Zeolite cellulose hemostatic sponge capable of enhancing hemostatic capability and preparation method of zeolite cellulose hemostatic sponge

Abstract

The invention provides a preparation method of zeolite cellulose hemostatic sponge with enhanced hemostatic capability. The preparation method specifically comprises the following steps: step 1, pretreating a regenerated cellulose sponge block; 2, preparing a precursor solution and mixing the precursor solution with the pretreated sponge block; step 3, microwave hydrothermal synthesis; step 4, post-treatment and ion exchange; 5, fixing the sponge after cleaning to obtain the zeolite cellulose hemostatic sponge. According to the preparation method, firstly, a standard cylindrical pretreated sponge block is obtained through cutting pretreatment, then uniform loading of zeolite crystals in a three-dimensional porous matrix is achieved by optimizing reaction kinetics, and the hemostatic sponge obtained through the preparation method has high porosity (larger than 90%) and mechanical adaptability (the compression modulus ranges from 15 kPa to 60 kPa); and the hemostasis and repair requirements of complex cavities such as through wounds are met.

Description

Technical Field

[0001] This invention belongs to the field of medical materials technology, and relates to hemostatic sponges, specifically to a zeolite cellulose hemostatic sponge with enhanced hemostatic ability and its preparation method. Background Technology

[0002] Traumatic hemorrhage is a core challenge in the global trauma emergency care field. Statistics show that approximately 30% of trauma-related deaths stem from uncontrolled bleeding. Deep vascular injuries caused by penetrating wounds (such as gunshot wounds and sharp object puncture wounds) are particularly problematic in pre-hospital hemostasis due to the depth and narrowness of the wound, the hidden bleeding point, and the difficulty in applying pressure. Traditional hemostatic materials (such as gauze and kaolin dressings) are limited by their two-dimensional planar structure, making it difficult to effectively fill irregular wound cavities. They also generally suffer from insufficient mechanical strength and the risk of embolism due to particle shedding. In recent years, hemostatic agents based on zeolite molecular sieves have attracted attention due to their high specific surface area and cation exchange capacity. However, traditional zeolite-based materials (such as the early QuikClot) exhibit significant exothermic reactions, easily leading to tissue burns, and are mostly in particulate form, making them difficult to fix in penetrating wounds, further limiting their application. How to construct novel hemostatic materials that combine three-dimensional adaptive filling capabilities with a stable biological interface has become a pressing scientific challenge in this field.

[0003] In recent years, cellulose-based materials have become an ideal choice for constructing hemostatic carriers due to their excellent biocompatibility and biodegradability. Previous studies have shown that researchers have anchored chalcogenide (CHA) onto the surface of cotton fibers using an in-situ growth method, significantly improving the bonding strength between zeolite and the carrier and reducing the risk of particle detachment. Other researchers have built nanoporous zeolite-cellulose nanofiber gelatin to alleviate the exothermic problem. However, existing composite systems still have certain problems: First, the material form is mostly gauze or film, which cannot fit the deep and narrow cavities of penetrating wounds, making it difficult to achieve precise coverage and compression of deep bleeding points; second, the preparation of zeolite mostly relies on traditional hydrothermal methods, with reaction cycles as long as 12-24 hours, and the zeolite loading and dispersibility are difficult to control synergistically, easily leading to uneven local coagulation efficiency and failing to achieve continuous coagulation cascade activation, making it difficult to cope with the continuous bleeding of penetrating wounds. These defects together make it difficult for current cellulose-zeolite composite systems to meet the requirements of efficient and safe hemostasis for penetrating wounds. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a zeolite cellulose hemostatic sponge with enhanced hemostatic ability and its preparation method, thereby solving the technical problem that the hemostatic effect of existing hemostatic materials in penetrating injuries needs to be further improved.

[0005] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: A method for preparing a zeolite cellulose hemostatic sponge with enhanced hemostatic ability, the method specifically includes the following steps: Step 1: Pre-treatment of regenerated cellulose sponge blocks: The regenerated cellulose sponge block was ultrasonically washed in water, and then compressed and cut using a mold to obtain a standard cylindrical pretreated sponge block.

[0006] Step 2: Prepare the precursor solution and mix it with the pretreated sponge block: Sodium hydroxide and aluminum hydroxide are dissolved in water, heated to 90°C and magnetically stirred until transparent to obtain mixture A; sodium hydroxide, 30% colloidal silica, and the pretreated sponge block obtained in step one are mixed with water and ultrasonically dispersed to obtain mixture B; then mixture A is poured into mixture B to obtain a mixture of precursor solution and pretreated sponge block, i.e., reactant.

[0007] Step 3, Microwave hydrothermal synthesis: The reactants obtained in step two are transferred to a polytetrafluoroethylene-lined reactor. The polytetrafluoroethylene-lined reactor containing the reactants is then placed in a microwave synthesizer to carry out the synthesis reaction, resulting in a reacted sponge.

[0008] Step 4, Post-treatment and Ion Exchange: The sponge obtained in step 3 was sequentially rinsed with deionized water, soaked in 0.2M calcium chloride aqueous solution, and ultrasonically cleaned to obtain a cleaned sponge.

[0009] Step 5: Fix the sponge after cleaning: The cleaned sponge obtained in step four is compressed into a mold and then transferred together into a freeze dryer. After freeze-drying for 12 hours, zeolite cellulose hemostatic sponge is obtained.

[0010] The present invention also has the following technical features: Specifically, in step one, the ultrasonic washing is performed three times, with each ultrasonic washing session lasting 10 minutes.

[0011] In step one, the pretreated standard cylindrical sponge block has a diameter of 1 cm and a length of 5 cm.

[0012] Specifically, in step two, the magnetic stirring time is 30 minutes and the magnetic stirring speed is 500 rpm.

[0013] In step two, the ultrasonic dispersion time is 30 seconds and the ultrasonic dispersion frequency is 40 kHz.

[0014] In step two, the molar ratio of sodium hydroxide: aluminum hydroxide: 30% colloidal silica: water in the precursor solution is 9:0.7:2:331, 9:0.7:4:331, 9:0.7:8:331, 9:0.7:10:331, or 9:0.7:14:331.

[0015] Specifically, in step three, the temperature of the synthesis reaction is 120°C, and the reaction time includes 0.5h, 1h, 2h, 4h, or 8h.

[0016] Specifically, in step four, the deionized water rinsing is performed five times.

[0017] In step four, the specific conditions for soaking in the 0.2M calcium chloride aqueous solution are: soaking for 12 hours under oscillation conditions of 150 rpm and temperature conditions of 25°C.

[0018] In step four, the ultrasonic cleaning frequency is 40kHz, the ultrasonic cleaning is performed 3 times, and the ultrasonic cleaning time for each time is 5 minutes.

[0019] Specifically, in step five, the cold trap temperature of the freeze dryer is -70°C, and the vacuum degree of the freeze dryer is 1 Pa.

[0020] The present invention also protects a zeolite cellulose hemostatic sponge prepared by the method described above for preparing a zeolite cellulose hemostatic sponge with enhanced hemostatic ability.

[0021] Compared with the prior art, the present invention has the following technical effects: (I) The present invention first obtains a standard cylindrical pretreated sponge block by cutting and pretreatment, and then achieves uniform loading of zeolite crystals in a three-dimensional porous matrix by optimizing reaction kinetics. The hemostatic sponge obtained by this preparation method has both high porosity (>90%) and mechanical adaptability (compression modulus 15~60kPa), which meets the hemostasis and repair needs of complex cavities such as penetrating wounds.

[0022] (II) The present invention uses microwave hydrothermal method, which shortens the synthesis time from 24 hours to 8 hours compared with the traditional hydrothermal method, thus improving the reaction efficiency.

[0023] (III) The present invention uses a pre-treated sponge block in the shape of a standard cylinder to replace two-dimensional planar materials such as gauze in the prior art, thereby improving the hemostasis efficiency of hemostatic materials when used for penetrating wounds.

[0024] (IV) This invention investigates the effects of zeolite on pretreated sponge blocks with different amounts of zeolite loading or no zeolite loading, physical adsorption of zeolite on pretreated sponge blocks, and different synthesis times during microwave hydrothermal synthesis by growing zeolite in situ on standard cylindrical pretreated sponge blocks. It was found that when the molar ratio of 30% colloidal silica was 8 and the synthesis time during microwave hydrothermal synthesis was 8 hours, the prepared hemostatic material had a good hemostatic effect. Compared with the prior art of growing zeolite in situ on gauze, this invention improves the hemostatic effect of the hemostatic material for penetrating wounds.

[0025] (V) The hemostatic mechanism of this invention is that zeolite is grown in situ on a hemostatic sponge prepared from cellulose, achieving efficient hemostasis through a dual mechanism. On the one hand, the inherent hydrophilicity and three-dimensional porous structure of cellulose material allow it to rapidly absorb liquid and swell upon contact with blood, thereby applying physical pressure to the bleeding point and achieving initial occlusion. On the other hand, the zeolite modified with calcium ion exchange can effectively activate the coagulation reaction, promote the conversion of prothrombin to thrombin, and accelerate thrombus formation. Based on the above characteristics, this zeolite-cellulose hemostatic sponge shows significant application potential in the treatment of massive bleeding caused by penetrating injuries. Attached Figure Description

[0026] Figure 1 The images show the surface zeolite of the zeolite cellulose hemostatic sponges obtained after microwave hydrothermal synthesis under different reaction time conditions. Figures (a), (b), (c), (d), and (e) are the scanning electron microscopy test results of the zeolite cellulose hemostatic sponges obtained in Examples 5 to 1, respectively.

[0027] Figure 2 The XRD results are for zeolite cellulose hemostatic sponges obtained by microwave hydrothermal synthesis under different reaction time conditions in Comparative Example 1 and Examples 1 to 4.

[0028] Figure 3 The following are scanning electron microscopy (SEM) results of sponges prepared by synthesizing 30% colloidal silica with different molar ratios under the condition of a synthesis reaction time of 8 h. In the figure, (a) is Comparative Example 1, (b) is Example 6, (c) is Example 7, (d) is Example 8, (e) is Example 1, and (f) is Example 9.

[0029] Figure 4 The image shows the EDS energy spectrum analysis of spCel-Z2 obtained in Example 8. In the image, (a) is the distribution of O element, (b) is the distribution of C element, (c) is the distribution of Si element, (d) is the distribution of Na element, (e) is the distribution of Al element and (f) is the distribution of Ca element.

[0030] Figure 5The image shows the EDS energy spectrum analysis of spCel-Z3 obtained in Example 1. In the image, (a) is the distribution of O element, (b) is the distribution of C element, (c) is the distribution of Si element, (d) is the distribution of Na element, (e) is the distribution of Al element and (f) is the distribution of Ca element.

[0031] Figure 6 The image shows the EDS energy spectrum analysis of spCel-Z4 obtained in Example 9. In the image, (a) is the distribution of O element, (b) is the distribution of C element, (c) is the distribution of Si element, (d) is the distribution of Na element, (e) is the distribution of Al element and (f) is the distribution of Ca element.

[0032] Figure 7 The images show the clarity of deionized water after immersion in water and ultrasonication of different samples. In the images, (a) represents spCel-Z2, (b) represents spCel-Z3, (c) represents spCel-Z4, and (d) represents spCel.

[0033] Figure 8 The images show the expansion of spCel in Comparative Example 2 after drying, compression, and liquid absorption recovery. In the figure, (a) is after drying and compression, and (b) is after liquid absorption recovery.

[0034] Figure 9 The images show the expansion of spCel-Z2 in Example 8 after drying, compression, and liquid absorption recovery. In the image, (a) is after drying and compression, and (b) is after liquid absorption recovery.

[0035] Figure 10 These are the feature images after 3D reconstruction of spCel, spCel-Z2, spCel-Z3, and spCel-Z4, respectively.

[0036] Figure 11 The graph shows the macroscopic porosity results for spCel, spCel-Z2, spCel-Z3, and spCel-Z4.

[0037] Figure 12 The graph shows the water absorption test results for spCel, spCel-Z2, spCel-Z3, and spCel-Z4.

[0038] Figure 13 The graph shows the test results of the compressive modulus of spCel, spCel-Z2, spCel-Z3 and spCel-Z4 under 80% deformation.

[0039] Figure 14The figures show the contact angle test results for spCel, spCel-Z2, spCel-Z3, and spCel-Z4. (a1), (b1), (c1), and (d1) are before the water droplet contacts; (a2), (b2), (c2), and (d2) are after the water droplet contacts.

[0040] Figure 15 To illustrate the biocompatibility of different materials, (a) represents spCel, (b) represents spCel-Z2, (c) represents spCel-Z3, and (d) represents spCel-Z4.

[0041] Figure 16 The hemolysis rates of different materials are shown in the figure. (a) represents spCel, (b) represents spCel-Z2, (c) represents spCel-Z3, and (d) represents spCel-Z4.

[0042] Figure 17 The figure shows the enrichment of platelets and red blood cells in different materials. Column (a) represents platelets and column (b) represents red blood cells.

[0043] Figure 18 Coagulation function index for different materials.

[0044] Figure 19 Images of tail-cutting hemorrhage in rats treated with different materials, along with the amount of bleeding and the hemostasis time statistics; in the figure, (a1), (a2), (a3), (a4), and (a5) are images of the untreated group, gauze group, gelatin sponge group, spCel group, and spCel-Z2 treated group, respectively; (b) is a graph of bleeding volume; and (c) is a graph of hemostasis time.

[0045] Figure 20 Images of liver hemorrhage in rats treated with different materials, along with the amount of bleeding and the hemostasis time. In the image, (a1), (a2), (a3), (a4), and (a5) are images of the untreated group, gauze group, gelatin sponge group, spCel group, and spCel-Z2 treated group, respectively. (b) is a graph of the amount of bleeding. (c) is a graph of the hemostasis time.

[0046] Figure 21 Images of femoral artery bleeding in rats treated with different materials, along with the amount of bleeding and the hemostasis time statistics; in the figure, (a1), (a2), (a3), (a4), and (a5) are images of the untreated group, gauze group, gelatin sponge group, spCel group, and spCel-Z2 treated group, respectively; (b) is a graph of bleeding volume; and (c) is a graph of hemostasis time.

[0047] Figure 22 Thermo-infrared images corresponding to images of femoral artery bleeding in rats treated with spCel-Z2.

[0048] The specific content of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Detailed Implementation

[0049] It should be noted that, unless otherwise specified, all the equipment, methods and raw materials in this invention are based on the equipment, methods and raw materials commonly known in the art in the prior art. For example, the testing methods and equipment for scanning electron microscopy are known scanning electron microscopy testing methods and equipment, the testing methods and equipment for XRD are known XRD testing methods and equipment, and the testing methods and equipment for energy dispersive spectroscopy are known testing methods and equipment.

[0050] The technical concept of this invention is as follows: Addressing the problems encountered by existing hemostatic materials when used for penetrating wounds, this invention designs and prepares a cellulose hemostatic sponge with in-situ grown zeolite, aiming to overcome the limitations of existing materials in penetrating wound hemostasis. Firstly, in terms of process optimization, a fixed cellulose sponge is prepared by cutting the foam. An innovative microwave hydrothermal method is used instead of the traditional hydrothermal method. By controlling the molar ratio of 30% colloidal silica (2-14) and the hydrothermal reaction time (0.5-8 h), rapid in-situ growth of zeolite in the cellulose matrix is ​​achieved. Then, a secondary calcium ion exchange process is used to increase the calcium ion binding amount in the zeolite framework, significantly enhancing its efficiency in activating the conversion of prothrombin to thrombin. This invention designs a novel method for preparing a cellulose hemostatic sponge and successfully grows calcium-type zeolite in situ, improving the application potential of the cellulose hemostatic sponge. For a successful hemostatic sponge sample, macroscopic performance testing is performed to verify its compression deformation recovery ability. Next, its physical properties, porosity and water absorption capacity are tested. Finally, scanning electron microscopy is used to further explore its microscopic three-dimensional pore structure and elemental composition.

[0051] In this invention, the regenerated cellulose sponge block is a commercially available regenerated cellulose sponge block (Jiangsu Hengfu New Material Technology Co., Ltd.).

[0052] In this invention, unless otherwise specified, deionized water is used, which is the type of deionized water commonly known in the art.

[0053] In this invention, room temperature refers to the ambient temperature during the production process, which is typically within the range of 20±10℃.

[0054] Following the above technical solution, specific embodiments of the present invention are given below. It should be noted that the present invention is not limited to the following specific embodiments, and all equivalent modifications made on the basis of the technical solution of the present invention fall within the protection scope of the present invention.

[0055] Example 1: This embodiment provides a method for preparing a zeolite cellulose hemostatic sponge with enhanced hemostatic ability, which specifically includes the following steps: Step 1: Pre-treatment of regenerated cellulose sponge blocks: The regenerated cellulose sponge block was ultrasonically washed in ultrapure water three times, with each ultrasonic wash lasting 10 minutes. The regenerated cellulose sponge block was then compressed and cut using a mold to obtain a standard cylindrical pretreated sponge block with a diameter of 1 cm and a length of 5 cm.

[0056] In this embodiment, the ultrapure water used is the commonly known ultrapure water in the art.

[0057] In this embodiment, ultrasonic washing is used to remove residual impurities from the regenerated cellulose sponge block.

[0058] In this embodiment, the mold is a commonly used mold known in the art, so that the pre-processed sponge block after cutting is a standard cylindrical shape with a diameter of 1cm and a length of 5cm.

[0059] Step 2: Prepare the precursor solution and mix it with the pretreated sponge block: Sodium hydroxide and aluminum hydroxide were dissolved in deionized water, heated to 90°C, and magnetically stirred at 500 rpm for 30 minutes until completely transparent, to obtain mixture A. Sodium hydroxide, 30% colloidal silica, the pretreated sponge block obtained in step one, and deionized water were mixed and ultrasonically dispersed at a frequency of 40 kHz for 30 seconds to obtain mixture B. Then, mixture A was quickly poured into mixture B to obtain a mixture of precursor solution and pretreated sponge block, i.e., reactant. The molar ratio of sodium hydroxide: aluminum hydroxide: 30% colloidal silica: water in the precursor solution was 9:0.7:10:331.

[0060] In this embodiment, sodium hydroxide is sodium hydroxide commonly known in the art; aluminum hydroxide is aluminum hydroxide commonly known in the art; 30% colloidal silica refers to the fact that the mass of solid silica particles accounts for 30% of the total mass in the entire colloidal dispersion system, and 30% colloidal silica is 30% colloidal silica commonly known in the art.

[0061] In this embodiment, the precursor solution refers to the solution prepared from mixture A and mixture B, excluding all substances from the pretreated sponge block.

[0062] Step 3, Microwave hydrothermal synthesis: The reactants obtained in step two were transferred to a polytetrafluoroethylene-lined reactor. The polytetrafluoroethylene-lined reactor containing the reactants was then placed in a microwave synthesizer for synthesis. The synthesis reaction temperature was 120°C and the synthesis reaction time was 8 hours, resulting in the obtained sponge.

[0063] In this embodiment, the polytetrafluoroethylene-lined reactor is a commonly used polytetrafluoroethylene-lined reactor known in the art; the microwave synthesizer is a commonly used microwave synthesizer of model CEM Mars6.

[0064] Step 4, Post-treatment and Ion Exchange: The reacted sponge obtained in step 3 was sequentially rinsed with deionized water, soaked in 0.2M calcium chloride aqueous solution, and ultrasonically cleaned to obtain a cleaned sponge. The deionized water rinsing was performed 5 times. The specific conditions for soaking in 0.2M calcium chloride aqueous solution were: 150 rpm oscillation and 25°C for 12 hours. The ultrasonic cleaning frequency was 40 kHz, and the ultrasonic cleaning was performed 3 times, with each ultrasonic cleaning lasting 5 minutes.

[0065] In this embodiment, the purpose of rinsing with deionized water is to remove unreacted precursors, and the purpose of soaking in 0.2M calcium chloride aqueous solution and ultrasonic cleaning is to remove loose zeolite particles.

[0066] In this embodiment, the 0.2M calcium chloride aqueous solution is a commonly used 0.2M calcium chloride aqueous solution known in the art.

[0067] Step 5: Fix the sponge after cleaning: The cleaned sponge obtained in step four is compressed into a mold and then transferred together into a freeze dryer. The cold trap temperature of the freeze dryer is -70℃ and the vacuum degree of the freeze dryer is 1Pa. After freeze-drying for 12 hours, zeolite cellulose hemostatic sponge is obtained.

[0068] In this embodiment, the freeze dryer used is a commonly known freeze dryer of model LGJ-12A.

[0069] In this embodiment, the sponge compression mold is a commonly used sponge compression mold known in the art, so that all the sponges that have absorbed water maintain the shape of a standard cylinder after being compressed and freeze-dried by the mold, and all the standard cylindrical sponges that have been compressed and freeze-dried by the mold have the same length and the same diameter.

[0070] The zeolite cellulose hemostatic sponge with enhanced hemostatic ability was prepared using the method described in this embodiment. The zeolite cellulose hemostatic sponge obtained using the preparation method described in this embodiment is designated as spCel-Z3.

[0071] Example 2: This embodiment provides a method for preparing a zeolite cellulose hemostatic sponge with enhanced hemostatic ability. This preparation method is basically the same as the preparation method in Example 1, except that in step three, the synthesis reaction time is changed from 8 hours to 4 hours.

[0072] The zeolite cellulose hemostatic sponge with enhanced hemostatic ability was prepared using the method described in this embodiment.

[0073] Example 3: This embodiment provides a method for preparing a zeolite cellulose hemostatic sponge with enhanced hemostatic ability. This preparation method is basically the same as the preparation method in Example 1, except that in step three, the synthesis reaction time is changed from 8 hours to 2 hours.

[0074] The zeolite cellulose hemostatic sponge with enhanced hemostatic ability was prepared using the method described in this embodiment.

[0075] Example 4: This embodiment provides a method for preparing a zeolite cellulose hemostatic sponge with enhanced hemostatic ability. This preparation method is basically the same as the preparation method in Example 1, except that in step three, the synthesis reaction time is changed from 8 hours to 1 hour.

[0076] The zeolite cellulose hemostatic sponge with enhanced hemostatic ability was prepared using the method described in this embodiment.

[0077] Example 5: This embodiment provides a method for preparing a zeolite cellulose hemostatic sponge with enhanced hemostatic ability. The preparation method is basically the same as the preparation method in Example 1, except that in step three, the synthesis reaction time is changed from 8 hours to 0.5 hours.

[0078] The zeolite cellulose hemostatic sponge with enhanced hemostatic ability was prepared using the method described in this embodiment.

[0079] The surface morphology of the zeolite cellulose hemostatic sponges prepared in Examples 1 to 5 was observed using SEM. Figure 1 As shown, when the synthesis reaction time in step three is in the range of 0.5 to 4 hours, only discrete nanoparticles are observed on the surface of the zeolite cellulose hemostatic sponge, and no complete zeolite crystal structure is formed; however, when the synthesis reaction time in step three is extended to 8 hours, the cubic lattice characteristics of typical type A zeolite can be clearly identified.

[0080] In this invention, the type A zeolite is the type A zeolite commonly known in the art, and the cubic lattice characteristics of a typical type A zeolite are the cubic lattice characteristics of a typical type A zeolite commonly known in the art.

[0081] like Figure 2As shown, XRD was used to further detect the crystallinity of the generated zeolite, which is a common method for determining whether zeolite has been formed. Compared with the blank control group, i.e., the Control obtained in Comparative Example 1, the zeolite cellulose hemostatic sponges synthesized in Examples 1 to 4 with reaction times of 1 to 4 hours showed the typical characteristic peak 2θ of cellulose at around 20°. Only the peaks generated after 8 hours corresponded to the characteristic peaks of typical type A zeolite, located at 12.5°, 16.1°, 21.7°, 27.1°, 29.9°, and 34.2°, respectively, which proves the successful preparation of zeolite.

[0082] Based on Examples 1 to 5 and Comparative Example 1, the optimal time for the synthesis reaction in step three is 8 hours.

[0083] Example 6: This embodiment provides a method for preparing a zeolite cellulose hemostatic sponge with enhanced hemostatic ability. The preparation method is basically the same as the preparation method in Example 1, except that in step two, the molar ratio of sodium hydroxide: aluminum hydroxide: 30% colloidal silica: water in the precursor solution is changed from 9:0.7:10:331 to 9:0.7:2:331.

[0084] The zeolite cellulose hemostatic sponge with enhanced hemostatic ability was prepared using the method described in this embodiment.

[0085] Example 7: This embodiment provides a method for preparing a zeolite cellulose hemostatic sponge with enhanced hemostatic ability. The preparation method is basically the same as the preparation method in Example 1, except that in step two, the molar ratio of sodium hydroxide: aluminum hydroxide: 30% colloidal silica: water in the precursor solution is changed from 9:0.7:10:331 to 9:0.7:4:331.

[0086] The zeolite cellulose hemostatic sponge with enhanced hemostatic ability was prepared using the method described in this embodiment.

[0087] Example 8: This embodiment provides a method for preparing a zeolite cellulose hemostatic sponge with enhanced hemostatic ability. The preparation method is basically the same as the preparation method in Example 1, except that in step two, the molar ratio of sodium hydroxide: aluminum hydroxide: 30% colloidal silica: water in the precursor solution is changed from 9:0.7:10:331 to 9:0.7:8:331.

[0088] The zeolite cellulose hemostatic sponge with enhanced hemostatic ability was prepared using the method described in this embodiment. The zeolite cellulose hemostatic sponge obtained using the preparation method described in this embodiment is denoted as spCel-Z2.

[0089] Example 9: This embodiment provides a method for preparing a zeolite cellulose hemostatic sponge with enhanced hemostatic ability. The preparation method is basically the same as the preparation method in Example 1, except that in step two, the molar ratio of sodium hydroxide: aluminum hydroxide: 30% colloidal silica: water in the precursor solution is changed from 9:0.7:10:331 to 9:0.7:14:331.

[0090] The zeolite cellulose hemostatic sponge with enhanced hemostatic ability was prepared using the method described in this embodiment. The zeolite cellulose hemostatic sponge obtained using the preparation method described in this embodiment is designated as spCel-Z4.

[0091] Comparative Example 1: This embodiment provides a method for preparing a zeolite cellulose hemostatic sponge with enhanced hemostatic ability. The preparation method is basically the same as the preparation method in Example 1, except that in step two, the molar ratio of sodium hydroxide: aluminum hydroxide: 30% colloidal silica: water in the precursor solution is changed from 9:0.7:10:331 to 9:0.7:0:331.

[0092] The zeolite cellulose hemostatic sponge with enhanced hemostatic ability was prepared using the method described in this embodiment. The zeolite cellulose hemostatic sponge obtained using the method described in this embodiment is denoted as Control.

[0093] Comparative Example 2: This embodiment provides a method for preparing physically adsorbed zeolite sponges, which includes the following steps: Step one is exactly the same as the method and raw materials in Step one of Example 1.

[0094] Step 2: The zeolite from the material synthesis process is centrifuged, subjected to calcium ion exchange, and washed to prepare calcium-type zeolite. Then, the calcium-type zeolite is dispersed in water, and the pretreated sponge block obtained in Step 1 is soaked in it at 25°C for 12 hours. After that, it is dried for later use to obtain a physically adsorbed zeolite sponge, denoted as spCel.

[0095] Figure 3As shown, under the conditions of a fixed microwave hydrothermal synthesis temperature of 120℃ and a synthesis reaction time of 8 h, the effect of the concentration of 30% colloidal silica in the precursor solution (molar ratios of 30% colloidal silica of 0, 2, 4, 8, 10, and 14, respectively) on the zeolite loading efficiency was systematically investigated through Comparative Example 1, Example 1, and Examples 6 to 9. Scanning electron microscopy analysis showed that when the molar ratio of 30% colloidal silica was greater than or equal to 8, regular zeolite crystals began to form on the surface of the zeolite cellulose hemostatic sponge, and the crystal density showed a positive correlation with the increase of the concentration of 30% colloidal silica in the precursor solution.

[0096] To quantitatively analyze the elemental distribution characteristics, energy dispersive spectroscopy (EDS, accelerating voltage 20 kV) was used to characterize spCel-Z2, spCel-Z3, and spCel-Z4. The results are as follows: Figures 4 to 6 As shown in the figure, energy dispersive spectroscopy (EDS) imaging results show that the silicon and aluminum atoms in the zeolite crystal region are highly consistent with the main constituent elements in typical type A zeolites. After Ca²⁺ ion exchange treatment, the calcium enrichment effect on the zeolite surface is significantly greater than that of the unmodified zeolite cellulose hemostatic sponge (Control) obtained in Comparative Example 1, demonstrating the advantage of zeolite in calcium ion enrichment.

[0097] 120 mg of each of spCel, spCel-Z2, spCel-Z3, and spCel-Z4 was placed in 10 mL of deionized water and soaked for the same amount of time at room temperature. Then, each sample was ultrasonically treated for 5 minutes using an ultrasonic cleaner (KQ218, Jiangsu Kunshan Ultrasonic Instrument Co., Ltd.). Digital photographs of the samples were then taken, and the binding strength of the zeolites was compared by observing the clarity of the deionized water. The test was repeated twice to reduce error. The test results are as follows: Figure 7 As shown in the figure, the deionized water containing the physically adsorbed zeolite sponge (spCel) became turbid, while the deionized water containing spCel-Z2, spCel-Z3, and spCel-Z4 remained clear, demonstrating the advantages of zeolite growth. This structural characteristic endows the material with excellent mechanical stability, maintaining a certain zeolite retention rate even after ultrasonic treatment, effectively avoiding the risk of zeolite particles detaching at the wound site.

[0098] Figure 8 and Figure 9 The images shown are characteristic images of the morphological recovery of spCel and spCel-Z2, respectively. The dried spCel and spCel-Z2 were compressed into discs and then placed in water. It was found that both the control group (spCel) and the zeolite-loaded sample group (spCel-Z2) recovered their original shape within approximately 1 second, demonstrating that although the water absorption decreased slightly, it did not affect the strong shape memory properties of spCel-Z2.

[0099] spCel, spCel-Z2, spCel-Z3, and spCel-Z4 were cut into cylinders 1 cm long and 1 cm in diameter, and placed in an Xradia 610 Versa X-Ray Microscope for analysis. The porosity of different materials was then further calculated using Dragonfly software. Figures 10 to 11 As shown, three-dimensional reconstructions were performed on spCel, spCel-Z2, spCel-Z3, and spCel-Z4, respectively. Quantitative analysis using Dragonfly software showed that the zeolite loading did not significantly change the macroscopic porosity of the pretreated sponge blocks. The macroscopic porosity of spCel was 92.3% ± 1.2%, while that of spCel-Z2, spCel-Z3, and spCel-Z4 was 90.8% ± 1.5%.

[0100] In this invention, both the Dragonfly software and the quantitative analysis method for macroscopic porosity are commonly used software and methods known in the art. The Xradia 610 Versa X-Ray Microscope instrument is a commonly used instrument known in the art.

[0101] The water absorption capacity of spCel, spCel-Z2, spCel-Z3, and spCel-Z4 was tested. First, the weight W1 of each dry sample was weighed. Then, each sample was soaked in water for the same amount of time. Afterward, the four samples were removed and weighed again until no water droplets remained, and the weight was recorded as W2. Based on... Perform calculations. For example... Figure 12 The water absorption test results show that the water absorption rate of spCel is 2400%±150%. With the introduction of zeolite, the water absorption rate of spCel-Z2, spCel-Z3 and spCel-Z4 decreased slightly, but still remained at around 1800%, which is better than most commercially available hemostatic sponge materials.

[0102] Mechanical strength tests were performed on spCel, spCel-Z2, spCel-Z3, and spCel-Z4 samples using a GMT-4503GD universal testing machine equipped with a 50N compression element. Before testing, each sample was molded into a cylinder with a height of 12 mm and a diameter of 10 mm and immersed in deionized water for 5 minutes. The samples were compressed at a constant strain rate of 0.5 mm / min until 80% of the volumetric strain (relative to the initial size) was reached, and then immediately restored to zero strain at the same rate. This loading-unloading cycle was repeated 10 times for each experimental group to evaluate cyclic deformation behavior. The results are as follows: Figure 13As shown, the compressive modulus of the material under 80% deformation increases in a concentration-dependent manner with respect to 30% colloidal silica, from 17.2±1.5 kPa for spCel to 60.3±4.8 kPa for spCel-Z2, spCel-Z3 and spCel-Z4.

[0103] In this invention, the GMT-4503GD universal testing machine and the method for uniaxial compression testing are both commonly used equipment and methods known in the art.

[0104] like Figure 14 As shown, the contact angle test revealed that due to the instantaneous expansion (<0.5 seconds) of the four materials spCel, spCel-Z2, spCel-Z3 and spCel-Z4 upon contact with liquid, static contact angle data could not be obtained. However, the dynamic wetting process showed their superhydrophilic properties, and the zeolite loading did not have a significant impact on wettability.

[0105] Biocompatibility tests were performed on spCel, spCel-Z2, spCel-Z3, and spCel-Z4 respectively. Figure 15 As shown, the biocompatibility test results indicated that sponge materials with different concentrations of 30% colloidal silica had no effect on cell viability after 24 hours or 48 hours, with cell survival rates all greater than 80%. Similar results were observed for spCel-Z2 and spCel-Z3, but the cell viability of the fourth group of materials decreased significantly after 48 hours, with a cell survival rate of only 75% at 2.5 mg / ml, and the lowest reaching 66%, making it unsuitable for subsequent animal experiments.

[0106] Unless otherwise specified, all test methods in this invention are commonly known in the art, such as the biocompatibility test method.

[0107] like Figure 16 The hemolysis percentages of spCel, spCel-Z2, spCel-Z3, and spCel-Z4 are shown in the figures, grouped into 1.25 mg / mL, 2.5 mg / mL, 5 mg / mL, and 10 mg / mL groups. The hemolysis rate of spCel was significantly higher in the 10 mg / mL group. The hemolysis percentages of spCel-Z2, spCel-Z3, and spCel-Z4 were significantly higher in the 5 mg / mL and 10 mg / mL groups compared to the 1.25 mg / mL and 2.5 mg / mL groups. However, the hemolysis rate of spCel-Z2 was 2.9%, far below the 5% threshold. The hemolysis rates of spCel-Z3 and spCel-Z4 were 4.6% and 4.5%, respectively, which are close to the 5% threshold and may pose some risk.

[0108] Erythrocyte and platelet adsorption electron microscopy experiments were performed on the gauze group, gelatin sponge group, and hemostatic material groups (spCel, spCel-Z2, spCel-Z3, and spCel-Z4). The sponges were cut into 15mg discs (1–1.5 mm) and evenly placed at the bottom of each well in a 24-well plate, then irradiated with UV for 30 min. Whole blood was centrifuged at 2000 rpm for 10 min to obtain erythrocytes; the supernatant was aspirated, and the sponges were centrifuged at 3500 rpm for 10 min to obtain platelets. The erythrocytes and platelets were then dropped onto the sponges and incubated at 37°C for 1 h, until the volume of erythrocytes and platelets was sufficient to wet the material. The samples were washed three times with sterile physiological saline to remove any adhering erythrocytes and platelets. 1 ml of 2.5% glutaraldehyde was added to fix the samples, and the incubation was 37°C for 2 h. The samples were then dehydrated using a gradient of ethanol (25%, 50%, 75%, and 100% for 15 min each), followed by tert-butanol for 15 min. The samples were then freeze-dried and observed using SEM. Figure 17 As shown in the scanned electron microscopy results, the hemostatic material group had a good enrichment effect on red blood cells and platelets, which was significantly better than the gauze group and the gelatin sponge group.

[0109] The coagulation function index of each sponge material in the hemostatic material groups (spCel, spCel-Z2, spCel-Z3, and spCel-Z4) was determined. 1.15 mg of sponge material was placed in a 2 ml centrifuge tube (Eppendorf tube), ensuring it was laid flat at the bottom. Four Eppendorf tubes were prepared for each group, and incubation was performed for four different times. 100 μL of whole blood was added to the sponge material, followed by 10 μL of 10 Mm CaCl2, ensuring full contact between the three components. Incubation was performed at 37°C for 30, 60, 90, and 120 seconds. 1 ml of deionized water was carefully added to the Eppendorf tube, slowly from the bottom using a 1 ml syringe. The sample was photographed, and the supernatant was carefully aspirated. The absorbance was measured at 540 nm. The absorbance of the deionized water was also measured simultaneously. The same tests were performed on the gauze and gelatin sponge groups as on the hemostatic material groups. Figure 18 As shown, at different time points, the blood clotting index (BCI) of the gauze group and the gelatin sponge group was significantly higher than that of the hemostatic material group, while there was no significant difference in the hemostatic material group, and it remained at around 10%.

[0110] In this invention, during biological experiments, the hemostatic material group only contains spCel and spCel-Z2; excluding all other tests in the biological experiments, the hemostatic material group includes spCel, spCel-Z2, spCel-Z3, and spCel-Z4. Figures 19 to 21 Control-1 in the text is the control group, also known as the untreated group, meaning that no materials were used to treat the wound.

[0111] All animal experiments in this invention were approved by the Animal Ethics Committee of the Air Force Medical University. The animal experiments were divided into five groups with different treatment methods: an untreated group (no material was used to treat the wound), a gauze group, a gelatin sponge group, and a hemostatic material group. The hemostatic material group included two different treatment methods: spCel and spCel-Z2. Three mice were used in each treatment group to conduct tail-cutting hemostasis experiments, a non-compression bleeding rat liver defect model, and a femoral artery transection hemostasis experiment, thus constructing three different models to measure the hemostatic effect of different materials. All rats weighed approximately 160g and were anesthetized with 1.5–2ml of 5wt% chloral hydrate intraperitoneally. The following experiments were then conducted: After disinfection of the rat tails at 5–6 weeks of age, the tails were cut 4cm from the base. Immediately afterward, the materials with different treatment methods were placed at the wound site, ensuring direct contact between the materials and the wound surface. Hemostasis time and bleeding volume were recorded. Open the abdominal cavity and separate the right lobe of the liver. Make a 1cm incision in the middle of the lobe and immediately place materials prepared using different methods between the incisions. Record the time of hemostasis and the amount of bleeding. Open the groin area, separate the femoral artery, cut it, and immediately apply materials prepared using different methods to achieve hemostasis. Record the time of bleeding and the amount of bleeding for each procedure.

[0112] like Figure 19 As shown, in the rat tail transection model, only the hemostatic material groups (spCel and spCel-Z2) showed a statistically significant difference in blood loss compared to the untreated group, with a significant reduction in bleeding volume. The effect of spCel-Z2 was more pronounced. The gauze group and gelatin sponge group showed no statistically significant difference compared to the untreated group (Control-1). Regarding hemostasis time, all materials shortened the hemostasis time compared to the untreated group, with the hemostatic material plus zeolite group (spCel-Z2) showing a significantly shorter hemostasis time. The hemostasis times for the untreated group, gauze group, gelatin sponge group, spCel, and spCel-Z2 were 113±7, 93±9, 89±8, 56±4, and 48±3 s, respectively. The bleeding volumes for the untreated group, gauze group, gelatin sponge group, spCel, and spCel-Z2 were 995±127, 807±169, 494±169, 88±54, and 55±17 mg, respectively.

[0113] like Figure 20As shown, in the rat liver rupture model, the hemostatic material groups (spCel and spCel-Z2) significantly reduced blood loss compared to the untreated group and the gauze group, with spCel-Z2 showing better efficacy. While the gelatin sponge group had some effect on reducing bleeding, it was less effective than the hemostatic material groups. The gauze, gelatin sponge, and hemostatic material groups showed significant statistical differences compared to the untreated group. Similarly, hemostasis time also showed the same trend. spCel-Z2 achieved the shortest hemostasis time, approximately one-third that of the untreated group. The gelatin sponge and spCel groups showed some reduction in bleeding compared to the untreated group. The gauze group showed no statistically significant difference in bleeding volume or hemostasis time compared to the untreated group. The hemostasis times for the untreated group, gauze group, gelatin sponge group, spCel, and spCel-Z2 were 165±18, 147±15, 114±16, 72±8, and 51±6 seconds, respectively. The blood loss for the untreated group, gauze group, gelatin sponge group, spCel, and spCel-Z2 was 1786±412, 1577±145, 1026±228, 241±110, and 98±33 mg, respectively.

[0114] Figure 21 As shown, in the rat femoral artery transection model, the hemostatic material group exhibited a significant hemostatic effect, with a marked reduction in blood loss. The gauze and gelatin sponge groups showed some reduction compared to the untreated group, but the effect was not as good as the hemostatic material groups (i.e., spCel and spCel-Z2). Regarding hemostasis time, the gauze, gelatin sponge, and hemostatic material groups all had shorter hemostasis times than the untreated group, with the hemostatic material group showing a significantly shorter hemostasis time than the gauze and gelatin sponge groups. The hemostasis times for the untreated group, gauze group, gelatin sponge group, spCel, and spCel-Z2 were 145±15, 102±8, 97±7, 66±5, and 50±3 seconds, respectively. The bleeding amounts in the untreated group, gauze group, gelatin sponge group, spCel and spCel-Z2 were 2859±486, 2505±169, 2478±171, 2411387±138 and 933±193 mg, respectively.

[0115] Figure 22 As shown, observation with a thermal imaging camera reveals that spCel-Z2 does not exhibit significant temperature rise, effectively preventing the potential exothermic effect of zeolite.

Claims

1. A method for preparing a zeolite cellulose hemostatic sponge with enhanced hemostatic ability, characterized in that, The preparation method specifically includes the following steps: Step 1: Pre-treatment of regenerated cellulose sponge blocks: The regenerated cellulose sponge block was ultrasonically washed in water, and then compressed and cut using a mold to obtain a standard cylindrical pretreated sponge block. Step 2: Prepare the precursor solution and mix it with the pretreated sponge block: Sodium hydroxide and aluminum hydroxide are dissolved in water, heated to 90°C and magnetically stirred until transparent to obtain mixture A; sodium hydroxide, 30% colloidal silica, and the pretreated sponge block obtained in step one are mixed with water and ultrasonically dispersed to obtain mixture B; then mixture A is poured into mixture B to obtain a mixture of precursor solution and pretreated sponge block, i.e., reactant. Step 3, Microwave hydrothermal synthesis: The reactants obtained in step two are transferred to a polytetrafluoroethylene-lined reactor. Then, the polytetrafluoroethylene-lined reactor containing the reactants is placed in a microwave synthesizer to carry out the synthesis reaction, and the reacted sponge is obtained. Step 4, Post-treatment and Ion Exchange: The sponge obtained in step 3 was rinsed with deionized water, soaked in 0.2M calcium chloride aqueous solution, and ultrasonically cleaned in sequence to obtain a cleaned sponge. Step 5: Fix the sponge after cleaning: The cleaned sponge obtained in step four is compressed into a mold and then transferred together into a freeze dryer. After freeze-drying for 12 hours, zeolite cellulose hemostatic sponge is obtained.

2. The method for preparing the zeolite cellulose hemostatic sponge with enhanced hemostatic ability as described in claim 1, characterized in that, In step one, the ultrasonic cleaning is performed three times, and each ultrasonic cleaning session lasts for 10 minutes. In step one, the pretreated standard cylindrical sponge block has a diameter of 1 cm and a length of 5 cm.

3. The method for preparing the zeolite cellulose hemostatic sponge with enhanced hemostatic ability as described in claim 1, characterized in that, In step two, the magnetic stirring time is 30 minutes and the magnetic stirring speed is 500 rpm. In step two, the ultrasonic dispersion time is 30 seconds and the ultrasonic dispersion frequency is 40 kHz. In step two, the molar ratio of sodium hydroxide: aluminum hydroxide: 30% colloidal silica: water in the precursor solution is 9:0.7:2:331, 9:0.7:4:331, 9:0.7:8:331, 9:0.7:10:331, or 9:0.7:14:

331.

4. The method for preparing the zeolite cellulose hemostatic sponge with enhanced hemostatic ability as described in claim 1, characterized in that, In step three, the temperature of the synthesis reaction is 120°C, and the reaction time includes 0.5h, 1h, 2h, 4h, or 8h.

5. The method for preparing the zeolite cellulose hemostatic sponge with enhanced hemostatic ability as described in claim 1, characterized in that, In step four, the deionized water rinsing is performed five times. In step four, the specific conditions for soaking in the 0.2M calcium chloride aqueous solution are: soaking for 12 hours under oscillation conditions of 150 rpm and a temperature of 25°C; In step four, the ultrasonic cleaning frequency is 40kHz, the ultrasonic cleaning is performed 3 times, and the ultrasonic cleaning time for each time is 5 minutes.

6. The method for preparing the zeolite cellulose hemostatic sponge with enhanced hemostatic ability as described in claim 1, characterized in that, In step five, the cold trap temperature of the freeze dryer is -70°C, and the vacuum degree of the freeze dryer is 1 Pa.

7. A zeolite cellulose hemostatic sponge prepared by the method for preparing a zeolite cellulose hemostatic sponge with enhanced hemostatic ability as described in any one of claims 1 to 6.

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