like a partition between two hydrophilic oriented layers, hindering the communication between the two hydrophilic oriented layers, resulting in excessively high local blood concentration and a decrease in the hemostatic ability of the hemostatic sponge. Therefore, in this technical solution, the preferred mass ratio of hexadecyltrimethoxysilane to chitosan is determined to be 1:1 to 1:3. In a more preferred embodiment, the mass ratio of hexadecyltrimethoxysilane to chitosan is 1:1 to 1:2.

[0025] Furthermore, the height ratio of the first suspension to the frozen solids is 2:1 to 4:1. Experiments have shown that a too-thick hydrophobic random layer can affect blood absorption, while a too-thin layer reduces the pressure applied to the blood. Therefore, preferably, the height ratio of the first suspension to the frozen solids is 2:1 to 4:1. In some more preferred embodiments, the height ratio of the first suspension to the frozen solids is 2:1 to 3:1.

[0026] Furthermore, the preparation of the Panax notoginseng polysaccharide-stabilized cerium oxide includes the following steps: dissolving Panax notoginseng polysaccharide, adding cerium nitrate hexahydrate and ammonia to the solution, and dialysis after the reaction is completed to obtain the Panax notoginseng polysaccharide-stabilized cerium oxide.

[0027] In some preferred embodiments, Panax notoginseng polysaccharide was weighed and dissolved in deionized water. The solution was stirred continuously under constant temperature conditions. Then, cerium nitrate hexahydrate was slowly added to the solution, and the reaction continued while maintaining the same temperature and stirring conditions. Subsequently, ammonia was added dropwise to the above mixed solution, and the reaction continued under constant temperature conditions. After the reaction was complete, the resulting solution was transferred to a dialysis bag and dialyzed continuously to completely remove unreacted substances. Finally, the solution was freeze-dried to obtain Panax notoginseng polysaccharide-stabilized cerium oxide.

[0028] Furthermore, in the first suspension, the mass ratio of the Panax notoginseng polysaccharide-stabilized cerium oxide to chitosan is 1:2 to 1:10.

[0029] Notoginseng polysaccharide-stabilized cerium oxide not only promotes rapid hemostasis but also facilitates cell migration and angiogenesis during repair and regeneration. Within a reasonable range, more notoginseng polysaccharide-stabilized cerium oxide can better promote hemostasis and regeneration. However, excessive notoginseng polysaccharide-stabilized cerium oxide can lead to toxicity and structural instability and difficulty in forming the sponge during preparation. Therefore, preferably, the mass ratio of notoginseng polysaccharide-stabilized cerium oxide to chitosan is 1:2 to 1:10. In some preferred embodiments, the mass ratio of notoginseng polysaccharide-stabilized cerium oxide to chitosan is 1:2 to 1:5.

[0030] Furthermore, the preparation of the bacterial cellulose stabilized silver nanoparticles includes the following steps: after washing the bacterial cellulose, NaIO4 is added for oxidation in the dark; the oxidized bacterial cellulose reacts with silver nitrate solution in the dark, and is then dialyzed; the dialyzed product is dissolved in a mixed solution of NaOH and urea, subjected to freeze-thaw treatment, dialyzed again, and then freeze-dried to obtain the bacterial cellulose stabilized silver nanoparticles.

[0031] In some preferred embodiments, bacterial cellulose is washed with pure water, shredded, and then treated with NaOH solution to remove impurities. After repeated washing and squeezing, it is dialyzed in pure water. Subsequently, it is mechanically pulverized, NaIO4 is added in proportion, and it is oxidized in the dark, pulverized again, and dialyzed. Next, the oxidized bacterial cellulose is reacted with silver nitrate solution, and dialyzed after the reaction. Finally, the product is dissolved using a mixed solution of NaOH and urea, and after freeze-thaw treatment to ensure complete dissolution, it is dialyzed again and freeze-dried to obtain bacterial cellulose-stabilized silver nanoparticles.

[0032] Furthermore, in the first suspension, the mass ratio of the bacterial cellulose-stabilized silver nanoparticles to chitosan is 1:5 to 1:10.

[0033] Bacterial cellulose-stabilized silver nanoparticles exhibit excellent antibacterial properties, which increase with increasing loading. However, once the loading reaches a certain level, it affects cell viability. Therefore, in this technical solution, the mass ratio of bacterial cellulose-stabilized silver nanoparticles to chitosan is 1:5 to 1:10. More preferably, the mass ratio of bacterial cellulose-stabilized silver nanoparticles to chitosan is 1:5 to 1:8.

[0034] Further, the mold is placed in a cold trap for freezing, with the bottom of the mold placed above the copper block for directional freezing of the first suspension located on one side of the frozen solid; subsequently, the top of the mold is placed above the copper block for directional freezing of the first suspension located on the other side of the frozen solid.

[0035] Furthermore, after the first suspensions on both sides of the frozen solid are frozen, they are soaked in deionized water, then placed in a cold trap to freeze again, and finally freeze-dried to obtain the heterogeneous wettable hemostatic sponge.

[0036] Another objective of this invention is to provide a heterogeneous wetting hemostatic sponge, which is prepared by any of the aforementioned preparation methods. The heterogeneous wetting hemostatic sponge includes a first hydrophilic oriented layer and a second hydrophilic oriented layer with an oriented pore structure. A hydrophobic random layer with a disordered pore structure is disposed between the first hydrophilic oriented layer and the second hydrophilic oriented layer. The first hydrophilic oriented layer and the second hydrophilic oriented layer contain cerium oxide stabilized by Panax notoginseng polysaccharide and nano-silver stabilized by bacterial cellulose.

[0037] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0038] 1. The heterogeneous wettable hemostatic sponge of this invention has a superhydrophilic-hydrophobic-superhydrophilic three-layer structure. During hemostasis, the hydrophilic oriented layer contacts the wound, and its oriented pore structure greatly improves the blood absorption rate, enabling rapid absorption of blood from the wound site. Furthermore, chitosan and Panax notoginseng polysaccharide synergistically activate coagulation factors, which can also accelerate coagulation. The hydrophobic random layer can apply pressure to inhibit the continued longitudinal diffusion of blood, further concentrate blood components, promote blood clot formation, improve hemostasis efficiency, and avoid the problem of a large amount of blood being absorbed before bleeding stops.

[0039] 2. In the three-layer structure of the heterogeneous wettable hemostatic sponge of the present invention, although the middle hydrophobic random layer is hydrophobic, it still has the ability to absorb blood. When the amount of bleeding in the hydrophilic oriented layer that is attached to the wound is too large, the excess blood can still move through the hydrophobic random layer to the hydrophilic oriented layer that is attached to the wound for coagulation, thus avoiding the situation of excessive blood at the wound due to the pressure applied by the hydrophobic random layer.

[0040] 3. This invention adjusts the hydrophobicity of the hydrophobic random layer by adjusting the amount of hexadecyltrimethoxysilane, so that the heterogeneous wetting hemostatic sponge can inhibit the longitudinal diffusion of blood, apply a certain pressure to the wound to reduce blood loss, increase blood concentration and coagulation at the physical level, and accelerate the hemostasis process.

[0041] 4. In this invention, Panax notoginseng polysaccharide is used to stabilize cerium oxide, which can effectively regulate the immune microenvironment to promote the repair process, reduce the expression of CD86, a marker of M1 macrophages, actively activate platelets and initiate the immune repair program, and promote cell migration and angiogenesis. At the same time, unlike other polysaccharides, Panax notoginseng polysaccharide can improve microcirculation, avoid excessive local blood concentration and local necrosis, and promote macrophage polarization from M1 to M2, regulate the immune microenvironment to accelerate healing, and significantly improve the healing and repair effect of wounds.

[0042] 5. The chitosan and Panax notoginseng polysaccharide of the present invention stabilize cerium oxide and synergistically activate coagulation factors to accelerate coagulation, thereby improving hemostasis efficiency at the biochemical level. At the same time, the hydrophobic random layer slows down the absorption of blood, further concentrates blood components, and promotes blood clot formation, thus significantly improving the hemostasis efficiency of the hemostatic sponge. Attached Figure Description

[0043] The accompanying drawings, which are included to provide a further understanding of embodiments of the invention and form part of this application, do not constitute a limitation thereof. In the drawings:

[0044] Figure 1 This is a schematic diagram of a specific embodiment of the present invention;

[0045] Figure 2 The characterization results of cerium oxide stabilization by Panax notoginseng polysaccharide in a specific embodiment of the present invention are shown;

[0046] Figure 3 The characterization results of bacterial cellulose-stabilized silver nanoparticles in a specific embodiment of the present invention are shown;

[0047] Figure 4 This is a schematic diagram of the structure of the heterogeneous wetting hemostatic sponge in a specific embodiment of the present invention;

[0048] Figure 5 The toxicity of hemostatic sponges loaded with different amounts of Panax notoginseng polysaccharide-stabilized cerium oxide is shown in specific embodiments of the present invention.

[0049] Figure 6 The toxicity of hemostatic sponges loaded with different amounts of bacterial cellulose-stabilized silver nanoparticles is shown in specific embodiments of the present invention.

[0050] Figure 7 The images show hemostasis and coagulation of sponges stabilized with Panax notoginseng polysaccharide and Bletilla striata polysaccharide, respectively, in a specific embodiment of the present invention.

[0051] Figure 8 The coagulation index of hemostatic sponges with different amounts of hexadecyltrimethoxysilane added is shown in a specific embodiment of the present invention;

[0052] Figure 9 The contact angles of the first hydrophilic orientation layer (upper layer), the hydrophobic random layer, and the second hydrophilic orientation layer (lower layer) of the hemostatic sponge 1 in a specific embodiment of the present invention are shown.

[0053] Figure 10 The superoxide anion and hydrogen peroxide scavenging capabilities of different hemostatic sponges in specific embodiments of the present invention are shown.

[0054] Figure 11 The antibacterial properties of different hemostatic sponges against Escherichia coli and Staphylococcus aureus are shown in specific embodiments of the present invention.

[0055] Figure 12 The water absorption rates of different hemostatic sponges in specific embodiments of the present invention are shown;

[0056] Figure 13 The blood absorption rates of different hemostatic sponges in specific embodiments of the present invention are shown;

[0057] Figure 14 The images and coagulation indices of different hemostatic sponges in specific embodiments of the present invention are shown.

[0058] Figure 15The figures show the number of red blood cells and platelets adhered to by different hemostatic sponges in specific embodiments of the present invention;

[0059] Figure 16 Images of different hemostatic sponges used to stop bleeding in the liver, along with the amount of blood loss and the time required for hemostasis, are shown in specific embodiments of the present invention.

[0060] Figure 17 Images and healing rates of different hemostatic sponges on wounds are shown in specific embodiments of the present invention;

[0061] Figure 18 This illustrates the regulation of the inflammatory macrophage marker CD86 by different hemostatic sponges in specific embodiments of the present invention;

[0062] Figure 19 The illustration shows pathological sections of the liver after treatment with different hemostatic sponges in a specific embodiment of the present invention. Detailed Implementation

[0063] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments and accompanying drawings. The illustrative embodiments and descriptions of the present invention are only used to explain the present invention and are not intended to limit the present invention.

[0064] All raw materials used in this invention are not particularly limited in their source; they can be purchased commercially or prepared using conventional methods well-known to those skilled in the art. The purity of all raw materials used in this invention is not particularly limited; however, analytical grade or purity requirements conventional in the field of hemostatic materials are preferred. All raw materials used in this invention have brand names and abbreviations that are conventional in the field, and each brand name and abbreviation is clearly defined within its relevant application. Those skilled in the art can obtain these materials from commercial sources or prepare them using conventional methods based on the brand name, abbreviation, and corresponding application.

[0065] The terms "first," "second," etc., used in this invention (e.g., first suspension, second suspension, first hydrophilic orientation layer, second hydrophilic orientation layer, etc.) are merely for clarity of description and are not intended to limit any order or emphasize importance. Furthermore, the term "connection" as used herein, unless otherwise specified, can refer to a direct connection or an indirect connection via other groups.

[0066] I. Preparation of hemostatic sponges

[0067]

Example 1

[0068] (1) Preparation of Panax notoginseng polysaccharide stabilized cerium oxide

[0069] Weigh 12 g of Panax notoginseng polysaccharide and dissolve it in 240 mL of deionized water. Stir continuously at 5000 rpm for 20 min at a constant temperature of 60 °C. Then, slowly add 0.34 g of cerium nitrate hexahydrate to the solution, maintaining the same temperature and stirring conditions for another 20 min. Add 60 mL of ammonia water dropwise to the above mixture, and continue the reaction at a constant temperature of 60 °C for 12 h. After the reaction is complete, transfer the resulting solution to a dialysis bag with a molecular weight cutoff of 1000. Change the dialysis solution every 6 h and continue dialysis for 72 h to completely remove unreacted substances. Finally, freeze-dry to obtain Panax notoginseng polysaccharide-stabilized cerium oxide.

[0070] Figure 2 The UV-Vis spectra of Panax notoginseng polysaccharide-stabilized cerium oxide (PPCe) and Panax notoginseng polysaccharide (PP) are shown in the figure. After successful loading of cerium oxide, the spectrum of Panax notoginseng polysaccharide-stabilized cerium oxide showed the characteristic absorption peak of cerium oxide.

[0071] (2) Preparation of bacterial cellulose-stabilized silver nanoparticles

[0072] Bacterial cellulose was washed with pure water, chopped, and then treated with 0.1 mol / L NaOH solution at 100 °C for 3 h to remove impurities. After repeated washing and squeezing, it was dialyzed in pure water for 3 days. Subsequently, it was mechanically pulverized, and NaIO4 was added in the appropriate ratio. It was then oxidized at 60 °C in the dark for 3 h, pulverized again, and dialyzed. Next, the oxidized bacterial cellulose was reacted with freshly prepared silver ammonia solution (silver nitrate and pure water were prepared at a ratio of 0.68 g: 20 mL) at 60 °C in the dark for 3 h, and dialyzed for 3 days after the reaction. Finally, the product was dissolved in a mixed solution of 12% NaOH and 8% urea, and subjected to a freeze-thaw treatment at -20 °C to ensure complete dissolution. After dialyzing for 3 days again, it was freeze-dried to obtain bacterial cellulose-stabilized silver nanoparticles.

[0073] Figure 3 The UV-Vis spectra of bacterial cellulose stabilized silver nanoparticles (OBCAg) and bacterial cellulose (BC) are shown in comparison. Similarly, after loading silver nanoparticles, the spectrum of bacterial cellulose stabilized silver nanoparticles shows the characteristic absorption peak of silver nanoparticles.

[0074] (3) Preparation of heterogeneous wetting hemostatic sponge

[0075] 500 mg chitosan, 200 mg Panax notoginseng polysaccharide-stabilized cerium oxide, and 100 mg bacterial cellulose-stabilized silver nanoparticles were dissolved in 50 mL of deionized water, and 240 μL of hydrochloric acid was added. The mixture was stirred at 60 °C for 2 h to dissolve. Next, 2.4 mL of 1M NaOH solution was added to the solution to obtain the first suspension. The suspension was dispersed at 9000 rpm for 3 min using a high-speed disperser, followed by centrifugation at 4000 rpm for 10 min. Then, 200 μL of epichlorohydrin (ECH) was added, and the mixture was thoroughly mixed and crosslinked at 37 °C for 4 h.

[0076] 500 mg of chitosan and 500 mg of hexadecyltrimethoxysilane were dissolved in 50 mL of deionized water, and 240 μL of hydrochloric acid were added. The mixture was stirred at 60 °C for 2 h to dissolve. Then, 2.4 mL of 1M NaOH solution was added to the solution to obtain a second suspension. Similarly, the suspension was dispersed at 9000 rpm for 3 min using a high-speed disperser, followed by centrifugation at 4000 rpm for 10 min. Then, 200 μL of epichlorohydrin (ECH) was added, and the mixture was thoroughly mixed and crosslinked at 37 °C for 4 h.

[0077] A second suspension was added to a polytetrafluoroethylene (PTFE) mold and frozen at -20 °C to obtain a frozen solid. This was then removed, and the first suspension, frozen solid, and the first suspension were added sequentially to another PTFE mold. The mold was then placed in a -12 °C cold trap with the bottom end of the mold above a copper block and frozen for 30 minutes. After freezing, the mold was inverted so that the top end was above the copper block, and frozen again for 30 minutes. Subsequently, the mold was soaked in deionized water for two days to remove unreacted impurities. After soaking, it was again placed in a -12 °C cold trap and frozen. Finally, it was freeze-dried to obtain hemostatic sponge 1.

[0078] like Figure 4 As shown, the middle layer of the prepared heterogeneous wettable hemostatic sponge is a hydrophobic random layer with disordered pore structure formed by the frozen solid after freezing the second suspension. The upper and lower layers of the hydrophobic random layer are superhydrophilic oriented layers with oriented pore structure formed by the directional freezing of the first suspension. The superhydrophilic oriented layers are loaded with cerium oxide stabilized by Panax notoginseng polysaccharide and silver nanoparticles stabilized by bacterial cellulose.

[0079]

Example 2

[0080] In this embodiment, the preparation method is similar to that in Example 1. The difference is that in step (3), when preparing the first suspension, not Panax notoginseng polysaccharide is added to stabilize cerium oxide. Instead, 500 mg of chitosan and 100 mg of bacterial cellulose stabilized nano-silver are dissolved in 50 mL of deionized water to finally obtain hemostatic sponge 2.

[0081]

Example 3

[0082] In this embodiment, the preparation method is similar to that in Example 1. The difference is that in step (3), when preparing the first suspension, the amount of Panax notoginseng polysaccharide stabilizing cerium oxide added to the deionized water is adjusted to 50 mg, and the hemostatic sponge 3 is finally obtained.

[0083]

Example 4

[0084] In this embodiment, the preparation method is similar to that in Example 1. The difference is that in step (3), when preparing the first suspension, the amount of Panax notoginseng polysaccharide stabilizing cerium oxide added to the deionized water is adjusted to 100 mg, and finally the hemostatic sponge 4 is obtained.

[0085]

Example 5

[0086] In this embodiment, the preparation method is similar to that in Example 1. The difference is that in step (3), when preparing the first suspension, the amount of Panax notoginseng polysaccharide stabilizing cerium oxide added to the deionized water is adjusted to 300 mg, and finally the hemostatic sponge 5 is obtained.

[0087]

Example 6

[0088] In this embodiment, the preparation method is similar to that in Example 1. The difference is that in step (3), when preparing the first suspension, bacterial cellulose stabilized nano-silver is not added. Instead, 500 mg chitosan and 200 mg Panax notoginseng polysaccharide stabilized cerium oxide are dissolved in 50 mL of deionized water to finally obtain hemostatic sponge 6.

[0089]

Example 7

[0090] In this embodiment, the preparation method is similar to that in Example 1. The difference is that in step (3), when preparing the first suspension, the amount of bacterial cellulose stabilized nano-silver added to the deionized water is adjusted to 25 mg, and the hemostatic sponge 7 is finally obtained.

[0091]

Example 8

[0092] In this embodiment, the preparation method is similar to that in Example 1. The difference is that in step (3), when preparing the first suspension, the amount of bacterial cellulose stabilized nano-silver added to the deionized water is adjusted to 50 mg, and the hemostatic sponge 8 is finally obtained.

[0093]

Example 9

[0094] In this embodiment, the preparation method is similar to that in Example 1. The difference is that in step (3), when preparing the first suspension, the amount of bacterial cellulose stabilized nano-silver added to the deionized water is adjusted to 200 mg, and the hemostatic sponge 9 is finally obtained.

[0095]

Example 10

[0096] In this embodiment, the preparation method is similar to that in Example 1. The difference is that in step (3), when preparing the first suspension, cerium oxide stabilized by Panax notoginseng polysaccharide and nano-silver stabilized by bacterial cellulose are not added. Instead, 500 mg of chitosan is added to deionized water to finally obtain hemostatic sponge 10.

[0097]

Example 11

[0098] In this embodiment, the preparation method is similar to that in Example 1. The difference is that in step (3), when preparing the second suspension, hexadecyltrimethoxysilane is not added, but only 500 mg of chitosan is added to deionized water to finally obtain hemostatic sponge 11.

[0099]

Example 12

[0100] In this embodiment, the preparation method is similar to that in Example 1, except that in step (3), when preparing the second suspension, the amount of hexadecyltrimethoxysilane added to the deionized water is adjusted to 250 mg, and the hemostatic sponge 12 is finally obtained.

[0101]

Example 13

[0102] In this embodiment, the preparation method is similar to that in Example 1. The difference is that in step (3), directional freezing is not used. Instead, the first suspension, the second suspension, and the first suspension are added sequentially to the polytetrafluoroethylene mold and then placed in a freezer at -20 ℃ for freezing to obtain hemostatic sponge 13.

[0103]

Example 14

[0104] In this embodiment, the preparation method is similar to that in Example 1. The difference is that in step (3), when preparing the first and second suspensions, cerium oxide stabilized by Panax notoginseng polysaccharide, nano-silver stabilized by bacterial cellulose, and hexadecyltrimethoxysilane are not added. Directional freezing is not used. Instead, the first suspension, the second suspension, and the first suspension are added sequentially to the polytetrafluoroethylene mold and frozen in a refrigerator at -20 ℃ to obtain hemostatic sponge 14.

[0105]

Example 15

[0106] In this embodiment, the preparation method is similar to that in Example 1, except that in step (3), when preparing the second suspension, the amount of hexadecyltrimethoxysilane added to the deionized water is adjusted to 1000 mg, and the hemostatic sponge 15 is finally obtained.

[0107] II. Performance Testing of Hemostatic Sponges

[0108]

Example 16

[0109] In this embodiment, the cell survival rate of hemostatic sponges containing different contents of cerium oxide stabilized by Panax notoginseng polysaccharide or nano-silver stabilized by bacterial cellulose was tested.

[0110] like Figure 5 As shown, when the loading of cerium oxide stabilized by Panax notoginseng polysaccharide was below 200 mg, none of the samples exhibited cytotoxicity. However, when the loading of cerium oxide stabilized by Panax notoginseng polysaccharide was further increased to 300 mg, cell viability significantly decreased to below 50%.

[0111] Similarly, such as Figure 6 As shown, when the loading of bacterial cellulose-stabilized silver nanoparticles was below 100 mg, none of the samples exhibited cytotoxicity. However, when the loading of bacterial cellulose-stabilized silver nanoparticles was further increased to 200 mg, cell viability significantly decreased to below 60%.

[0112] Therefore, considering the effects of antibacterial and anti-inflammatory capabilities, toxicity, and the influence of loading amount on the hydrophilicity of the orientation layer, in some preferred embodiments, the loading amount of cerium oxide stabilized by Panax notoginseng polysaccharide is 150-250 mg, and more preferably, the loading amount of cerium oxide stabilized by Panax notoginseng polysaccharide is 180-220 mg. In some preferred embodiments, the loading amount of silver nanoparticles stabilized by bacterial cellulose is 50-150 mg, and more preferably, the loading amount of silver nanoparticles stabilized by bacterial cellulose is 80-120 mg.

[0113]

Example 17

[0114] In this embodiment, to compare the hemostatic abilities of sponges loaded with Panax notoginseng polysaccharide-stabilized cerium oxide and Bletilla striata polysaccharide-stabilized cerium oxide, hemostatic sponge 16 and hemostatic sponge 17 were prepared respectively. The preparation methods of the two sponges were similar to those in Example 1. The difference was that hemostatic sponge 16 only added the first suspension to the mold and placed it in a freezer at -20 ℃; while hemostatic sponge 17 replaced the Panax notoginseng polysaccharide-stabilized cerium oxide in the first suspension with 200 mg of Bletilla striata polysaccharide-stabilized cerium oxide to prepare the first suspension, and after adding the first suspension to the mold, it was placed in a freezer at -20 ℃.

[0115] like Figure 7As shown, the hemostatic effect of sponges stabilized with Panax notoginseng polysaccharide at the same loading level was lower than that of sponges stabilized with Bletilla striata polysaccharide, indicating that sponges stabilized with Panax notoginseng polysaccharide exhibit superior hemostatic ability. Mechanistic analysis suggests that this may be because Panax notoginseng polysaccharide can better activate the coagulation process and coagulation pathway at high blood concentrations.

[0116]

Example 18

[0117] The amount of hexadecyltrimethoxysilane added affects the hydrophobicity of the hydrophobic random layer, and thus the hemostatic ability of the hemostatic sponge. Therefore, in this embodiment, the hemostatic index of hemostatic sponges with different amounts of hexadecyltrimethoxysilane added was tested.

[0118] Specifically, equal weight samples were incubated at 37 °C for 5 min, 50 μL of anticoagulated whole blood was added and incubated at 37 °C for another 5 min, then 3 mL of deionized water was added. The absorbance was measured after incubation for 5 min, and the BCI was calculated.

[0119] The test results are as follows Figure 8 As shown, the BCI index of the hemostatic sponge continuously decreased with increasing hexadecyltrimethoxysilane (HDTMS) content, indicating that the hydrophobicity of the hydrophobic random layer can provide appropriate pressure to improve coagulation. However, with the HDTMS content increasing to 1000 mg, the hydrophobicity of the hydrophobic random layer further increased, significantly reducing the hemostatic sponge's blood absorption capacity and increasing the coagulation index. Furthermore, the hydrophobic random layer also significantly reduced the connectivity of its two hydrophilic oriented layers, resulting in decreased hemostatic ability when there is a large amount of blood.

[0120] In some preferred embodiments, the amount of hexadecyltrimethoxysilane added to the hydrophobic random layer of the hemostatic sponge is 400-800 mg. More preferably, the amount of hexadecyltrimethoxysilane added is 400-600 mg.

[0121]

Example 19

[0122] In this embodiment, the water contact angles of the first and second hydrophilic oriented layers and the hydrophobic random layer of the hemostatic sponge 1 prepared in Example 1 were tested. The experimental results are as follows: Figure 9 As shown, the first and second hydrophilic oriented layers are both superhydrophilic, while the hydrophobic random layer in the middle is hydrophobic, indicating that a heterogeneous wetting hemostatic sponge has been obtained.

[0123]

Example 20

[0124] The addition of Panax notoginseng polysaccharides to the hydrophilic orientation layer stabilizes cerium oxide, enabling the utilization of cerium oxide based on Ce. 3+ / Ce 4+Its unique redox properties exhibit excellent enzyme-mimicking activity, effectively scavenging excess ROS and reducing ROS accumulation at wound sites. Furthermore, the dynamic oxygen reduction cycle cascade formed in cerium oxide significantly enhances O2... ·- And the removal efficiency of H2O2.

[0125] In this embodiment, the antioxidant capacity of hemostatic sponge 1, hemostatic sponge 2 and hemostatic sponge 14 were tested. Among them, hemostatic sponge 2 and hemostatic sponge 14 did not contain Panax notoginseng polysaccharide to stabilize cerium oxide.

[0126] The test results are as follows Figure 10 and Figure 11 As shown, hemostatic sponge 1 possesses excellent O2... ·- It has a clearing ability, with a clearance rate of over 90%. Simultaneously, the hemostatic sponge 1 also has the ability to clear H2O2, with a clearance rate of over 95%.

[0127] In this embodiment, the antibacterial properties of hemostatic sponge 1, hemostatic sponge 6 and hemostatic sponge 14 were also tested. Among them, hemostatic sponge 6 and hemostatic sponge 14 did not contain bacterial cellulose stabilized nano-silver.

[0128] E. coli and S. aureus were selected as typical bacterial representatives to study the broad-spectrum antibacterial activity of sponges. The experimental results are as follows: Figure 10 As shown, the antibacterial activity of hemostatic sponge 6, which only added Panax notoginseng polysaccharide to stabilize cerium oxide, was slightly better than that of hemostatic sponge 14, but the antibacterial activity of both was low. Hemostatic sponge 1, on the other hand, exhibited excellent antibacterial effects, with inhibition rates of over 95% and 96% against E. coli and S. aureus, respectively.

[0129]

Example 21

[0130] In this embodiment, the absorption performance of hemostatic sponges 1, 13, and 14 for water and blood was tested. Figure 12 and 13 As shown, thanks to the loose and porous structure of chitosan, all three types of hemostatic sponges exhibit high porosity, water absorption rate, and blood absorption rate. However, by forming a hemostatic sponge 1 with an oriented pore structure through directional freezing, the porosity of the material can be further improved, resulting in higher water and blood absorption rates and faster absorption speeds.

[0131]

Example 22

[0132] In this embodiment, the coagulation properties of hemostatic sponge 1, hemostatic sponge 13, and hemostatic sponge 14 were tested.

[0133] like Figure 14 As shown, GS is gelatin sponge, Celox TMThese are commercially available hemostatic materials. The GS group had the highest BCI value, followed by hemostatic sponge 13 and hemostatic sponge 14, with Celox... TM Group 1 can reduce BCI to some extent, but hemostatic sponge 1 showed the lowest BCI value. This indicates that the superhydrophilicity brought about by the oriented pore structure of hemostatic sponge 1, combined with the heterogeneous wettability of the non-orienting hydrophobic layer, can significantly improve the hemostatic ability of the hemostatic sponge. At the same time, the Panax notoginseng polysaccharide introduced into the hydrophilic oriented layer can also help improve the hemostatic performance.

[0134] Figure 15 The number of red blood cells and platelets adhered to the sponge was also shown. As shown in the figure, hemostatic sponge 1 exhibited the highest adhesion amount compared to other sponges and commercial hemostatic materials, further validating the above results.

[0135]

Example 23

[0136] In this embodiment, the in vivo hemostatic ability of hemostatic sponge 1, hemostatic sponge 13 and hemostatic sponge 14 was tested.

[0137] like Figure 16 As shown in the figure, a 5 mm diameter and 3 mm depth hole was punched in the rat liver using a sterile punch, and different materials were applied for hemostasis. The hemostasis time and blood loss of each group were measured and compared. The results are shown in the figure; the GS group showed persistent bleeding, similar to the Celox group. TM Compared with other hemostatic sponge groups, hemostatic sponge 1 showed a significantly shorter hemostasis time and a significantly reduced blood loss. This demonstrates that in vivo data further validates that the oriented pore structure and heterotrophic wettability of hemostatic sponge 1 can significantly improve the sponge's hemostatic ability.

[0138]

Example 24

[0139] In this embodiment, hemostatic sponge 1, hemostatic sponge 14, and commercial 3M were tested. TM The body's ability to repair itself.

[0140] Specifically, diabetic rats were randomly divided into four groups. A 10 mm diameter full-thickness skin defect was created on the back using a circular perforator, and different materials were applied to each group for treatment. For example... Figure 17 As shown in the figure, statistical analysis of healing rates indicates that the healing effect of each sponge group is significantly better than that of 3M. TM The healing rates of the two groups, the hemostatic sponge group and the group with hemostatic sponge, reached over 95%, indicating that the introduction of Panax notoginseng polysaccharide-stabilized cerium oxide can effectively regulate the immune microenvironment to promote the repair process, reduce the expression of the M1 macrophage marker CD86, promote cell migration and angiogenesis, and thus significantly promote the wound healing process. Figure 18 At the same time, such as Figure 19As shown in the in vivo kidney slices, the hemostatic sponge also exhibits excellent biocompatibility.

[0141] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a heterogeneous wetting hemostatic sponge, characterized in that, Includes the following steps: Chitosan, Panax notoginseng polysaccharide-stabilized cerium oxide, and bacterial cellulose-stabilized silver nanoparticles were dissolved in an acidic aqueous solution to obtain a first solution. An alkali was then added to the first solution to obtain a first suspension. Chitosan and hexadecyltrimethoxysilane were dissolved in an acidic aqueous solution to obtain a second solution. An alkali was then added to the second solution to obtain a second suspension. The second suspension is frozen to obtain a frozen solid. Then, the first suspension, the frozen solid, and the first suspension are added to the mold in sequence, and the first suspension on both sides of the frozen solid is frozen in a directional manner to obtain the heterogeneous wettable hemostatic sponge.

2. The method for preparing a heterogeneous wetting hemostatic sponge according to claim 1, characterized in that, In the second suspension, the mass ratio of hexadecyltrimethoxysilane to chitosan is 1:1 to 1:

3.

3. The method for preparing a heterogeneous wetting hemostatic sponge according to claim 1, characterized in that, The height ratio of the first suspension to the frozen solid is 2:1 to 4:

1.

4. The method for preparing a heterogeneous wetting hemostatic sponge according to claim 1, characterized in that, The preparation of the cerium oxide stabilized by Panax notoginseng polysaccharide includes the following steps: After dissolving Panax notoginseng polysaccharide, cerium nitrate hexahydrate and ammonia were added to the solution. After the reaction was completed, the solution was dialyzed to obtain the stabilized cerium oxide of Panax notoginseng polysaccharide.

5. The method for preparing a heterogeneous wetting hemostatic sponge according to claim 4, characterized in that, In the first suspension, the mass ratio of the cerium oxide stabilized by Panax notoginseng polysaccharide to chitosan is 1:2 to 1:

10.

6. The method for preparing a heterogeneous wetting hemostatic sponge according to claim 1, characterized in that, The preparation of the bacterial cellulose-stabilized silver nanoparticles includes the following steps: After washing the bacterial cellulose, NaIO4 was added for oxidation in the dark. Oxidized bacterial cellulose reacts with silver nitrate solution in the dark, followed by dialysis. The dialyzed product was dissolved in a mixed solution of NaOH and urea, subjected to freeze-thaw treatment, dialyzed again, and then freeze-dried to obtain the bacterial cellulose-stabilized silver nanoparticles.

7. The method for preparing a heterogeneous wetting hemostatic sponge according to claim 6, characterized in that, In the first suspension, the mass ratio of bacterial cellulose-stabilized silver nanoparticles to chitosan is 1:5 to 1:

10.

8. The method for preparing a heterogeneous wetting hemostatic sponge according to claim 1, characterized in that, The mold is placed in a cold trap for freezing, with the bottom of the mold placed above the copper block for directional freezing of the first suspension located on one side of the frozen solid; subsequently, the top of the mold is placed above the copper block for directional freezing of the first suspension located on the other side of the frozen solid.

9. The method for preparing a heterogeneous wetting hemostatic sponge according to claim 1, characterized in that, After the first suspensions on both sides of the frozen solid are frozen, they are soaked in deionized water, then placed in a cold trap to freeze again, and finally freeze-dried to obtain the heterogeneous wettable hemostatic sponge.

10. A heterogeneous wetting hemostatic sponge, characterized in that, The heterogeneous wettable hemostatic sponge, prepared by any one of claims 1 to 9, comprises a first hydrophilic oriented layer and a second hydrophilic oriented layer having an oriented pore structure, wherein a hydrophobic random layer having a disordered pore structure is disposed between the first hydrophilic oriented layer and the second hydrophilic oriented layer, wherein the first hydrophilic oriented layer and the second hydrophilic oriented layer contain cerium oxide stabilized by Panax notoginseng polysaccharide and silver nanoparticles stabilized by bacterial cellulose.

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