Self-crosslinking visceral hemostatic powder as well as preparation method and application thereof

Abstract

The invention discloses self-crosslinking visceral hemostatic powder as well as a preparation method and application thereof. The preparation method comprises the following steps: dissolving carboxymethyl chitosan and sodium alginate grafted dopamine in a solvent, and stirring until the carboxymethyl chitosan and the sodium alginate grafted dopamine are completely dissolved; dissolving oxidized guar gum in a solvent, and stirring to completely dissolve the oxidized guar gum; freeze-drying the two obtained solutions, and mixing to obtain mixed powder of carboxymethyl chitosan, sodium alginate grafted dopamine and oxidized guar gum; and adding an activating agent into the mixed powder, and uniformly mixing to obtain the self-crosslinking visceral hemostatic powder. The self-crosslinking visceral hemostatic powder prepared by the invention has good biocompatibility, excellent free radical scavenging ability and antibacterial ability, and can be applied to safe and efficient hemostasis and wound healing of deep incompressible visceral hemorrhage wounds.

Description

Technical Field

[0001] This invention relates to a powder composite, specifically a self-crosslinked visceral hemostatic powder, its preparation method, and its application, belonging to the field of biomedical materials technology. Background Technology

[0002] Currently, existing hemostatic materials are categorized into mechanical compression type, coagulation factor activating type, mineral-based material type, mucosal adhesion type, and expandable internal packing type. Mechanical compression hemostatic materials promote the natural coagulation process by providing a physical barrier and absorbing blood, concentrating platelets and coagulation factors. Representative materials include traditional gauze, cotton, and bandages. However, mechanical compression hemostatic materials are only suitable for superficial wounds and surgical wound oozing. Coagulation factor activating hemostatic materials bypass some of the coagulation cascade reactions, directly supplementing or activating key coagulation factors to rapidly form an artificial clot. Representative materials include fibrin glue, thrombin powder, and gelatin sponges containing coagulation factors. However, the hemostatic effect of coagulation factor activating hemostatic materials is not ideal for complex and mixed traumatic bleeding. Furthermore, animal-derived thrombin or fibrinogen may induce an immune response. Mineral-based hemostatic materials have extremely high water absorption, rapidly absorbing water from the blood, concentrating platelets and coagulation factors. Simultaneously, the exothermic reaction may promote protein denaturation and vascular occlusion. Representative materials include zeolite-based dressings. However, zeolite-based materials can generate heat exceeding 90°C, potentially causing secondary burns to tissues. Mucosal adhesive hemostatic materials form a strong sealing layer on the wound surface through physicochemical adhesion or polymerization, isolating blood from the external environment; cyanoacrylate-based materials are representative. However, cyanoacrylate adhesives are unsuitable for frequently moving areas such as joints, as well as wounds that are shallow, require drainage, or are infected. Expandable internal packing hemostatic materials expand after penetrating deep into the wound or cavity, applying physical pressure to the bleeding point and blocking blood flow; expandable hemostatic sponges are representative. However, expandable internal packing hemostatic materials adhere tightly to the wound, causing secondary damage and rebleeding upon removal. Excessive expansion may compress surrounding healthy tissue, affecting blood supply.

[0003] Hydrogels, as representative polymers with hydrophilic properties, have a structure similar to the natural extracellular matrix. Their excellent biocompatibility, strong adhesion, high tensile strength, and good self-healing properties make them representative of novel hemostatic products. However, hydrogels with fixed shapes are difficult to cover wounds with complex shapes, resulting in lower hemostatic efficiency for deep, incompressible visceral wounds. Therefore, there is an urgent need to develop hemostatic materials that are fast-acting, have strong adhesion to moist tissues, are easy to use, safe and absorbable, low in cost, and universally effective for various types of bleeding. This is the ultimate goal of current research and development. Summary of the Invention

[0004] The technical problem to be solved by this invention is that existing hemostatic materials cannot be applied to incompressible visceral bleeding.

[0005] To address the above problems, this invention provides a method for preparing a self-crosslinking visceral hemostatic powder, characterized by comprising the following steps: Step A: Dissolve guar gum in a solvent and stir until completely dissolved; Step B: Add an oxidizing agent to the guar gum solution to oxidize the guar gum, and dry it after the reaction to obtain oxidized guar gum powder; Step C: Dissolve sodium alginate and dopamine in a solvent and stir until completely dissolved; Step D: Add activator A to sodium alginate solution to graft dopamine, and react to obtain sodium alginate-grafted dopamine; Step E: Dissolve carboxymethyl chitosan in a solvent and stir until completely dissolved; Step F: Dissolve sodium alginate grafted with dopamine in carboxymethyl chitosan solution and stir until completely dissolved; Step G: Dry the solution obtained in step F to obtain carboxymethyl chitosan / sodium alginate grafted dopamine powder; Step H: Mix the two powders obtained in Step B and Step G evenly to obtain a mixed powder. Then add activator B to the mixed powder and mix evenly to obtain a self-crosslinked visceral hemostatic powder.

[0006] Preferably, the solvent in step A is any one of distilled water, PBS (pH = 7.4) and physiological saline (w / v = 0.9%), the mass concentration of the dissolved guar gum is 0.1~5%, and the dissolution temperature of the guar gum is 30~80℃.

[0007] Preferably, the oxidant in step B is any one of sodium periodate, potassium permanganate, and hydrogen peroxide, the mass concentration of the oxidant is 0.1% to 10%, the reaction temperature is 30 to 80°C, the reaction time is 5 to 72 h, and the mass ratio of the oxidant to carboxymethyl chitosan is 0.3 to 1.5:1.

[0008] Preferably, the solvent in step C is any one of distilled water, PBS (pH = 7.4), and physiological saline (w / v = 0.9%); the mass concentration of the dissolved sodium alginate is 0.5~10%; and the dissolution temperature of the sodium alginate is 30~80℃.

[0009] Preferably, the activator in step D is any one or two of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, the mass concentration of activator A in the mixed solution is 0.1% to 10%, the mass concentration of dopamine is 0.01% to 1%, and the mass ratio of dopamine to sodium alginate is 0.01 to 0.5:1.

[0010] Preferably, the reaction temperature in step D is 10~80℃, and the reaction time is 1~30h.

[0011] Preferably, the solvent in step E is any one of distilled water, PBS (pH = 7.4), or physiological saline (w / v = 0.9%), the mass concentration of the dissolved carboxymethyl chitosan is 0.5-15%, and the dissolution temperature of the carboxymethyl chitosan is 30-80℃.

[0012] Preferably, in step F, the mass concentration of sodium alginate grafted with dopamine after dissolution is 0.05%–0.5%, and the mass ratio of sodium alginate grafted with dopamine to carboxymethyl chitosan is 0.01–0.5:1. The dissolution temperature is 30–80°C.

[0013] Preferably, the drying method of the solution in step G is either baking or freeze drying.

[0014] Preferably, the activator B in step H is any one or two of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, and the mass ratio of activator B in the mixed powder is 5% to 40%, and the mass ratio of carboxymethyl chitosan / sodium alginate grafted dopamine powder to oxidized guar gum powder is 0.1 to 3:1.

[0015] The present invention also provides a self-crosslinking visceral hemostatic powder prepared by the above method, and the application of the self-crosslinking visceral hemostatic powder in the preparation of materials for incompressible visceral bleeding wounds.

[0016] This invention adds an activator to carboxymethyl chitosan, sodium alginate grafted with dopamine, and oxidized guar gum powder. Taking sodium periodate as the oxidant and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide as activators, a method for preparing hemostatic powder is provided: Carboxymethyl chitosan, sodium alginate grafted with dopamine, and oxidized guar gum powder are mixed evenly, then 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide are added, and then mixed evenly to form hemostatic powder.

[0017] This invention utilizes the property of activating the carboxyl groups in carboxymethyl chitosan and sodium alginate with an activator, enabling them to undergo cross-linking reactions with the amino groups of carboxymethyl chitosan, and the property of cross-linking reactions between the amino groups of carboxymethyl chitosan and the aldehyde groups of oxidized guar gum. It provides a method for preparing a self-cross-linked gel by grafting dopamine and oxidized guar gum onto carboxymethyl chitosan and sodium alginate under the activation of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide.

[0018] This invention features a simple process, short product preparation time, and the resulting product exhibits excellent biocompatibility in both in vitro and in vivo simulation experiments. This invention selects carboxymethyl chitosan, sodium alginate, and oxidized guar gum as matrices. Carboxymethyl chitosan is a modified chitosan derivative that retains the excellent biocompatibility and biodegradability of chitosan; its degradation products (amino sugars) can be absorbed or metabolized by the human body. Sodium alginate is a natural polysaccharide with excellent water absorption and swelling properties and superior biocompatibility. Guar gum is a natural high-molecular-weight polysaccharide (galactomannan) extracted from guar bean seeds. It possesses unique physicochemical properties and biological characteristics. The carboxyl groups in carboxymethyl chitosan and sodium alginate are activated using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, enabling them to crosslink with carboxymethyl chitosan. Simultaneously, the amino groups of carboxymethyl chitosan can also undergo a crosslinking reaction with the aldehyde groups of oxidized guar gum, preparing a self-crosslinked visceral hemostatic powder. Experiments show that the hemostatic powder prepared in this invention can rapidly absorb tissue fluid or blood after contact with the wound, concentrate clotting factors, coagulate blood cells, and undergo a self-crosslinking reaction, ultimately forming a stable hydrogel that adheres to the wound surface, thus achieving rapid hemostasis.

[0019] Compared with existing technologies, the technological advancements of this invention are significant. Existing hemostatic materials are primarily hydrogels. Because hydrogels already contain a large amount of water during preparation, their swelling rate is lower than that of hemostatic powders made from the same raw materials. Hemostatic powders can be injected into deep visceral bleeding sites using auxiliary equipment, completely covering irregular wounds. More importantly, when the hemostatic powder comes into contact with the wound surface, it undergoes repeated water absorption and self-crosslinking to form a hydrogel. This not only provides a moist environment for wound healing but also forms a protective barrier on the wound surface. This hemostatic powder retains the inherent biocompatibility and high fluid absorption capacity of hydrogels, while also possessing a larger specific surface area, a faster rate of body fluid absorption, and excellent compressibility and resilience. These characteristics make it particularly suitable for deep, incompressible visceral bleeding wounds, rapidly absorbing blood and providing a protective barrier, while also being easier to manipulate and adhere to the wound surface in complex cavities such as the abdominal and thoracic cavities. The hemostatic powder prepared by this invention has a simple process, is readily available, has good free radical scavenging and antibacterial capabilities, and can adapt to different shapes and surfaces. It can overcome the shortcomings of existing hemostatic powders, such as their limited effect and inability to be applied to wounds with bleeding from incompressible internal organs. It is expected to be applied to the field of hemostasis and wound healing of deep, incompressible internal organs and has certain clinical application value.

[0020] This invention also provides the application of the above-mentioned hemostatic powder as a safe and efficient antibacterial and anti-inflammatory biomaterial. The self-crosslinking visceral hemostatic powder prepared by this invention has good biocompatibility, excellent free radical scavenging ability and antibacterial ability, and can be applied to the safe and efficient treatment of hemostasis and wound healing of deep incompressible visceral bleeding wounds. Attached Figure Description

[0021] Figure 1 a is an FESEM image of the self-crosslinked visceral hemostatic powder in Example 7; Figure 1 b is a FESEM image of the hydrogel formed by the self-crosslinking of the self-crosslinking visceral hemostatic powder after absorbing PBS in Example 7; Figure 2 Dynamic time-scan rheological analysis of the self-crosslinking visceral hemostatic powder absorbing PBS and forming a gel in Example 8; Figure 3 a is the compression strain-stress curve in the mechanical property test results of the hydrogel formed by the self-crosslinked visceral hemostatic powder after absorbing PBS in Example 9; Figure 3 b represents the average compressive stress and compressive modulus in the mechanical property test results of the hydrogel formed by the self-crosslinked visceral hemostatic powder after absorbing PBS in Example 9. Figure 4 The in vitro biosafety of the self-crosslinked visceral hemostatic powder prepared in Example 6; wherein, Figure 4 'a' represents the hemolysis test result. Figure 4 b represents the survival rate of fibroblasts after co-culturing with self-crosslinked visceral hemostatic powder; Figure 4 cf represents the DEAD / LIVE staining result: Figure 4 c represents the result without material treatment and the result after treatment with self-crosslinking visceral hemostatic powder at a concentration of 2.5 mg / mL. Figure 4 d), 5mg / mL ( Figure 4 e), 10 mg / mL ( Figure 4 f); Figure 5 Examples 12 and 13 show the swelling ratio, swelling kinetics, and in vitro degradation curves of the self-crosslinked visceral hemostatic powder. Figure 5 'a' represents the swelling ratio in physiological saline. Figure 5 b represents the swelling ratio in PBS. Figure 5 c represents the swelling kinetics curve in physiological saline. Figure 5 d represents the swelling kinetics curve in PBS. Figure 5 e represents the in vitro degradation curve in physiological saline. Figure 5 f is the in vitro degradation curve in PBS; Figure 6 The results of the in vitro antibacterial experiments of the self-crosslinked visceral hemostatic powder in Examples 14 and 15 are as follows: Figure 6 a represents the proliferation of Escherichia coli colonies on the agar plate. Figure 6 b represents the inhibition rate of the self-crosslinked visceral hemostatic powder against Escherichia coli. Figure 6 c represents the proliferation of Staphylococcus aureus colonies on the agar plate. Figure 6 d represents the inhibition rate of the self-crosslinked visceral hemostatic powder against Staphylococcus aureus; Figure 7 The results show the free radical scavenging performance of the self-crosslinked visceral hemostatic powder prepared in Example 6; wherein, Figure 7 a represents the result of DPPH· removal. Figure 7 b represents the result of clearing PTIO·; Figure 7 c represents the result of ABTS· removal; Figure 8 The results of routine blood tests were used to assess the in vivo biocompatibility of the self-crosslinked visceral hemostatic powder prepared in Example 5. Figure 8 a represents hemoglobin (HGB). Figure 8 b represents hematocrit (HCT). Figure 8 c represents mean corpuscular hemoglobin (MCH). Figure 8 d represents the mean corpuscular hemoglobin concentration (MCHC). Figure 8 e represents the mean corpuscular volume (MCV). Figure 8 f stands for platelet count (PLT). Figure 8 g represents the red blood cell count (RBC). Figure 8 h represents the red blood cell distribution width (RDW). Figure 8 i represents the white blood cell count (WBC); Figure 9 The in vivo biosafety blood biochemical test results of the self-crosslinked visceral hemostatic powder prepared in Example 5 are as follows: Figure 9 a represents total bilirubin (TB), urea (Urea), and creatinine (Crea). Figure 9 b represents alanine aminotransferase (ALT) and aspartate aminotransferase (AST); Figure 10 The results of hematoxylin-eosin (H&E) staining for the in vivo biosafety of the self-crosslinked visceral hemostatic powder prepared in Example 5; Figure 11 The in vitro coagulation results of the self-crosslinked visceral hemostatic powder prepared in Example 5 are as follows. Figure 11 Image a shows the release of hemoglobin from uncoagulated red blood cells during the coagulation process. Figure 11 b represents the coagulation index; Figure 12 The results of promoting wound healing of the self-crosslinked visceral hemostatic powder prepared in Example 5; wherein, Figure 12 a represents the healing status of the wound on the mouse's back. Figure 12 b shows the staining results of skin tissue after wound healing (hematoxylin-eosin, Masson's red, Sirius red); Figure 13 The results of liver hemostasis using the self-crosslinked visceral hemostatic powder prepared in Example 5 are as follows. Figure 13 Photo 'a' shows the process of liver hemostasis. Figure 13 b represents the total blood loss during the liver's hemostasis process. Figure 13 c represents the time it takes for the liver to stop bleeding. Detailed Implementation

[0022] To make the present invention more apparent and understandable, preferred embodiments are described in detail below with reference to the accompanying drawings. Example 1

[0023] 5.0 g of guar gum was dissolved in 500 mL of deionized water and stirred until homogeneous at 50 °C. Then, 5.0 g of sodium periodate was dispersed in the above solution, and the mixture was reacted at 50 °C in the dark for 12 h. Subsequently, 3 mL of ethylene glycol was added to the reaction system to terminate the reaction. After the reaction was completed, the solution was dialyzed against deionized water for 72 h, and finally freeze-dried to obtain oxidized guar gum. Example 2

[0024] 5.0 g of guar gum was dissolved in 500 mL of deionized water and stirred thoroughly at 50 °C. Then, 3.0 g of sodium periodate was dispersed in the above solution, and the mixture was reacted at 50 °C in the dark for 12 h. Subsequently, 3 mL of ethylene glycol was added to the reaction system to terminate the reaction. After the reaction was completed, the solution was dialyzed against deionized water for 72 h, and finally freeze-dried to obtain oxidized guar gum.

[0025] Example 3 1.0 g of sodium alginate was dissolved in 40 mL of deionized water and stirred until homogeneous at 50 °C. Then, 0.02 g of dopamine hydrochloride was dispersed in the above solution. Subsequently, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide (0.1 g and 0.1 g respectively) were added to the reaction system, and the reaction was carried out at 50 °C for 12 h. After the reaction was completed, the solution was dialyzed against deionized water for 72 h, and finally freeze-dried to obtain sodium alginate-grafted dopamine.

[0026] Example 4 1.0 g of sodium alginate was dissolved in 40 mL of deionized water and stirred until homogeneous at 50 °C. Then, 0.05 g of dopamine hydrochloride was dispersed in the above solution. Subsequently, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide (0.1 g and 0.1 g respectively) were added to the reaction system, and the reaction was carried out at 50 °C for 12 h. After the reaction was completed, the solution was dialyzed against deionized water for 72 h, and finally freeze-dried to obtain sodium alginate-grafted dopamine.

[0027] Example 5 Take 1.0 g of carboxymethyl chitosan and 0.2 g of sodium alginate-grafted dopamine, dissolve them in 70 mL of deionized water, and stir evenly at 50 °C to obtain a mixed solution of carboxymethyl chitosan / sodium alginate-grafted dopamine. Freeze-dry using a lyophilizer to obtain carboxymethyl chitosan / sodium alginate-grafted dopamine powder. Take 2.26 g of carboxymethyl chitosan / sodium alginate-grafted dopamine powder and 1.55 g of oxidized guar gum powder, and mix evenly. Then add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide (0.47 g and 0.47 g respectively) to obtain a self-crosslinked visceral hemostatic powder.

[0028] Example 6 Take 1.0 g of carboxymethyl chitosan and 0.2 g of sodium alginate-grafted dopamine, dissolve them in 70 mL of deionized water, and stir evenly at 50 °C to obtain a mixed solution of carboxymethyl chitosan / sodium alginate-grafted dopamine. Freeze-dry using a lyophilizer to obtain carboxymethyl chitosan / sodium alginate-grafted dopamine powder. Take 1.55 g of carboxymethyl chitosan / sodium alginate-grafted dopamine powder and 1.55 g of oxidized guar gum powder, and mix evenly. Then add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide (0.47 g, 0.47 g) to obtain a self-crosslinked visceral hemostatic powder.

[0029] Example 7 The morphology of the self-crosslinked visceral hemostatic powder in Example 5 before and after PBS absorption was analyzed. The samples were analyzed using a ZeissSigma 300 field emission scanning electron microscope. The electron micrographs show that the self-crosslinked visceral hemostatic powder without PBS absorption exhibits a uniform granular structure. Figure 1 a and Figure 1 (b) Upon absorption of PBS, the self-crosslinked visceral hemostatic powder transforms into a hydrogel with a three-dimensional porous structure, characterized by densely distributed and small-sized pores. This three-dimensional porosity enables it to absorb tissue fluid / blood, promoting the transport of cellular nutrients and thus facilitating hemostasis and healing.

[0030] Example 8 The gelation process of self-crosslinked visceral hemostatic powder was studied by observing the final storage modulus (G') and final loss modulus (G'') in dynamic time-scan rheological experiments. Dynamic rheological studies were conducted using a rotational rheometer (MARS Ⅲ HAAKE) with parallel plates (P20 TiL, 20 mm diameter). The self-crosslinked visceral hemostatic powder in Example 5 was subjected to time-scan oscillation experiments at a frequency of 1 Hz, a gap of 1 mm, and a strain of 10%. The corresponding hemostatic powder and PBS were injected onto the plate, with the gap adjusted to 1 mm. The scan measurements of the hydrogel are expressed as G' and G''. The gel point was determined when G' exceeded G''. Due to intermolecular self-crosslinking, G' increased rapidly after the hemostatic powder absorbed PBS, indicating that the efficiency of the hemostatic powder in self-crosslinking to form a hydrogel is very high. Figure 2 ).

[0031] Example 9 The self-crosslinked visceral hemostatic powder prepared in Example 5 was mechanically evaluated using a Zwick Roell Z2.5 TH universal testing machine with a 2.5 kN sensor. In the compression test, the self-crosslinked visceral hemostatic powder was first gelled by absorbing PBS in a cylindrical mold with a diameter of 11 mm and a thickness of 3 mm, and then subjected to a compression test at a strain rate of 1 mm / min. Figure 3a). Within a strain range of 10%–20%, the compressive modulus was recorded using the linear fitting value of the stress-strain curve. In Example 5, the maximum compressive stress and compressive modulus of the self-crosslinked visceral hemostatic powder were 341.12 kPa and 32.49 kPa, respectively. Figure 3 b).

[0032] Example 10 Study on the blood compatibility of self-crosslinked visceral hemostatic powder (Example 5). Example 5 was prepared by pre-absorbing PBS and forming a gel for later use. 2 mL of whole blood was centrifuged (5000 rpm, 5 minutes) and washed three times with PBS to obtain red blood cells. The obtained red blood cells were stored in 50 mL of PBS for further use. In the hemolysis test, 0.5 mL of the above mouse red blood cells were placed in a 5.0 mL centrifuge tube and incubated for 2 hours at 37°C with (1) 2 mL PBS (negative control), (2) 2 mL deionized water (positive control), and (3) different mass / volume fractions of the hydrogel obtained from Example 5 with absorbed PBS (0.36, 0.72, 1.44 g / mL, in 2 mL PBS). The absorbance of the supernatant at 541 nm was collected (using a Shimadzu UV-3600 UV-Vis-NIR spectrometer), and the hemolysis rate of the red blood cells was calculated. Figure 4 As shown in Figure a, the calculated sponge hemolysis rates were all less than 5%. The supernatant images show that the red blood cell supernatant incubated with the self-crosslinked hydrogel of the hemostatic powder and PBS was clear and transparent. However, the blood treated with deionized water appeared distinctly red due to positive hemolysis. These results indicate that the self-crosslinked visceral hemostatic powder has good blood compatibility.

[0033] Example 11 First, the self-crosslinked visceral hemostatic powder obtained in Example 5 was absorbed into PBS to obtain a self-crosslinked hydrogel. Then, the hydrogel was soaked in cell culture medium to obtain extracts of different concentrations. Fibroblasts were seeded in 96-well plates and cultured overnight with 100 μL of cell culture medium. The culture medium was discarded, and 100 μL of extracts of different concentrations (2.5, 5, 10 mg / mL) were added. The control group received only 100 μL of cell culture medium (viability set at 100%). The cells were incubated in a CO2 incubator for 24 hours, and cell viability was quantitatively and qualitatively assessed using the CCK-8 and LIVE / DEAD cell viability assay kits. Figure 4 As shown in b, the self-crosslinked visceral hemostatic powder did not affect cell survival. Similar to the control group ( Figure 4 c) Cells treated with the self-crosslinked visceral hemostatic powder were all stained green by the LIVE / DEAD reagent (live cells were stained green), and almost no cells were stained red (dead cells were stained red). Figure 4df). CCK-8 and LIVE / DEAD cell staining results showed that the prepared self-crosslinked visceral hemostatic powder had good cell compatibility.

[0034] Example 12 To investigate the swelling capacity of the hemostatic powder, physiological saline was added to the self-crosslinked visceral hemostatic powder obtained in Example 5 until excess, and the mixture was soaked at 37°C for 24 hours. The hydrogel was then gently wiped to remove surface saline and weighed. Finally, the hydrogel was freeze-dried, and its initial mass was weighed to determine the swelling ratio. Figure 5 a). Furthermore, the swelling kinetics of the hemostatic powder in physiological saline were investigated ( Figure 5 (c) This demonstrates that the hemostatic powder is rapidly absorbed into the liquid; after soaking in physiological saline for 5 minutes, the swelling rate reached 45.65 g / g. The swelling rate test of the self-crosslinked visceral hemostatic powder in PBS followed the same procedure as the above-mentioned physiological saline test. After storage in PBS for 5 minutes, the swelling rate of the hemostatic powder reached 32.69 g / g. Figure 5 b and Figure 5 d) Example 13 First, the self-crosslinked visceral hemostatic powder obtained in Example 5 was absorbed into PBS to obtain a hydrogel. Then, the hydrogel was placed in 10 mL of lysozyme (1×10⁻⁶). 4 Incubate the hydrogel in physiological saline or PBS (U / mL) at 37°C with continuous shaking for 28 days. Change the culture medium every other day. At each time point, remove the hydrogel from the culture medium, gently rinse with deionized water or simulated body fluid, and lyophilize. Weigh the lyophilized sponge to calculate the degradation rate. The study found that the hemostatic powder retained approximately 40.6% of its original mass after 28 days of degradation in physiological saline containing lysozyme. Figure 5 e). After degradation in PBS containing lysozyme for 28 days, the hemostatic powder retained approximately 40.53% of its original mass ( Figure 5 f) Example 14 First, bacteria (Escherichia coli and Staphylococcus aureus) were activated in liquid culture medium and incubated in a tri-gas incubator at 37°C for 24 hours at a rotation speed of 120 rpm. Then, the activated bacterial suspension was added to sterile liquid culture medium, and the OD value of the bacterial suspension at 600 nm was adjusted to approximately 0.1. Self-crosslinking visceral hemostatic powder (prepared in Example 5) was pre-absorbed and self-crosslinked into a hydrogel, and placed in a test tube containing 5 mL of the bacterial suspension. The gel was incubated in a tri-gas incubator at 37°C for 24 hours. Under the same conditions, an untreated bacterial suspension was used as a positive control. Subsequently, the co-incubated bacterial suspension (10 μL) was diluted and spread onto agar plates, and incubated in a tri-gas incubator at 37°C for 24 hours. The number of bacteria growing on the agar plates was counted. Figure 6 a and Figure 6As shown in Figure c, after 24 hours of incubation, the colony growth of the two bacteria demonstrated that the self-crosslinked visceral hemostatic powder has a certain inhibitory effect on Gram-negative and Gram-positive bacteria, with inhibition rates of 89.1% and 89.23%, respectively. Figure 6 b and Figure 6 d).

[0035] Example 15 First, self-crosslinking visceral hemostatic powder (prepared in Example 5) was pre-absorbed to obtain self-crosslinking hydrogels. Hydrogels of different mass fractions were then placed in solutions of DPPH (hydrogel mass fraction: 10, 15, 20 mg / mL), PTIO (hydrogel mass fraction: 40, 60, 80 mg / mL), and ABTS (hydrogel mass fraction: 15, 30, 60 mg / mL). These solutions were then incubated at 37°C for different times. The absorbance (A1) of the supernatant was measured using a UV-Vis-NIR spectrophotometer (Shimadzu UV-3600, Japan). The absorbance of the original solutions was measured as A0 (DPPH: 519 nm, PTIO: 557 nm, and ABTS: 734 nm). The clearance efficiencies of the self-crosslinked visceral hemostatic powder against DPPH (hydrogel concentration: 20 mg / mL), PTIO (hydrogel concentration: 80 mg / mL), and ABTS (hydrogel concentration: 60 mg / mL) were 94.82%, 66.52%, and 97.33%, respectively. Figure 7 ac).

[0036] Example 16 The self-crosslinked visceral hemostatic powder obtained in Example 5 was implanted subcutaneously into the back of Kunming mice. Complete blood count and blood biochemistry tests were performed on the Kunming mice on days 3, 7, and 14. The results showed that the mice's complete blood count (…) Figure 8 AI) and blood biochemistry ( Figure 9 a and Figure 9 (b) All indicators were within the standard range. Histological studies were then conducted on the important organs (heart, liver, spleen, lung, and kidney) of the mice. Compared with the control group, the self-crosslinked visceral hemostatic powder did not produce any adverse effects on the mice. Figure 10 This indicates that the self-crosslinked visceral hemostatic powder has good in vivo safety.

[0037] Example 17 The in vitro coagulation ability of the self-crosslinked visceral hemostatic powder was studied. 50 μL of anticoagulated mouse whole blood was mixed with 10 μL of 0.1 M CaCl2 aqueous solution and dropped onto the surface of the self-crosslinked visceral hemostatic powder (prepared in Example 5). Separately, 50 μL of anticoagulated mouse whole blood was mixed with 10 μL of 0.1 M CaCl2 aqueous solution and dropped onto gauze or allowed to coagulate naturally in air. All samples were stored at 37°C for 30, 60, and 180 s. Finally, 10 mL of deionized water was added to release hemoglobin from uncoagulated mouse red blood cells. The absorbance of the supernatant at 545 nm was collected using a UV-Vis-NIR spectrophotometer (Shimadzu UV-3600 UV-Vis-NIR spectrometer, Japan), and the coagulation index was calculated. Figure 11 As shown in Figure a, the supernatant of the gauze group and the natural coagulation group was red, while the supernatant of the blood treated with the self-crosslinked visceral hemostatic powder group was lighter in color, indicating that the blood treated with the self-crosslinked visceral hemostatic powder had already coagulated. At each culture time point, the dynamic coagulation index of the self-crosslinked visceral hemostatic powder group was lower than that of the natural coagulation group and the gauze group. Figure 11 b).

[0038] Example 18 The in vivo hemostatic ability of self-crosslinked visceral hemostatic powder was studied. Six healthy Kunming mice (50 ± 5g) were randomly divided into an experimental group and a control group (n=3). A dorsal wound model was established: after anesthesia, a hole with a diameter of about 10mm was punched in the back of the Kunming mice. The experimental group was immediately sprayed with the self-crosslinked visceral hemostatic powder (0.2g) prepared in Example 6, while the control group only had the wound exposed without any intervention. Figure 12 a). Wound healing was observed and photographed at days 0, 3, 7, 10, and 14. Results showed that the wound healing rate in the experimental group was significantly faster than that in the control group. Skin samples from the backs of Kunming mice taken on day 14 were stained with hematoxylin-eosin, masson's red, and Sirius red. Figure 12 b). Hematoxylin-eosin staining histological analysis showed that the experimental group mice had a thinner dermis, a flatter epidermis, more orderly collagen arrangement, and increased skin regeneration at the wound site. In contrast, the control group mice exhibited scarring characteristics such as epidermal loss and dermal thickening. Furthermore, Masson's red and Sirius red staining showed a significant reduction in collagen fibers in the experimental group compared to the control group. These results indicate that the self-crosslinked visceral hemostatic powder prepared in this invention possesses excellent wound healing promoting properties.

[0039] Example 19 The in vivo hemostatic ability of self-crosslinked visceral hemostatic powder was studied. Six healthy SD rats (300 ± 20 g) were randomly divided into an experimental group and a control group (n=3). A liver hemorrhage model was established: after anesthesia, the liver was lacerated with surgical scissors, creating a 3 mm deep and 1 cm long laceration. The experimental group was immediately sprayed with the self-crosslinked visceral hemostatic powder (1 g) prepared in Example 5, while the control group only had the wound exposed without any hemostatic intervention. Figure 13 a). Observe the bleeding from the wound, measure the bleeding time, and weigh the blood loss. The results showed that the hemostasis time in the experimental group was 46.3 s, while that in the control group was 166 s, which was significantly shorter ( Figure 13 c), the amount of bleeding in the experimental group (1.1g) was significantly less than that in the control group (3.1g). Figure 13 b). The above results indicate that the self-crosslinking visceral hemostatic powder prepared in this invention has excellent in vivo hemostatic properties.

[0040] The above descriptions are merely some embodiments of the present invention. Those skilled in the art can make various modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. A method for preparing a self-crosslinking visceral hemostatic powder, characterized in that, Includes the following steps: Step A: Dissolve guar gum in a solvent and stir until completely dissolved; Step B: Add an oxidizing agent to the guar gum solution to oxidize the guar gum, and dry it after the reaction to obtain oxidized guar gum powder; Step C: Dissolve sodium alginate and dopamine in a solvent and stir until completely dissolved; Step D: Add an activator to a sodium alginate solution to graft dopamine, and react to obtain sodium alginate-grafted dopamine; Step E: Dissolve carboxymethyl chitosan in a solvent and stir until completely dissolved; Step F: Dissolve sodium alginate grafted with dopamine in carboxymethyl chitosan solution and stir until completely dissolved; Step G: Dry the solution obtained in step F to obtain carboxymethyl chitosan / sodium alginate grafted dopamine powder; Step H: Mix the two powders obtained in Step B and Step G evenly to obtain a mixed powder. Then add activator B to the mixed powder and mix evenly to obtain a self-crosslinked visceral hemostatic powder.

2. The method for preparing the self-crosslinking visceral hemostatic powder as described in claim 1, characterized in that, The solvent in steps A, C, and E is any one of distilled water, phosphate buffer solution, and physiological saline. The pH of the phosphate buffer solution is 7.4, and the mass concentration (w / v) of the physiological saline is 0.9%. The mass concentration of the dissolved guar gum is 0.1-5%, and the dissolution temperature of the guar gum is 30-80°C.

3. The method for preparing the self-crosslinking visceral hemostatic powder as described in claim 1, characterized in that, In step B, the oxidant is any one of sodium periodate, potassium permanganate, and hydrogen peroxide, the mass concentration of the oxidant is 0.1% to 10%, the reaction temperature is 30 to 80°C, the reaction time is 5 to 72 hours, and the mass ratio of the oxidant to carboxymethyl chitosan is 0.3 to 1.5:

1.

4. The method for preparing the self-crosslinking visceral hemostatic powder as described in claim 1, characterized in that, In step C, the mass concentration of the dissolved sodium alginate is 0.5% to 10%, and the dissolution temperature of the sodium alginate is 30 to 80°C.

5. The method for preparing the self-crosslinking visceral hemostatic powder as described in claim 1, characterized in that, The activator A in step D is any one or two of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide. The mass concentration of the activator A in the mixed solution is 0.1% to 10%, the mass concentration of the dopamine is 0.01% to 1%, the mass ratio of the dopamine to sodium alginate is 0.01 to 0.5:1, the reaction temperature in step D is 10 to 80°C, and the reaction time is 1 to 30 hours.

6. The method for preparing the self-crosslinking visceral hemostatic powder as described in claim 1, characterized in that, In step E, the mass concentration of the dissolved carboxymethyl chitosan is 0.5% to 15%, and the dissolution temperature of the carboxymethyl chitosan is 30 to 80°C.

7. The method for preparing the self-crosslinking visceral hemostatic powder as described in claim 1, characterized in that, In step F, the mass concentration of sodium alginate grafted with dopamine after dissolution is 0.05% to 0.5%, the mass ratio of sodium alginate grafted with dopamine to carboxymethyl chitosan is 0.01 to 0.5:1, and the dissolution temperature is 30 to 80°C. The drying method for the solution in step G can be either oven drying or freeze drying.

8. The method for preparing the self-crosslinking visceral hemostatic powder as described in claim 1, characterized in that, The activator B in step H is any one or two of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide. The mass ratio of the activator B in the mixed powder is 5% to 40%, and the mass ratio of the carboxymethyl chitosan / sodium alginate grafted dopamine powder to oxidized guar gum powder is 0.1 to 3:

1.

9. The self-crosslinking visceral hemostatic powder obtained by the method according to any one of claims 1-8.

10. Use of the hemostatic powder according to claim 9 in the preparation of hemostatic materials and / or wound healing materials for deep, incompressible visceral bleeding wounds.

Images