A quaternized pullulan-cellulose composite hemostatic sponge containing snail mucus, its preparation method and application

Abstract

This invention discloses a quaternized pullulan / cellulose composite hemostatic sponge containing snail mucus, its preparation method, and its application, belonging to the field of biomedical materials technology. The quaternized pullulan / cellulose composite hemostatic sponge containing snail mucus is obtained by cross-linking hydroxypropyl methylcellulose, quaternized pullulan polysaccharide, and snail mucus powder with a cross-linking agent; the mass ratio of hydroxypropyl methylcellulose, quaternized pullulan polysaccharide, and snail mucus is 1.9-2.1:1:3.8-4.2. The quaternized pullulan / cellulose composite hemostatic sponge containing snail mucus of this invention can solve the problems of insufficient porosity, poor wet tissue adhesion, single hemostatic mechanism, and low functional integration of existing hemostatic materials.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, and relates to a quaternized pullulan-cellulose composite hemostatic sponge containing snail mucus, its preparation method and application, specifically to a quaternized pullulan-cellulose composite hemostatic sponge containing snail mucus, its preparation method and its application in the preparation of medical materials with functions of rapid hemostasis in vivo and in vitro, promoting wound healing and preventing wound infection. Background Technology

[0002] Uncontrolled bleeding is one of the leading causes of death in trauma, war injuries, and surgery. Ideal hemostatic materials should possess multiple functions, including rapid clotting, strong wet tissue adhesion, excellent mechanical properties, good biocompatibility, and anti-infection capabilities. However, existing commercially available hemostatic materials, such as gelatin sponges, polyvinyl alcohol (PVA) sponges, and chitosan (CS) sponges, generally suffer from insufficient porosity, poor wet tissue adhesion, a single hemostatic mechanism, and low functional integration, making them insufficient to meet the clinical needs of active massive bleeding. Summary of the Invention

[0003] The purpose of this invention is to provide a quaternized pullulan-cellulose composite hemostatic sponge containing snail mucus, in order to solve the problems of insufficient porosity, poor wet tissue adhesion, single hemostatic mechanism and low functional integration of existing hemostatic materials.

[0004] Another objective of this invention is to provide a method for preparing the aforementioned composite hemostatic sponge, which is simple in process, mild in conditions, and easy to scale up for production.

[0005] Another object of the present invention is to provide the application of the aforementioned composite hemostatic sponge in the preparation of medical materials with functions of rapid hemostasis in vivo and in vitro, promoting wound healing and preventing wound infection.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A quaternized pullulan-cellulose composite hemostatic sponge containing snail mucus is obtained by cross-linking hydroxypropyl methylcellulose, quaternized pullulan polysaccharide, and snail mucus powder with a cross-linking agent. Preferably, the mass ratio of hydroxypropyl methylcellulose, quaternized pullulan, and snail mucus is 1.9-2.1:1:3.8-4.2; Preferably, the quaternized pullulan polysaccharide has a degree of substitution of 0.3-0.31 and a molecular weight of 92-93 kDa; Preferably, the snail mucus powder is derived from the white jade snail, and the preparation method consists of the following steps: collecting mucus, treating with liquid nitrogen, freeze-drying, and grinding; More preferably, the method for collecting the mucus is to collect the mucus by mechanically stimulating the surface of the foot of the white jade snail; More preferably, the liquid nitrogen temperature in the liquid nitrogen treatment is -197°C to -196°C, and the treatment time is 2-2.5 min; More preferably, the freeze-drying process is carried out at a temperature of -42°C to -38°C, a pressure of 0.09-0.11 mbar, and a time of 12-12.5 h. More preferably, when storing the snail mucus powder, the storage temperature is -22°C to -18°C; Preferably, the crosslinking agent is an aqueous solution of glutaraldehyde; More preferably, the crosslinking agent is an aqueous solution of glutaraldehyde with a volume concentration of 48-50%.

[0007] The present invention also provides a method for preparing the aforementioned quaternized pullulan-cellulose composite hemostatic sponge containing snail mucus, wherein an aqueous solution of hydroxypropyl methylcellulose is mixed with an aqueous solution of quaternized pullulan polysaccharide, snail mucus powder is added, a crosslinking agent is added to obtain a crosslinking system, the crosslinking system is stirred and freeze-dried to obtain the composite hemostatic sponge; Preferably, the mass-volume concentration of the hydroxypropyl methylcellulose aqueous solution is 1.9-2.1%; Preferably, the mass-volume concentration of the quaternized pullulan polysaccharide aqueous solution is 1.9-2.1%; Preferably, in the crosslinking system, the volume concentration of the effective component of the crosslinking agent is 0.48-0.52%; Preferably, the stirring temperature is room temperature, and the stirring time is 47-49 hours; Preferably, the freeze-drying temperature is -82°C to -78°C, and the time is 47-49 hours.

[0008] The present invention also provides the application of the aforementioned quaternized pullulan-cellulose composite hemostatic sponge containing snail mucus in the preparation of medical materials with functions of rapid hemostasis in vivo and in vitro, promoting wound healing and preventing wound infection.

[0009] Compared with the prior art, the present invention has the following significant technical advancements and beneficial effects: 1. The composite hemostatic sponge of this invention utilizes a triple synergistic hemostatic mechanism of snail mucus, quaternized pullulan, and hydroxypropyl methylcellulose, significantly improving hemostatic efficiency. Snail mucus is a natural glycoprotein-glycosaminoglycan complex, rich in polar amino acids, uronic acids, and glycosaminoglycans. Its biochemical composition is highly similar to the extracellular matrix, possessing the potential to promote cell proliferation, tissue repair, and wet adhesion. However, there are currently no reports on constructing a high-porosity, highly adhesive, multifunctional hemostatic sponge by combining snail mucus with quaternized pullulan and hydroxypropyl methylcellulose in a ternary composite. This invention constructs a synergistic hemostatic system with a physical-electrostatic-biochemical triple mechanism through a ternary composite of snail mucus, quaternized pullulan, and hydroxypropyl methylcellulose. The physical mechanism involves the snail mucus and quaternized pullulan forming a stable electrostatic network through a polyelectrolyte complex effect, inhibiting structural collapse during freeze-drying and achieving a sponge porosity of up to 128.18%, enabling ultra-rapid blood absorption and coagulation factor enrichment. The electrostatic mechanism involves the composite sponge surface maintaining strong cationicity, with a zeta potential reaching +31.53 mV, rapidly capturing negatively charged red blood cells and platelets through electrostatic attraction. The biochemical mechanism involves the natural glycoproteins and glycosaminoglycans in snail mucus mimicking key components of the extracellular matrix, activating platelets and promoting efficient fibrin network construction.

[0010] 2. The composite hemostatic sponge of the present invention has a coagulation index as low as 8.25%, which is significantly better than that of single components (QP: 20.61%, HPMC: 24.21%, SM: 65.74%) and commercially available gelatin hemostatic sponges, polyvinyl alcohol hemostatic sponges, and chitosan hemostatic sponges. Through rat liver parenchymal defect models, femoral artery transection models, and tail amputation models, it was confirmed that the hemostasis time of the composite hemostatic sponge of the present invention was shortened to 36.29s, 45.46s, and 56.07s, respectively, and the blood loss was reduced by more than 60% compared with the control group. It is rich in polar amino acids such as aspartic acid (12.8%) and glutamic acid (11.3%), as well as uronic acid, which can form extensive hydrogen bonds and electrostatic interactions at the material-tissue interface. The wet tissue adhesion strength of the composite hemostatic sponge of the present invention reaches 27.1kPa, which is about 197% higher than that of the control group without SM (9.13kPa), effectively resisting dynamic blood flow impact and preventing sponge displacement.

[0011] 3. The composite hemostatic sponge of the present invention has excellent mechanical properties and structural stability. The rigid cellulose skeleton of hydroxypropyl methylcellulose and the quaternized pullulan polysaccharide-snail mucus polyelectrolyte complex form a dense cross-linked interpenetrating network, which enables the sponge to withstand about 0.7 MPa stress without structural fracture under 90% compressive strain, thus meeting the needs of clinical pressure hemostasis and tissue deformation adaptation.

[0012] 4. In the composite hemostatic sponge of the present invention, the glycoproteins and quaternary ammonium cations of snail mucus endow the material with antibacterial ability. Agar plate counting showed that the composite hemostatic sponge at a concentration of 5 mg / mL and above had 100% bactericidal activity against Escherichia coli and Staphylococcus aureus, which can effectively prevent secondary wound infection.

[0013] 5. The composite hemostatic sponge of the present invention exhibits excellent biocompatibility and healing potential. The erythrocyte adhesion rate of the composite hemostatic sponge reaches 67.18%, the platelet adhesion rate reaches 76.83%, and the hemolysis rate is less than 2.0%, meeting the blood compatibility standards for medical materials. CCK-8 assays and Calcein-AM / PI live / dead staining show that the composite hemostatic sponge is non-toxic to L929 fibroblasts, and the cell survival rate exceeds 105% after 72 hours, with cells exhibiting a dense fusion state. In vivo histopathological examination (H&E staining) shows that after implantation of the composite hemostatic sponge of the present invention into rats for 1-4 weeks, no obvious inflammation or necrosis was observed in the heart, liver, spleen, lungs, kidneys, and skin, confirming its good biosafety and biodegradability. Attached Figure Description

[0014] Figure 1 SEM images of composite hemostatic sponges obtained when the mass ratio of HPMC:QP:SM is 2:1:0, 2:1:1, 2:1:2, 2:1:4, 2:1:6, and 2:1:8, respectively. Figure 2 The stress-strain curves of the composite hemostatic sponge obtained when the mass ratio of HPMC:QP:SM is 2:1:4 and commercially available gel, PVA, and Cs are shown. Figure 3 The infrared spectra of the composite hemostatic sponge obtained when the mass ratio of HPMC:QP:SM is 2:1:4 and the raw materials HPMC, QP, and SM are shown. Figure 4 Scanning electron microscopy images of red blood cells and white blood cells adhering to the surface of a composite hemostatic sponge obtained when the mass ratio of HPMC:QP:SM is 2:1:4; In the picture, Figure 4 (a) is a scanning electron microscope image of red blood cells adhering to the surface of a composite hemostatic sponge obtained when the mass ratio of HPMC:QP:SM is 2:1:4; Figure 4 (b) is a scanning electron microscope image of leukocytes adhering to the surface of a composite hemostatic sponge obtained when the mass ratio of HPMC:QP:SM is 2:1:4; Figure 5 Photographs of antibacterial plates of composite hemostatic sponge against Escherichia coli and Staphylococcus aureus at different concentrations; Figure 6 Macroscopic photograph of an in vivo hemostasis experiment; Figure 7 These are H&E stained pathological sections of major organs and local tissues after implantation of composite hemostatic sponge. Detailed Implementation

[0015] The present invention will be further described in detail below with reference to specific embodiments. These embodiments are intended to illustrate the present invention by way of example, and are not intended to limit the scope of protection of the present invention.

[0016] Example 1: Extraction and component characterization of snail mucus 1. Snail mucus extraction Clean white jade snails with a shell length of 6cm were selected, and mucus was collected from the surface of their feet by gentle mechanical stimulation. The collected mucus was immediately placed in liquid nitrogen for treatment (liquid nitrogen temperature -196℃, treatment time 2min), and then freeze-dried at -40°C and 0.1mbar for 12 hours. The mixture was then ground into powder to obtain freeze-dried SM powder, which was then sealed and stored at -20°C.

[0017] 2. Amino acid composition analysis Take 100 mg of lyophilized SM powder, add 6 M hydrochloric acid under a nitrogen atmosphere, and hydrolyze at 110 °C for 24 hours. Remove residual hydrochloric acid by rotary evaporation of the hydrolysate, filter through a 0.22 μm filter membrane, redissolve with 0.02 M hydrochloric acid, and detect using an amino acid analyzer.

[0018] The results showed that SM was significantly enriched with polar and charged amino acids, with aspartic acid accounting for 12.8%, glutamic acid for 11.3%, lysine for 5.01%, and arginine for 4.65%. These results further demonstrate that the high density of polar functional groups in SM provides abundant dynamic interfacial binding sites for wet adhesion.

[0019] 3. Monosaccharide composition analysis Weigh 100 mg of sample into a hydrolysis tube, add 1 mL of 72% sulfuric acid solution, incubate in a 30°C water bath for 1 hour, bring the volume to 10 mL, purge with nitrogen, and hydrolyze in a 110°C oven for 2 hours. After removal and cooling to room temperature, take 0.5 mL of the sample into a 5 mL centrifuge tube, adjust the pH to neutral, bring the volume to 1 mL, add 0.2 mL of 0.3 mol / L sodium hydroxide solution and 0.4 mL of PMP methanol solution, purge with nitrogen, incubate in a 70°C water bath for 60 minutes, and cool to room temperature. Add 0.2 mL of 0.3 mol / L hydrochloric acid solution, bring the volume to 2 mL with water, add 1.5 mL of chloroform, shake well, and allow to separate into layers. Discard the lower chloroform layer, filter the aqueous layer through a 0.45 μm filter membrane, and perform analysis. The determination conditions are as follows: Mobile phase A: 15% acetonitrile (acetonitrile diluted with 0.05 mol / L potassium dihydrogen phosphate solution, pH 6.8); Mobile phase B: 40% acetonitrile (acetonitrile diluted with 0.05 mol / L potassium dihydrogen phosphate solution, pH 6.8); Flow rate: 1.0 mL / min; column temperature: 25℃; injection volume: 20 μL; detection wavelength: 254 nm.

[0020] The results showed that glucose (226,786.53 mg / kg), mannose (8,353.27 mg / kg), and galacturonic acid (1,923.96 mg / kg) were the main carbohydrate components.

[0021] The results of amino acid and monosaccharide composition analysis show that the core bioactive components of SM are glycoproteins and glycosaminoglycans (GAGs). The uronic acid in the freeze-dried SM powder endows the SM powder with multi-anionic properties.

[0022] Example 2: Preparation of hemostatic sponge 1. Raw material preparation Prepare the following ingredients: Hydroxypropyl methylcellulose (hereinafter referred to as HPMC, Macklin Biochemical Co., Ltd.); Quaternized pullulan (hereinafter referred to as QP, degree of substitution = 0.305, molecular weight = 92.1kDa); The quaternized pullulan was prepared in the laboratory using the following method: 50g of pullulan was mixed with 500mL of a 0.3% sodium hydroxide aqueous solution and stirred at 4℃ for 1h to obtain a pullulan solution; 150g of 2,3-epoxypropyltrimethylammonium chloride (GTA) was mixed with 1500mL of a 0.3% sodium hydroxide aqueous solution and stirred at room temperature for 30min to obtain a cationic solution; the pullulan solution was stirred at 55℃ while the cationic solution was slowly added dropwise over 30min. After the addition was completed, stirring was continued for 24h. Then, glacial acetic acid was added to adjust the pH to 7, and the solution was transferred to a dialysis bag with a molecular weight cutoff of 8000. The dialysis bag was placed in water for dialysis for 60h, with the water changed every 6h. The dialysate in the dialysis bag was then freeze-dried at -45℃ to obtain the quaternized pullulan. In the preparation of the quaternized pullulan, the molecular weight of the pullulan used is 196.69 kDa; Snail mucus powder (SM powder, hereinafter referred to as SM, prepared according to Example 1); Glutaraldehyde aqueous solution (50%, v / v, Macklin Biochemical Co., Ltd.).

[0023] 2. Preparation method HPMC and QP were dissolved in deionized water to prepare 2.0% (w / v) solutions. The two solutions were mixed at a volume ratio of 2:1, and SM powder was added to each solution to make the mass ratio of HPMC:QP:SM 2:1:0, 2:1:1, 2:1:2, 2:1:4, 2:1:6 and 2:1:8, respectively. Glutaraldehyde was added dropwise to a final concentration of 0.5% (v / v). The mixture was stirred continuously at room temperature for 48 hours, then poured into molds, pre-frozen to -80°C, and freeze-dried at -80°C for 48 hours to obtain composite hemostatic sponges.

[0024] Example 3: Structural and performance characterization of porous hemostatic sponge 1. Porosity determination The porosity of the composite hemostatic sponge obtained in Example 2 was determined using the liquid displacement method. The results showed that the porosity exhibited a bell-shaped dependence with the proportion of SM. Specifically, when the HPMC:QP:SM ratio increased from 2:1:0 to 2:1:4, the porosity significantly increased from 80.65% to 128.18%; however, when the ratio continued to increase to 2:1:8, the porosity decreased to 65.51% due to phase separation and structural collapse. The specific porosity test results are as follows:

[0025] For comparison, gelatin hemostatic sponge (hereinafter referred to as Gel), polyvinyl alcohol hemostatic sponge (hereinafter referred to as PVA), and chitosan hemostatic sponge (hereinafter referred to as CS) were purchased from the market. Porosity was tested, and other properties, such as zeta potential, compressive mechanical properties, and blood coagulation index (BCI), were subsequently tested. The porosity test results are as follows:

[0026] The results above show that the porosity is highest when the mass ratio of HPMC:QP:SM is 2:1:4, and is much higher than that of commercially available gelatin hemostatic sponges, polyvinyl alcohol hemostatic sponges, and chitosan hemostatic sponges.

[0027] SEM analysis was performed on the composite hemostatic sponges obtained when the mass ratios of HPMC:QP:SM were 2:1:0, 2:1:1, 2:1:2, 2:1:4, 2:1:6, and 2:1:8, respectively. The SEM images are shown below. Figure 1 ,Depend on Figure 1 It can be seen that when the mass ratio of HPMC:QP:SM is 2:1:4, the resulting composite hemostatic sponge has the most loose and highly interconnected porous structure.

[0028] 2. Surface charge analysis Using a Zeta potentiometer, the raw materials HPMC and SM used in Example 2 exhibited weak negative charges of -0.875 mV and -0.891 mV, respectively, while raw material QP, due to its high density of quaternary ammonium groups, showed a strong positive charge of +35.2 mV. The optimal formulation (HPMC / QP / SM mass ratio of 2:1:4) still maintained a significant positive surface charge of +31.53 mV, providing a strong electrostatic driving force for capturing blood cells. Specific test results are as follows:

[0029] 3. Tissue adhesion properties The wet tissue adhesion strength of the composite hemostatic sponge obtained in Example 2 was evaluated using a shear test. When the mass ratio of HPMC:QP:SM was 2:1:0, the wet tissue adhesion strength of the composite hemostatic sponge was 9.13 kPa. With increasing SM content, the adhesion strength significantly increased, reaching a peak of 27.1 kPa when the mass ratio of HPMC:QP:SM was 2:1:4. As the mass ratio of HPMC:QP:SM continued to increase, the excess SM (2:1:6, 2:1:8) caused structural densification, leading to a decrease in adhesion strength. Specific results are as follows:

[0030] 4. Compressive mechanical properties A uniaxial compression test was performed on the composite hemostatic sponge obtained in Example 2 when the mass ratio of HPMC:QP:SM was 2:1:4. Simultaneously, uniaxial compression tests were also performed on commercially available gel, PVA, and Cs. The resulting pressure-strain curves are shown below. Figure 2 ,Depend on Figure 2 It can be seen that the composite hemostatic sponge obtained when the mass ratio of HPMC / QP / SM is 2:1:4 can withstand a compressive stress of about 0.7MPa at 90% strain while maintaining structural continuity, demonstrating excellent toughness and resistance to deformation.

[0031] 5. Infrared spectroscopy characterization Infrared spectroscopy analysis was performed on the composite hemostatic sponge obtained in Example 2 when the mass ratio of HPMC:QP:SM was 2:1:4. Infrared spectroscopy analysis was also performed on the raw materials HPMC, QP, and SM. The resulting infrared spectra are shown below. Figure 3 ,Depend on Figure 3 It can be seen that the composite hemostatic sponge is located at approximately 3350 cm. -1 The OH stretching vibration band widens to 1050cm. -1 Enhanced COC absorption at 1640 cm⁻¹ confirms that HPMC, QP, and glutaraldehyde underwent an acetalization cross-linking reaction; -1The absorption band at this point is a superposition of the amide I vibration of SM and the C=N stretching vibration of Schiff base, proving that SM was successfully incorporated and formed a stable chemical cross-linked network.

[0032] Example 4: Evaluation of in vitro hemostasis and biocompatibility 1. Blood Coagulation Index (BCI) Measurement The coagulation index (BCI) was used to determine the in vitro procoagulant properties of the composite hemostatic sponge obtained in Example 2. The specific test methods and results are as follows: First, 100 μL of fresh rabbit whole blood containing sodium citrate anticoagulant and 10 μL of 0.2 M CaCl2 were added dropwise to the surface of 25 mg of sample. Then, the mixture was incubated at 37 °C for 5 min. Next, 25 mL of deionized water was added, and the mixture was incubated for another 5 min at 37 °C using a constant-temperature shaker. Finally, the absorbance (OD) of the supernatant at 540 nm was measured using a UV-Vis spectrophotometer. sample No sample group served as a negative control (OD). control Each sample was tested three times, and the BCI was calculated using the following formula:

[0033] Meanwhile, the coagulation index (BCI) of raw materials QP, HPMC, SM, and commercially available gel, PVA, and CS was tested and calculated.

[0034] The calculated blood clotting index (BCI) results are as follows:

[0035] The results above show that the composite hemostatic sponge with the lowest BCI was obtained when the mass ratio of HPMC:QP:SM was 2:1:4, indicating that the mass ratio of HPMC:QP:SM was 2:1:4, which showed a significant synergistic effect.

[0036] 2. Blood cell adhesion performance assay Following the method described in "Preparation and Performance Study of Polyphenol-Modified Hemostatic Sponge" (Lin Jie, Master's Thesis, Beijing University of Chemical Technology, 2024), the erythrocyte adhesion rate and platelet adhesion rate of the composite hemostatic sponge obtained in Example 2 when the mass ratio of HPMC:QP:SM was 2:1:4 were tested. The erythrocyte adhesion rate and platelet adhesion rate of the raw materials HPMC, QP, and SM were also tested. The test results are as follows:

[0037]

[0038] The results in the table above show that when the mass ratio of HPMC:QP:SM is 2:1:4, the composite hemostatic sponge has an erythrocyte adhesion rate of 67.18% and a platelet adhesion rate of 76.83%, which is also better than that of each individual component.

[0039] Meanwhile, referring to the SEM observation method in "Preparation and Performance Study of Polyphenol-Modified Hemostatic Sponge" (Lin Jie, Master's Thesis, Beijing University of Chemical Technology, 2024), the adhesion morphology of erythrocytes and platelets on the surface of the composite hemostatic sponge obtained in Example 2 when the mass ratio of HPMC:QP:SM was 2:1:4 was studied. Specifically, 5% (v / v) erythrocyte suspension or 50% (v / v) anemic platelet plasma was dropped into the sample and incubated at 37°C for 1 h. Then, it was washed 3 times with PBS, and a 2.5% (v / v) glutaraldehyde PBS solution was prepared and fixed at 4°C for 1 h. It was then rinsed 3 times with deionized water, dehydrated with a gradient ethanol / PBS solution, dried at 37°C for 12 h, and used for SEM observation. The obtained SEM images are shown below. Figure 4 .

[0040] 3. Antibacterial performance test The inhibition rate of the composite hemostatic sponge obtained in Example 2 with a mass ratio of HPMC:QP:SM of 2:1:4 against Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive) was tested using the agar plate colony count method. The inhibition rate results were as follows when the concentration of the composite hemostatic sponge in the system was 1.25 mg / mL and 2.5 mg / mL:

[0041] When the concentration of the composite hemostatic sponge in the system was 5 mg / mL and 10 mg / mL, no viable colonies were detected, and the antibacterial rate reached 100%.

[0042] Photographs of antibacterial plates of the composite hemostatic sponge against Escherichia coli and Staphylococcus aureus at different concentrations are shown below. Figure 5 .

[0043] 4. Hemolysis test The hemolysis rate of the composite hemostatic sponge obtained in Example 2 with a mass ratio of HPMC:QP:SM of 2:1:4 was tested according to GB / T 14233.2-2005. Specifically, the hemolysis rate of the composite hemostatic sponge at different concentrations (1.25-10 mg / mL) in the system was tested. The test results showed that the hemolysis rate at a concentration of 10 mg / mL was still less than 2.0%, which meets the blood compatibility requirements for medical materials. Specific test results are as follows:

[0044] 5. Cytotoxicity evaluation The compatibility of the composite hemostatic sponge obtained in Example 2 with a mass ratio of HPMC:QP:SM of 2:1:4 to L929 fibroblasts was assessed using the CCK-8 assay and Calcein-AM / PI live / dead staining. Specifically, the compatibility of the composite hemostatic sponge with L929 fibroblasts at different concentrations (1.25-10 mg / mL) in the system was evaluated. The specific cell viability results are as follows:

[0045] The results in the table above show that the cell viability remained above 99% at all concentrations for 24, 48, and 72 hours, and showed an increasing trend over time. Example 5: Evaluation of in vivo hemostatic effect Three standardized hemorrhage models were established using SD rats (provided by Jinan Pengyue Experimental Animal Breeding Co., Ltd.): 1. Liver parenchymal defect model After creating a 5mm diameter liver parenchyma defect, a composite hemostatic sponge of the same volume (7mm×7mm×10mm) made of Gel, PVA, Cs, and HPMC:QP:SM in Example 2 with a mass ratio of 2:1:4 was placed on the wound, and the hemostasis time and blood loss were recorded. A blank control group was also set up, receiving no treatment; the hemostasis time and blood loss in the blank control group were recorded. The results are as follows:

[0046]

[0047] 2. Femoral artery transection model After transection of the femoral artery, a composite hemostatic sponge of the same volume (7mm×7mm×10mm) containing Gel, PVA, Cs, and the mass ratio of HPMC:QP:SM obtained in Example 2 (2:1:4) was placed on the wound, and hemostasis time and blood loss were recorded. A blank control group was also set up, receiving no treatment; hemostasis time and blood loss in the blank control group were recorded. The results are as follows:

[0048]

[0049] 3. Tail amputation model The tail was severed 2 cm from the tip. A composite hemostatic sponge of the same volume (7 mm × 7 mm × 10 mm) made of Gel, PVA, Cs, and HPMC:QP:SM in Example 2 with a mass ratio of 2:1:4 was placed on the wound, and the hemostasis time and blood loss were recorded. A blank control group was also set up, receiving no treatment. The hemostasis time and blood loss of the blank control group were recorded. The results are as follows:

[0050]

[0051] Macroscopic photographs of the in vivo hemostasis test are shown below. Figure 6 .

[0052] The above results show that the composite hemostatic sponge obtained when the mass ratio of HPMC:QP:SM is 2:1:4 achieved rapid hemostasis and effective blood loss control in all three active bleeding models, and its performance was significantly better than that of commercially available gel, PVA, and Cs.

[0053] Example 6: In vivo biosafety evaluation Five rats were used. One rat was sacrificed, and the other four were anesthetized. The hair on the backs of the rats was shaved and the area was disinfected with iodine. A 2 cm skin incision was made, and blunt dissection was performed to form a tissue capsule. The composite hemostatic sponge obtained in Example 2 with a HPMC:QP:SM mass ratio of 2:1:4 was then disinfected, and a 7mm × 7mm × 10mm piece was implanted into the tissue capsule. The incision was then sutured. Subsequently, one rat was sacrificed at 1, 2, 3, and 4 weeks. H&E-stained pathological sections of major organs and local tissues were prepared. (See H&E-stained pathological section images below.) Figure 7 .

[0054] Depend on Figure 7 It can be seen that after the composite hemostatic sponge was placed into the tissue capsule, no obvious morphological abnormalities, tissue necrosis or significant inflammatory cell infiltration were found at any observation time point. The sponge gradually degraded and was replaced by new tissue, indicating that it has excellent in vivo biocompatibility, reliable biodegradability and good biosafety.

Claims

1. A quaternized pullulan-cellulose composite hemostatic sponge containing snail mucus, characterized in that, It is obtained by cross-linking hydroxypropyl methylcellulose, quaternized pullulan polysaccharide, and snail mucus powder with a cross-linking agent; The mass ratio of hydroxypropyl methylcellulose, quaternized pullulan, and snail mucus was 1.9-2.1:1:3.8-4.

2. The degree of substitution of the quaternized pullulan polysaccharide is 0.3-0.31, and the molecular weight is 92-93 kDa; The snail mucus powder is derived from the white jade snail, and the preparation method consists of the following steps: collecting mucus, treating with liquid nitrogen, freeze-drying, and grinding. The preparation method of the quaternized pullulan-cellulose composite hemostatic sponge is as follows: hydroxypropyl methylcellulose aqueous solution and quaternized pullulan polysaccharide aqueous solution are mixed, snail mucus powder is added, and a crosslinking agent is added to obtain a crosslinking system. The crosslinking system is stirred and freeze-dried to obtain the composite hemostatic sponge. The mass-volume concentration of the hydroxypropyl methylcellulose aqueous solution is 1.9-2.1%; The quaternized pullulan polysaccharide aqueous solution has a mass-volume concentration of 1.9-2.1%; In the crosslinking system, the volume concentration of the effective component of the crosslinking agent is 0.48-0.52%.

2. The quaternized pullulan-cellulose composite hemostatic sponge containing snail mucus according to claim 1, characterized in that, The method for collecting the mucus is to collect the mucus by mechanically stimulating the surface of the foot of the white jade snail. The liquid nitrogen temperature in the liquid nitrogen treatment is -197℃ to -196℃, and the treatment time is 2-2.5 min; In the preparation of snail mucus powder, the freeze-drying temperature was -42℃ to -38℃, the freeze-drying pressure was 0.09-0.11 mbar, and the freeze-drying time was 12-12.5 h. When storing snail mucus powder, the storage temperature should be between -22°C and -18°C.

3. The quaternized pullulan-cellulose composite hemostatic sponge containing snail mucus according to claim 1, characterized in that, The crosslinking agent is an aqueous solution of glutaraldehyde.

4. The quaternized pullulan-cellulose composite hemostatic sponge containing snail mucus according to claim 1, characterized in that, In the preparation of the quaternized pullulan-cellulose composite hemostatic sponge, the stirring temperature is room temperature and the time is 47-49 hours. The freeze-drying temperature of the cross-linked system is -82℃ to -78℃, and the freeze-drying time is 47-49h.

5. The application of a quaternized pullulan-cellulose composite hemostatic sponge containing snail mucus as described in any one of claims 1-4 in the preparation of medical materials with functions of rapid hemostasis in vivo and in vitro, promoting wound healing, and preventing wound infection.

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