A composite hemostatic sponge for massive bleeding from deep and narrow wounds and its preparation method

Abstract

This invention discloses a composite hemostatic sponge for deep and narrow wounds with massive bleeding and its preparation method. The composite hemostatic sponge is prepared by low-temperature polymerization and cross-linking of gelatin, dopamine, quaternized chitosan, and oxidized dextran, followed by freeze-drying. The composite hemostatic sponge possesses advantages such as high hemostatic efficiency, water-triggered shape recovery behavior, high compressive strength, strong antibacterial activity, safety and non-toxicity, and good blood / biocompatibility. It can effectively prevent fatal bleeding from deep and narrow wounds by combining physical and chemical hemostasis. The composite hemostatic sponge prepared by this invention has widely available raw materials, a simple preparation method, and can be mass-produced, showing potential clinical application value in bleeding scenarios such as deep and narrow wounds and ballistic injuries.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials and biomedical applications, specifically relating to a composite hemostatic sponge for deep and narrow wounds with massive bleeding and its preparation method. Background Technology

[0002] Uncontrollable massive bleeding is a leading cause of death and injury in war, surgery, natural disasters, and major accidents, accounting for over 30% of trauma deaths globally each year. Therefore, using highly effective hemostatic products as a pre-hospital treatment method to improve patient survival is crucial. Traditional methods such as gauze pressure, surgical sutures, and electrocoagulation are time-consuming, complex, and prone to secondary injuries, especially in cases of bleeding from incompressible areas like the torso and liver, where rapid hemostasis is difficult. Currently, various hemostatic materials have been developed for early bleeding control in wounded soldiers to reduce mortality, such as kaolin combat gauze, fibrin glue, gelatin sponges, polysaccharide porous microspheres, viscous hydrogels, and oxidized cellulose. However, these materials still cannot address massive bleeding from deep, narrow, penetrating wounds caused by bullets or sharp objects. Several new FDA-approved hemostatic agents for deep, massive bleeding wounds have been developed. For example, RevMedx has developed a product called XStat. TM The hemostatic device is a syringe filled with a large amount of compressed microfiber sponges. The numerous microfiber sponges in the syringe can be injected into the wound cavity, and after absorbing blood, they expand, rapidly filling deep wounds and creating physical pressure on the wound walls. Although XStat TM It can overcome the shortcomings of traditional compression hemostatic agents in deep wounds. It achieves the purpose of controlling incompressible bleeding by rapidly expanding to fill deep wounds. However, since several microfibrillary sponges need to be removed from the wound one by one afterward, it will cause additional pain to patients in clinical applications such as visceral repair surgery, and will also increase the operation time and cost.

[0003] In recent years, some polymer sponges with shape memory function have been designed as alternative methods for treating deep wounds. Despite these advances, these technologies also have limitations. For example, the internal cross-linked network of these sponge materials has poor interconnectivity, low water and blood absorption efficiency, and takes tens of seconds to recover its original shape after injection into the wound, seriously affecting the hemostatic effect. In addition, these materials cannot activate the intrinsic coagulation pathway and lack antibacterial activity against bacterial infection. Therefore, there is an urgent need to develop a class of hemostatic materials with advantages such as high hemostatic efficiency, good water-triggered shape recovery behavior, and strong antibacterial activity for the treatment of non-compressible wounds. Patent CN202211711650.8 discloses a method for preparing a chitosan hemostatic sponge. This method mixes chitosan, acetic acid solution, and hydrophilic substances to obtain a hydrophilic chitosan solution, and prepares a chitosan hemostatic sponge with a bilayer structure through a combination of pre-freezing and freeze-drying. The hydrophilic layer is close to the skin, and the coagulation layer is on the outside, allowing blood to quickly penetrate the material upon contact and achieve rapid hemostasis with the help of chitosan. However, chitosan alone has significant limitations in its antibacterial properties; its rigid texture makes it difficult to conform to the wound, reducing the pressure exerted on blood vessels and increasing the space for bacterial growth. Patent CN202211007835.0 discloses a hemostatic gel and sponge based on silk fibroin, its preparation method, and its application. This method mixes lauroyl arginine ethyl ester hydrochloride and silk fibroin to prepare an asymmetric porous hemostatic sponge. This hemostatic sponge has loose pores at one end to absorb blood, while the denser end seals the wound. The interconnected porous structure allows for rapid blood absorption and exhibits good antibacterial properties. However, due to its low compressive strength, more material needs to be used when applied to deep wounds, and the material is prone to collapse under pressure from blood pressure and the wound wall, limiting its use.

[0004] Therefore, this invention provides a composite hemostatic sponge for deep and narrow wounds with massive bleeding and its preparation method. The composite hemostatic sponge is prepared by low-temperature polymerization and cross-linking of gelatin, dopamine, quaternized chitosan, and oxidized dextran, followed by freeze-drying. The composite hemostatic sponge possesses advantages such as high hemostatic efficiency, water-triggered shape recovery behavior, high compressive strength, strong antibacterial activity, safety and non-toxicity, and good blood / biocompatibility. By combining physical expansion hemostasis with chemical hemostasis, it can effectively prevent fatal bleeding from deep and narrow wounds, and is expected to have potential clinical application value in bleeding scenarios such as deep and narrow wounds and ballistic injuries. Summary of the Invention

[0005] The technical problem solved by this invention is to provide a composite hemostatic sponge for deep and narrow wounds with massive bleeding and its preparation method. Under the action of the oxidant NaIO4 and the catalytic system EDC and NHS, the raw materials gelatin, dopamine, quaternized chitosan, and oxidized dextran polymerize and crosslink at low temperatures through the formation of amide bonds, hydrogen bonds, and Schiff base bonds, thus generating a continuous and uniform porous network. This gives the material strong water absorption; upon contact with water, it expands back to its initial shape, providing physical pressure to the oral cavity wall during application. Simultaneously, the presence of multiple crosslinking mechanisms results in excellent mechanical properties. The intrinsic hemostatic properties of dopamine, quaternized chitosan, and oxidized dextran can activate endogenous hemostatic pathways, improving hemostatic efficiency through chemical hemostasis. Furthermore, the positively charged protonated amino groups on the quaternized chitosan molecular chain can not only physically crosslink blood cells to form an adhesion barrier but also extend to the bacterial cell membrane to kill bacteria. Therefore, the composite hemostatic sponge disclosed in this invention exhibits excellent mechanical properties, high hemostatic efficiency, rapid water-triggered recovery behavior, safety and non-toxicity, and ease of operation, and is expected to realize potential clinical application value in bleeding scenarios such as deep and narrow wounds and ballistic injuries.

[0006] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0007] A method for preparing a composite hemostatic sponge for massive bleeding from deep, narrow wounds includes the following steps:

[0008] (1) Dissolve gelatin in deionized water and stir evenly at a certain temperature to obtain solution S1;

[0009] (2) Dissolve quaternized chitosan in deionized water and stir evenly at a certain temperature to obtain solution S2;

[0010] (3) Synthesis of oxidized dextran: Dextran was dissolved in deionized water, sodium periodate (NaIO4) was added, and the mixture was stirred at room temperature in the dark. After the reaction was completed, diethylene glycol was added to quench the reaction, followed by dialyzing and freeze-drying to obtain oxidized dextran. Oxidized dextran was dissolved in PBS buffer (0.1 mol / L, pH = 7.4) to obtain solution S3;

[0011] (4) Dissolve dopamine in deionized water and stir until homogeneous at room temperature to obtain solution S4;

[0012] (5) Dissolve EDC in deionized water and stir evenly at room temperature to obtain solution S5; dissolve NHS in deionized water and stir evenly at room temperature to obtain solution S6;

[0013] (6) Dissolve the oxidant in deionized water and stir until homogeneous at room temperature to obtain solution S7;

[0014] (7) In an ice bath, solutions S2, S3, and S4 are added to solution S1 and stirred until homogeneous. Then, solutions S5, S6, and S7 are added sequentially to obtain precursor solution S8. The precursor solution S8 is polymerized at low temperature, then dialyzed and freeze-dried to obtain a composite hemostatic sponge for deep and narrow wounds with massive bleeding.

[0015] Preferably, the concentration of solution S1 in step (1) is 0.1-10 wt%, and the stirring temperature is 10-80℃.

[0016] Preferably, the concentration of solution S2 in step (2) is 0.1-20 wt%, and the stirring temperature is 10-70℃.

[0017] Preferably, the molar ratio of dextran to NaIO4 and diethylene glycol in step (3) is 20:1:1 to 1:1:1; the reaction time in the dark is 12-48 h; the quenching reaction time is 0.1-6 h; and the concentration of oxidized dextran in solution S3 is 0.1-20 wt%.

[0018] Preferably, the concentration of solution S4 in step (4) is 0.1-30 wt%.

[0019] Preferably, the concentration of solution S5 in step (5) is 0.01-3 wt%; and the concentration of solution S6 is 0.01-3 wt%.

[0020] Preferably, the oxidant in step (6) is NaIO4, and the concentration of solution S7 is 0.1-15 wt%.

[0021] Preferably, in step (7), the volume ratio of solution S1:S2:S3:S4 is 1:0.5:1:0.7-200:50:200:70; the volume ratio of solution S1 to solutions S5 and S6 is 1:0.1:0.1-500:50:50; the volume ratio of solution S4 to solution S7 is 1:1-1:9; the precursor solution S8 is subjected to low-temperature polymerization reaction at -80 to -20℃ for 24 to 48 hours, thawed, dialyzed, and freeze-dried to obtain a composite hemostatic sponge for deep and narrow wounds with massive bleeding.

[0022] Compared with existing technologies, this invention has the following technical advantages: The composite hemostatic sponge prepared by this invention, under the action of the oxidant NaIO4 and the catalytic system EDC and NHS, allows the raw materials gelatin, dopamine, quaternized chitosan, and oxidized dextran to polymerize and crosslink at low temperatures through the formation of amide bonds, hydrogen bonds, and Schiff base bonds. This imparts a continuous and uniform porous network structure to the composite sponge, enabling it to rapidly absorb and concentrate red blood cells and platelets in water and blood environments. Benefiting from this multi-mode crosslinking, its excellent compressive mechanical properties and rapid liquid-triggered morphological expansion recovery ability enable it to achieve physical compression hemostasis when applied to deep and narrow wounds. The intrinsic hemostatic properties of dopamine, quaternized chitosan, and oxidized dextran can activate endogenous hemostatic pathways, improving hemostatic efficiency through chemical hemostasis. This combination of physical and chemical hemostasis methods can effectively prevent fatal bleeding from deep and narrow wounds. Furthermore, the positively charged protonated amino groups on the quaternized chitosan molecular chain can not only physically cross-link blood cells to form an adhesion barrier, but also extend to the bacterial cell membrane to kill bacteria, thus endowing the composite sponge with good antibacterial activity. In summary, the composite hemostatic sponge designed in this invention has advantages such as dual-mode hemostasis, high hemostatic efficiency, strong mechanical properties, liquid-triggered recovery ability, good biocompatibility / blood compatibility, and ease of operation, and is expected to realize potential clinical application value in bleeding scenarios such as deep and narrow wounds and ballistic injuries. Attached Figure Description

[0023] Figure 1 This is a scanning electron microscope image of the composite hemostatic sponge prepared in Example 5 for massive bleeding from deep and narrow wounds.

[0024] Figure 2 The image shows a quantitative analysis of the compressive strength of the composite hemostatic sponges prepared in Example 5 and Comparative Example 1 for massive bleeding from deep and narrow wounds.

[0025] Figure 3 The maximum absorption rate of (A) water and (B) anticoagulated rabbit blood of the composite hemostatic sponges for deep and narrow wounds with massive bleeding prepared in Example 5 and Comparative Example 1.

[0026] Figure 4 The recovery time of the composite hemostatic sponges prepared in Example 5 and Comparative Example 1 for deep and narrow wound hemorrhage after triggering in (A) water and (B) anticoagulated rabbit blood.

[0027] Figure 5 The hemolysis rate test results are shown for the composite hemostatic sponges prepared in Example 5 and Comparative Example 1 for massive bleeding from deep and narrow wounds.

[0028] Figure 6 In vitro coagulation dynamics characterization of the composite hemostatic sponge prepared for deep and narrow wounds with massive bleeding, as used in Example 5 and the comparative examples.

[0029] Figure 7 The cell proliferation capacity of the composite hemostatic sponge prepared for deep and narrow wound hemorrhage prepared in Example 5 and Comparative Example 1 after co-culturing with NIH / 3T3 cells for 1, 3 and 7 days. Detailed Implementation

[0030] To further understand the present invention, preferred embodiments are described below with reference to examples. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, and not for limiting the scope of the claims. Furthermore, it should be understood that after reading the description of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0031] This invention discloses a composite hemostatic sponge for massive bleeding from deep, narrow wounds and its preparation method, comprising the following steps:

[0032] (1) Dissolve gelatin in deionized water and stir evenly at a certain temperature to obtain solution S1;

[0033] (2) Dissolve quaternized chitosan in deionized water and stir evenly at a certain temperature to obtain solution S2;

[0034] (3) Synthesis of oxidized dextran: Dextran was dissolved in deionized water, NaIO4 was added, and the mixture was stirred at room temperature in the dark. After the reaction was completed, diethylene glycol was added to quench the reaction, followed by dialyzing and freeze-drying to obtain oxidized dextran. Oxidized dextran was dissolved in PBS buffer (0.1 mol / L, pH = 7.4) to obtain solution S3;

[0035] (4) Dissolve dopamine in deionized water and stir until homogeneous at room temperature to obtain solution S4;

[0036] (5) Dissolve EDC in deionized water and stir evenly at room temperature to obtain solution S5; dissolve NHS in deionized water and stir evenly at room temperature to obtain solution S6;

[0037] (6) Dissolve the oxidant in deionized water and stir until homogeneous at room temperature to obtain solution S7;

[0038] (7) In an ice bath, solutions S2, S3, and S4 are added to solution S1 and stirred until homogeneous. Then, solutions S5, S6, and S7 are added sequentially to obtain precursor solution S8. The precursor solution S8 is polymerized at low temperature, then dialyzed and freeze-dried to obtain a composite hemostatic sponge for deep and narrow wounds with massive bleeding.

[0039] In this invention, firstly, a certain amount of gelatin is weighed and dissolved in deionized water, and stirred evenly at a certain temperature to obtain solution S1; the concentration of solution S1 is 0.1-8 wt%, and the stirring temperature is 10-70℃. More preferably, the concentration of solution S1 is 0.1-5 wt%, and the stirring temperature is 25-60℃. Secondly, a certain amount of quaternized chitosan is weighed and dissolved in deionized water, and stirred evenly at a certain temperature to obtain solution S2; the concentration of solution S2 is 0.1-15 wt%, and the stirring temperature is 10-60℃. More preferably, the concentration of solution S2 is 0.1-10 wt%, and the stirring temperature is 25-50℃.

[0040] Subsequently, the dextran was modified by dissolving it in deionized water, adding NaIO4, and stirring the mixture at room temperature in the dark. After the reaction was complete, diethylene glycol was added to quench the reaction, followed by dialyzing and freeze-drying to obtain oxidized dextran. The oxidized dextran was dissolved in PBS buffer (0.1 mol / L, pH = 7.4) to obtain solution S3. The molar ratio of dextran to NaIO4 and diethylene glycol was 8:1:1 to 1:1:1; the reaction time in the dark was 12-36 h; the quenching reaction time was 0.5-3 h; and the concentration of oxidized dextran solution S3 was 0.1-15 wt%. More preferably, the molar ratio of dextran to NaIO4 and diethylene glycol was 6:1:1 to 1:1:1; the reaction time in the dark was 12-30 h; the quenching reaction time was 0.5-2.5 h; and the concentration of oxidized dextran solution S3 was 0.1-10 wt%.

[0041] A certain amount of dopamine is weighed and dissolved in deionized water to obtain solution S4; the concentration of solution S4 is 0.1-20 wt%. More preferably, the concentration of solution S4 is 0.1-15 wt%. A certain amount of EDC and NHS are weighed and dissolved in deionized water to obtain solutions S5 and S6; the concentrations of solutions S5 and S6 are both 0.01-2 wt%. More preferably, the concentrations of solutions S5 and S6 are 0.01-1.5 wt% and 0.01-1 wt%, respectively. A certain amount of oxidant NaIO4 is weighed and dissolved in deionized water to obtain solution S7; the concentration of solution S7 is 0.1-10 wt%. More preferably, the concentration of solution S7 is 0.1-8 wt%.

[0042] Finally, in an ice bath, solutions S2, S3, and S4 were added to solution S1 and stirred until homogeneous. Then, solutions S5, S6, and S7 were added sequentially to obtain precursor solution S8. Precursor solution S8 was polymerized at low temperature, followed by dialysis and freeze-drying to obtain a composite hemostatic sponge for deep, narrow wounds with massive bleeding. The volume ratio of solutions S1:S2:S3:S4 was 1:0.5:1:0.7-200:50:200:70; the volume ratio of solution S1 to solutions S5 and S6 was 1:0.1:0.1-500:50:50; and the volume ratio of solution S4 to solution S7 was 1:1-1:9. Precursor solution S8 was polymerized at -80 to -20°C for 24-48 hours. More preferably, the volume ratio of solutions S1:S2:S3:S4 is 1:0.5:1:0.7-100:25:100:35; the volume ratio of solution S1 to solutions S5 and S6 is 1:0.1:0.1-300:30:30; the volume ratio of solution S4 to solution S7 is 1:1-1:6; and the precursor solution S8 is subjected to a low-temperature polymerization reaction at -80 to -36°C for 30 to 48 hours.

[0043] To further understand the present invention, preferred embodiments of the present invention are described below in conjunction with examples. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, and not for limiting the scope of the claims of the present invention.

[0044] Example 1

[0045] A composite hemostatic sponge for massive bleeding from deep, narrow wounds and its preparation method, comprising the following steps:

[0046] (1) Dissolve 0.3g of gelatin in 10mL of deionized water at 50℃ by stirring to obtain solution S1.

[0047] (2) Dissolve 0.25g of quaternized chitosan in 5mL of deionized water at 45℃ to obtain solution S2.

[0048] (3) Dissolve 12.5 g of dextran (100 kDa, 77 mmol glucose monomer) in 500 mL of deionized water to obtain a dextran solution. Then dissolve 16.19 g of sodium periodate (77 mmol) in 125 mL of deionized water and add it to the dextran solution. Stir and react in the dark for 24 h. Quench the reaction with an equimolar amount of diethylene glycol and stir for 2 h. Then, dialyze the reacted mixture in pure water for more than 3 days using a dialysis membrane. Remove the dextran solution from the dialysis bag and freeze-dry it to form a white oxidized dextran powder. Dissolve 0.8 g of oxidized dextran in 10 mL of PBS buffer (0.1 mol / L, pH = 7.4) to obtain solution S3.

[0049] (4) Dissolve 0.25g of dopamine in 5mL of deionized water to obtain solution S4.

[0050] (5) Weigh 0.3g of EDC and dissolve it in 4mL of deionized water to obtain solution S5; weigh 0.18g of NHS and dissolve it in 4mL of deionized water to obtain solution S6.

[0051] (6) In an ice bath, solutions S1, S2, S3, and S4 were stirred evenly in a volume ratio of 2:1:1:1. Then, solutions S5 and S6 were added sequentially at a volume of 0.25 times that of solution S1, and solution S7 was added at a volume of 3 times that of solution S4, finally yielding precursor solution S8. The obtained precursor solution S8 was polymerized at -80℃ for 36 hours, followed by dialysis and freeze-drying to obtain the composite hemostatic sponge GPOQ for deep and narrow wounds with massive bleeding.

[0052] Example 2

[0053] A composite hemostatic sponge for massive bleeding from deep, narrow wounds and its preparation method, comprising the following steps:

[0054] (1) Dissolve 0.3g of gelatin in 10mL of deionized water at 50℃ by stirring to obtain solution S1.

[0055] (2) Dissolve 0.25g of quaternized chitosan in 5mL of deionized water at 45℃ to obtain solution S2.

[0056] (3) Dissolve 12.5 g of dextran (100 kDa, 77 mmol glucose monomer) in 500 mL of deionized water to obtain a dextran solution. Then dissolve 16.19 g of sodium periodate (77 mmol) in 125 mL of deionized water and add it to the dextran solution. Stir and react in the dark for 24 h. Quench the reaction with an equimolar amount of diethylene glycol and stir for 2 h. Then, dialyze the reacted mixture in pure water for more than 3 days using a dialysis membrane. Remove the dextran solution from the dialysis bag and freeze-dry it to form a white oxidized dextran powder. Dissolve 0.8 g of oxidized dextran in 10 mL of PBS buffer (0.1 mol / L, pH = 7.4) to obtain solution S3.

[0057] (4) Dissolve 0.25g of dopamine in 5mL of deionized water to obtain solution S4.

[0058] (5) Weigh 0.3g of EDC and dissolve it in 4mL of deionized water to obtain solution S5; weigh 0.18g of NHS and dissolve it in 4mL of deionized water to obtain solution S6.

[0059] (6) In an ice bath, solutions S1, S2, S3, and S4 were stirred evenly in a volume ratio of 2:1:2:1. Then, solutions S5 and S6 were added sequentially at a volume of 0.25 times that of solution S1, and solution S7 was added at a volume of 3 times that of solution S4, finally yielding precursor solution S8. The obtained precursor solution S8 was polymerized at -80℃ for 36 hours, followed by dialysis and freeze-drying to obtain the composite hemostatic sponge GPOQ for deep and narrow wounds with massive bleeding.

[0060] Example 3

[0061] A composite hemostatic sponge for massive bleeding from deep, narrow wounds and its preparation method, comprising the following steps:

[0062] (1) Dissolve 0.3g of gelatin in 10mL of deionized water at 50℃ by stirring to obtain solution S1.

[0063] (2) Dissolve 0.25g of quaternized chitosan in 5mL of deionized water at 45℃ to obtain solution S2.

[0064] (3) Dissolve 12.5 g of dextran (100 kDa, 77 mmol glucose monomer) in 500 mL of deionized water to obtain a dextran solution. Then dissolve 16.19 g of sodium periodate (77 mmol) in 125 mL of deionized water and add it to the dextran solution. Stir and react in the dark for 24 h. Quench the reaction with an equimolar amount of diethylene glycol and stir for 2 h. Then, dialyze the reacted mixture in pure water for more than 3 days using a dialysis membrane. Remove the dextran solution from the dialysis bag and freeze-dry it to form a white oxidized dextran powder. Dissolve 0.8 g of oxidized dextran in 10 mL of PBS buffer (0.1 mol / L, pH = 7.4) to obtain solution S3.

[0065] (4) Dissolve 0.25g of dopamine in 5mL of deionized water to obtain solution S4.

[0066] (5) Weigh 0.3g of EDC and dissolve it in 4mL of deionized water to obtain solution S5; weigh 0.18g of NHS and dissolve it in 4mL of deionized water to obtain solution S6.

[0067] (6) In an ice bath, solutions S1, S2, S3, and S4 were stirred evenly in a volume ratio of 2:1:1:1. Then, solutions S5 and S6 were added sequentially at a volume of 0.25 times that of solution S1, and solution S7 was added at a volume of 2 times that of solution S4, finally yielding precursor solution S8. The obtained precursor solution S8 was polymerized at -80℃ for 36 hours, followed by dialysis and freeze-drying to obtain the composite hemostatic sponge GPOQ for deep and narrow wounds with massive bleeding.

[0068] Example 4

[0069] A composite hemostatic sponge for massive bleeding from deep, narrow wounds and its preparation method, comprising the following steps:

[0070] (1) Dissolve 0.25g of gelatin in 10mL of deionized water at 50℃ by stirring to obtain solution S1.

[0071] (2) Dissolve 0.25g of quaternized chitosan in 5mL of deionized water at 45℃ to obtain solution S2.

[0072] (3) Dissolve 12.5 g of dextran (100 kDa, 77 mmol glucose monomer) in 500 mL of deionized water to obtain a dextran solution. Then dissolve 16.19 g of sodium periodate (77 mmol) in 125 mL of deionized water and add it to the dextran solution. Stir and react in the dark for 24 h. Quench the reaction with an equimolar amount of diethylene glycol and stir for 2 h. Then, dialyze the reacted mixture in pure water for more than 3 days using a dialysis membrane. Remove the dextran solution from the dialysis bag and freeze-dry it to form a white oxidized dextran powder. Dissolve 0.8 g of oxidized dextran in 10 mL of PBS buffer (0.1 mol / L, pH = 7.4) to obtain solution S3.

[0073] (4) Dissolve 0.25g of dopamine in 5mL of deionized water to obtain solution S4.

[0074] (5) Weigh 0.3g of EDC and dissolve it in 4mL of deionized water to obtain solution S5; weigh 0.18g of NHS and dissolve it in 4mL of deionized water to obtain solution S6.

[0075] (6) In an ice bath, solutions S1, S2, S3, and S4 were stirred evenly in a volume ratio of 2:1:1:1. Then, solutions S5 and S6 were added sequentially at a volume of 0.25 times that of solution S1, and solution S7 was added at a volume of 3 times that of solution S4, finally yielding precursor solution S8. The obtained precursor solution S8 was polymerized at -80℃ for 36 hours, followed by dialysis and freeze-drying to obtain the composite hemostatic sponge GPOQ for deep and narrow wounds with massive bleeding.

[0076] Example 5

[0077] A composite hemostatic sponge for massive bleeding from deep, narrow wounds and its preparation method, comprising the following steps:

[0078] (1) Dissolve 0.25g of gelatin in 10mL of deionized water at 50℃ by stirring to obtain solution S1.

[0079] (2) Dissolve 0.25g of quaternized chitosan in 5mL of deionized water at 45℃ to obtain solution S2.

[0080] (3) Dissolve 12.5 g of dextran (100 kDa, 77 mmol glucose monomer) in 500 mL of deionized water to obtain a dextran solution. Then dissolve 16.19 g of sodium periodate (77 mmol) in 125 mL of deionized water and add it to the dextran solution. Stir and react in the dark for 24 h. Quench the reaction with an equimolar amount of diethylene glycol and stir for 2 h. Then, dialyze the reacted mixture in pure water for more than 3 days using a dialysis membrane. Remove the dextran solution from the dialysis bag and freeze-dry it to form a white oxidized dextran powder. Dissolve 0.8 g of oxidized dextran in 10 mL of PBS buffer (0.1 mol / L, pH = 7.4) to obtain solution S3.

[0081] (4) Dissolve 0.25g of dopamine in 5mL of deionized water to obtain solution S4.

[0082] (5) Weigh 0.3g of EDC and dissolve it in 4mL of deionized water to obtain solution S5; weigh 0.18g of NHS and dissolve it in 4mL of deionized water to obtain solution S6.

[0083] (6) In an ice bath, solutions S1, S2, S3, and S4 were stirred evenly in a volume ratio of 2:1:2:1. Then, solutions S5 and S6 were added sequentially at a volume of 0.25 times that of solution S1, and solution S7 was added at a volume of 3 times that of solution S4, finally yielding precursor solution S8. The obtained precursor solution S8 was polymerized at -80℃ for 36 hours, followed by dialysis and freeze-drying to obtain the composite hemostatic sponge GPOQ for deep and narrow wounds with massive bleeding.

[0084] Example 6

[0085] A composite hemostatic sponge for massive bleeding from deep, narrow wounds and its preparation method, comprising the following steps:

[0086] (1) Dissolve 0.25g of gelatin in 10mL of deionized water at 50℃ by stirring to obtain solution S1.

[0087] (2) Dissolve 0.25g of quaternized chitosan in 5mL of deionized water at 45℃ to obtain solution S2.

[0088] (3) Dissolve 12.5 g of dextran (100 kDa, 77 mmol glucose monomer) in 500 mL of deionized water to obtain a dextran solution. Then dissolve 16.19 g of sodium periodate (77 mmol) in 125 mL of deionized water and add it to the dextran solution. Stir and react in the dark for 24 h. Quench the reaction with an equimolar amount of diethylene glycol and stir for 2 h. Then, dialyze the reacted mixture in pure water for more than 3 days using a dialysis membrane. Remove the dextran solution from the dialysis bag and freeze-dry it to form a white oxidized dextran powder. Dissolve 0.8 g of oxidized dextran in 10 mL of PBS buffer (0.1 mol / L, pH = 7.4) to obtain solution S3.

[0089] (4) Dissolve 0.25g of dopamine in 5mL of deionized water to obtain solution S4.

[0090] (5) Weigh 0.3g of EDC and dissolve it in 4mL of deionized water to obtain solution S5; weigh 0.18g of NHS and dissolve it in 4mL of deionized water to obtain solution S6.

[0091] (6) In an ice bath, solutions S1, S2, S3, and S4 were stirred evenly in a volume ratio of 2:1:2:1. Then, solutions S5 and S6 were added sequentially at a volume of 0.25 times that of solution S1, and solution S7 was added at a volume of 2 times that of solution S4, finally yielding precursor solution S8. The obtained precursor solution S8 was polymerized at -80℃ for 36 hours, followed by dialysis and freeze-drying to obtain the composite hemostatic sponge GPOQ for deep and narrow wounds with massive bleeding.

[0092] Example 7

[0093] A composite hemostatic sponge for massive bleeding from deep, narrow wounds and its preparation method, comprising the following steps:

[0094] (1) Dissolve 0.25g of gelatin in 10mL of deionized water at 50℃ by stirring to obtain solution S1.

[0095] (2) Dissolve 0.25g of quaternized chitosan in 5mL of deionized water at 45℃ to obtain solution S2.

[0096] (3) Dissolve 12.5 g of dextran (100 kDa, 77 mmol glucose monomer) in 500 mL of deionized water to obtain a dextran solution. Then dissolve 16.19 g of sodium periodate (77 mmol) in 125 mL of deionized water and add it to the dextran solution. Stir and react in the dark for 24 h. Quench the reaction with an equimolar amount of diethylene glycol and stir for 2 h. Then, dialyze the reacted mixture in pure water for more than 3 days using a dialysis membrane. Remove the dextran solution from the dialysis bag and freeze-dry it to form a white oxidized dextran powder. Dissolve 0.8 g of oxidized dextran in 10 mL of PBS buffer (0.1 mol / L, pH = 7.4) to obtain solution S3.

[0097] (4) Dissolve 0.25g of dopamine in 5mL of deionized water to obtain solution S4.

[0098] (5) Weigh 0.3g of EDC and dissolve it in 4mL of deionized water to obtain solution S5; weigh 0.18g of NHS and dissolve it in 4mL of deionized water to obtain solution S6.

[0099] (6) In an ice bath, solutions S1, S2, S3, and S4 were stirred evenly in a volume ratio of 2:2:2:1. Then, solutions S5 and S6 were added sequentially at a volume of 0.25 times that of solution S1, and solution S7 was added at a volume of 3 times that of solution S4, finally yielding precursor solution S8. The obtained precursor solution S8 was polymerized at -80℃ for 36 hours, followed by dialysis and freeze-drying to obtain the composite hemostatic sponge GPOQ for deep and narrow wounds with massive bleeding.

[0100] Example 8

[0101] A composite hemostatic sponge for massive bleeding from deep, narrow wounds and its preparation method, comprising the following steps:

[0102] (1) Dissolve 0.25g of gelatin in 10mL of deionized water at 50℃ by stirring to obtain solution S1.

[0103] (2) Dissolve 0.25g of quaternized chitosan in 5mL of deionized water at 45℃ to obtain solution S2.

[0104] (3) Dissolve 12.5 g of dextran (100 kDa, 77 mmol glucose monomer) in 500 mL of deionized water to obtain a dextran solution. Then dissolve 16.19 g of sodium periodate (77 mmol) in 125 mL of deionized water and add it to the dextran solution. Stir and react in the dark for 24 h. Quench the reaction with an equimolar amount of diethylene glycol and stir for 2 h. Then, dialyze the reacted mixture in pure water for more than 3 days using a dialysis membrane. Remove the dextran solution from the dialysis bag and freeze-dry it to form a white oxidized dextran powder. Dissolve 0.8 g of oxidized dextran in 10 mL of PBS buffer (0.1 mol / L, pH = 7.4) to obtain solution S3.

[0105] (4) Dissolve 0.25g of dopamine in 5mL of deionized water to obtain solution S4.

[0106] (5) Weigh 0.3g of EDC and dissolve it in 4mL of deionized water to obtain solution S5; weigh 0.18g of NHS and dissolve it in 4mL of deionized water to obtain solution S6.

[0107] (6) In an ice bath, solutions S1, S2, S3, and S4 were stirred evenly in a volume ratio of 2:2:2:1. Then, solutions S5 and S6 were added sequentially at a volume of 0.25 times that of solution S1, and solution S7 was added at a volume of 2 times that of solution S4, finally yielding precursor solution S8. The obtained precursor solution S8 was polymerized at -80℃ for 36 hours, followed by dialysis and freeze-drying to obtain the composite hemostatic sponge GPOQ for deep and narrow wounds with massive bleeding.

[0108] Comparative Example 1 (without quaternized chitosan)

[0109] (1) Dissolve 0.25g of gelatin in 10mL of deionized water at 50℃ by stirring to obtain solution S1.

[0110] (2) Dissolve 12.5 g of dextran (100 kDa, 77 mmol glucose monomer) in 500 mL of deionized water to obtain a dextran solution. Then dissolve 16.19 g of sodium periodate (77 mmol) in 125 mL of deionized water and add it to the dextran solution. Stir and react in the dark for 24 h. Quench the reaction with an equimolar amount of diethylene glycol and stir for 2 h. Then, dialyze the reacted mixture in pure water for more than 3 days using a dialysis membrane. Remove the dextran solution from the dialysis bag and freeze-dry it to form a white oxidized dextran powder. Dissolve 0.8 g of oxidized dextran in 10 mL of PBS buffer (0.1 mol / L, pH = 7.4) to obtain solution S2.

[0111] (3) Dissolve 0.25g of dopamine in 5mL of deionized water to obtain solution S3.

[0112] (4) Weigh 0.3g of EDC and dissolve it in 4mL of deionized water to obtain solution S4; weigh 0.18g of NHS and dissolve it in 4mL of deionized water to obtain solution S5.

[0113] (5) In an ice bath, solutions S1, S2, and S3 were stirred evenly in a volume ratio of 2:2:1. Then, solutions S4 and S5 were added sequentially at a volume of 0.25 times that of solution S1, and solution S6 was added at a volume of 3 times that of solution S3, finally yielding precursor solution S7. The obtained precursor solution S7 was polymerized at -80℃ for 36 hours, followed by dialysis and freeze-drying to obtain the composite hemostatic sponge GPO.

[0114] Comparative Example 2 (reduced quaternized chitosan content)

[0115] (1) Dissolve 0.25g of gelatin in 10mL of deionized water at 50℃ by stirring to obtain solution S1.

[0116] (2) Dissolve 0.15g of quaternized chitosan in 5mL of deionized water at 45℃ by stirring to obtain solution S2.

[0117] (3) Dissolve 12.5 g of dextran (100 kDa, 77 mmol glucose monomer) in 500 mL of deionized water to obtain a dextran solution. Then dissolve 16.19 g of sodium periodate (77 mmol) in 125 mL of deionized water and add it to the dextran solution. Stir and react in the dark for 24 h. Quench the reaction with an equimolar amount of diethylene glycol and stir for 2 h. Then, dialyze the reacted mixture in pure water for more than 3 days using a dialysis membrane. Remove the dextran solution from the dialysis bag and freeze-dry it to form a white oxidized dextran powder. Dissolve 0.8 g of oxidized dextran in 10 mL of PBS buffer (0.1 mol / L, pH = 7.4) to obtain solution S3.

[0118] (4) Dissolve 0.25g of dopamine in 5mL of deionized water to obtain solution S4.

[0119] (5) Weigh 0.3g of EDC and dissolve it in 4mL of deionized water to obtain solution S5; weigh 0.18g of NHS and dissolve it in 4mL of deionized water to obtain solution S6.

[0120] (6) In an ice bath, solutions S1, S2, S3, and S4 were stirred evenly in a volume ratio of 2:1:2:1. Then, solutions S5 and S6 were added sequentially at a volume of 0.25 times that of solution S1, and solution S7 was added at a volume of 3 times that of solution S4, finally yielding precursor solution S8. The obtained precursor solution S8 was polymerized at -80℃ for 36 hours, followed by dialysis and freeze-drying to obtain the composite hemostatic sponge GPOQ.

[0121] The scanning electron microscope image of the composite hemostatic sponge prepared in Example 5 for massive bleeding from deep and narrow wounds is shown below. Figure 1 As shown in the figure. Microscopic observation at high and low magnification revealed that the obtained GPOQ composite hemostatic sponge has a highly continuous porous network structure. This structure is beneficial for enhancing the mechanical properties of the composite sponge, improving its ability to absorb liquid, accelerating blood clotting at the wound site, and providing a scaffold for cell growth and proliferation to accelerate the repair process.

[0122] The compressive strength of the composite hemostatic sponges prepared in Example 5 and Comparative Example 1 for massive bleeding from deep and narrow wounds is quantitatively as follows: Figure 2 As shown in the figure. The results showed that the compressive strengths of the GPOQ and GPO composite hemostatic sponges were 103.54±9.15 kPa and 82.42±7.23 kPa, respectively. The compressive strength of the composite sponge was improved after the introduction of quaternized chitosan, which is attributed to the fact that the amino groups of its side chains can form amide bonds with the carboxyl groups of gelatin and the aldehyde groups of oxidized dextran, respectively, to combine with Schiff bases, thereby increasing the cross-linking degree of the sponge network.

[0123] The maximum absorption rates of water and anticoagulated rabbit blood of the composite hemostatic sponges prepared in Example 5 and Comparative Example 1 for massive bleeding from deep and narrow wounds are as follows: Figure 3 As shown in the figure. Experimental results show that the maximum absorption rates of GPOQ and GPO composite hemostatic sponges in water were 5973.02% ± 229.13% and 5139.71% ± 308.47%, respectively. In anticoagulated rabbit blood, the maximum absorption rates were 5161.83% ± 312.69% and 4732.98% ± 284.41%, respectively. The porous network structure endows the composite sponge with a high absorption rate, enabling it to rapidly absorb blood and water from deep, narrow wounds with massive bleeding, concentrating platelets and red blood cells to accelerate hemostasis.

[0124] The recovery time of the composite hemostatic sponges prepared in Example 5 and Comparative Example 1 for massive bleeding from deep and narrow wounds after triggering in water and anticoagulated rabbit blood is as follows: Figure 4As shown in the figure. The results showed that the shape recovery times of GPOQ and GPO composite hemostatic sponges in water were 2.76±0.23s and 4.12±0.11s, respectively. In anticoagulated rabbit blood, the shape recovery times were 17.03±2.16s and 23.69±3.87s, respectively. The composite sponge prepared in Example 5 exhibited a faster shape recovery rate in both water and anticoagulated rabbit blood compared to Comparative Example 1. Due to the viscosity of blood and the adhesion and enrichment of blood cells and platelets by the sponge network during absorption, the recovery time of the composite sponge in the blood environment was prolonged compared to the water environment.

[0125] The hemolysis rate test results of the composite hemostatic sponges prepared in Example 5 and Comparative Example 1 for massive bleeding from deep and narrow wounds are shown in the figure below. Figure 5 As shown, both GPOQ and GPO composite hemostatic sponges exhibited a hemolysis rate of less than 5%, meeting national standards for medical materials and demonstrating good blood compatibility.

[0126] The in vitro coagulation dynamics of the composite hemostatic sponges prepared in Example 5 and Comparative Example 1 for massive bleeding from deep and narrow wounds are as follows: Figure 6 As shown. A lower BCI value indicates a better hemostatic effect of the material. Experimental results show that, within the specified time range, the BCI value of GPOQ (from 0.94% ± 0.01% to 0.71% ± 0.01%) exhibits a lower value than that of GPO (from 0.97% ± 0.03% to 0.72% ± 0.01%). In vitro coagulation dynamics experiments confirm that the composite hemostatic sponge prepared in Example 5 for deep, narrow wounds with massive bleeding has a more efficient hemostatic rate.

[0127] Biocompatibility characterization of the composite hemostatic sponges prepared in Example 5 and Comparative Example 1 for massive bleeding from deep and narrow wounds is as follows: Figure 7 As shown, mouse embryonic fibroblasts (NIH / 3T3) were seeded onto two different groups of composite hemostatic sponges and co-cultured for different numbers of days. Cytotoxicity was tested using the CCK-8 assay, and the absorbance of the solution at 450 nm was measured using an ELISA reader at different time intervals. The results showed that the composite hemostatic sponge prepared in this invention for deep, narrow wounds with massive bleeding exhibits excellent biocompatibility.

[0128] Table 1 shows the complete blood loss and hemostasis time in a rat liver perforation model. Medical gauze, Comparative Example 1, Comparative Example 2, and the composite hemostatic sponges prepared in Examples 1-8 were used in the rat liver perforation experiment. Table 1 shows that the composite hemostatic sponges provided in Examples 1-8 of this invention resulted in less blood loss and shorter hemostasis time in the rat liver perforation bleeding model compared to the gauze group, Comparative Example 1, and Comparative Example 2, demonstrating high hemostatic efficiency. Specifically, due to the introduction of quaternized chitosan, its positively charged protonated amino groups can adsorb, concentrate, and aggregate erythrocytes and platelets through electrostatic interactions, thereby forming an adhesion barrier and increasing the speed of scab formation at the wound site. Furthermore, the amino groups on its side chains can form amide bonds with the carboxyl groups of gelatin and the aldehyde groups of oxidized dextran, respectively, to combine with Schiff bases, creating a uniform porous network structure within the sponge, improving water and blood absorption capacity and better promoting the aggregation of erythrocytes and platelets. This composite sponge with excellent hemostatic properties is expected to be further applied in deep and narrow wounds, becoming a new generation of biomedical hemostatic materials.

[0129] Table 1. Experimental data of the composite hemostatic sponge prepared according to the present invention for massive bleeding from deep and narrow wounds in a rat liver perforation hemorrhage model.

[0130] Blood loss (mg) Hemostasis time (s) Medical gauze 739.65 162.49 Comparative Example 1 491.39 109.17 Comparative Example 2 422.56 83.72 Example 1 374.17 63.04 Example 2 342.08 58.21 Example 3 300.48 50.66 Example 4 298.52 46.39 Example 5 188.63 31.47 Example 6 197.70 38.20 Example 7 200.82 39.76 Example 8 236.98 47.48

[0131] As shown in Table 2, the composite hemostatic sponge prepared in this invention for deep and narrow wounds with massive bleeding exhibits excellent non-toxicity, non-allergenicity, and non-irritation. Combined with... Figure 7 The cytotoxicity test results show that after co-culturing the composite hemostatic material of this invention with cells for 1, 3, and 7 days, the cell viability exceeded 90%, indicating that the composite hemostatic sponge has good biocompatibility.

[0132] Table 2 shows the medical performance test results of the composite hemostatic sponges prepared in Examples 1-8 and Comparative Examples 1-2 of this invention for massive bleeding from deep and narrow wounds.

[0133]

[0134]

[0135] The above embodiments are provided only to help understand the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from its principles, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

[0136] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a composite hemostatic sponge for massive bleeding from deep, narrow wounds, characterized in that, Includes the following steps: (1) Dissolve gelatin in deionized water and stir evenly at a certain temperature to obtain solution S1; (2) Dissolve quaternized chitosan in deionized water and stir evenly at a certain temperature to obtain solution S2; (3) Synthesis of oxidized dextran: Dextran was dissolved in deionized water, sodium periodate was added, and the reaction was stirred at room temperature in the dark. After the reaction was completed, diethylene glycol was added to quench the reaction, dialyzed, and freeze-dried to obtain oxidized dextran; oxidized dextran was dissolved in PBS buffer to obtain solution S3; (4) Dissolve dopamine in deionized water and stir until homogeneous at room temperature to obtain solution S4; (5) Dissolve 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride EDC in deionized water and stir evenly at room temperature to obtain solution S5; dissolve N-hydroxysuccinimide NHS in deionized water and stir evenly at room temperature to obtain solution S6; (6) Dissolve the oxidant in deionized water and stir until homogeneous at room temperature to obtain solution S7; (7) In an ice bath, solutions S2, S3 and S4 are added to solution S1 and stirred until homogeneous. Then solutions S5, S6 and S7 are added in sequence to obtain precursor solution S8. The precursor solution S8 is polymerized at low temperature and then dialyzed and freeze-dried to obtain a composite hemostatic sponge for deep and narrow wounds with massive bleeding. The concentration of solution S1 in step (1) is 0.1–10 wt%, and the stirring temperature is 10–80 °C; The concentration of solution S2 in step (2) is 0.1–20 wt%, and the stirring temperature is 10–70 °C; The molar ratio of dextran to sodium periodate and diethylene glycol in step (3) is 20:1:1-1:1:1; the reaction time in the dark is 12-48 h; the quenching reaction time is 0.1-6 h; the concentration of the PBS buffer used is 0.1 mol / L, pH=7.4; and the concentration of oxidized dextran in solution S3 is 0.1-20 wt%. The concentration of solution S4 in step (4) is 0.1–30 wt% The concentration of solution S5 in step (5) is 0.01–3 wt%; the concentration of solution S6 is 0.01–3 wt%. The oxidant mentioned in step (6) is sodium periodate, and the concentration of solution S7 is 0.1–15 wt%. In step (7), the volume ratio of solutions S1:S2:S3:S4 is 1:0.25:1:0.35−1000:250:1000:350; the volume ratio of solution S1 to solutions S5 and S6 is 1:0.1:0.1−1000:100:100; the volume ratio of solution S4 to solution S7 is 1:1−1:10; the precursor solution S8 is subjected to low-temperature polymerization at -80 ~ -18 ℃ for 24~56 h, thawed, dialyzed, and freeze-dried to obtain a composite hemostatic sponge for deep and narrow wounds with massive bleeding.

2. A composite hemostatic sponge for deep and narrow wounds with massive bleeding, prepared by the method described in claim 1.

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