Multifunctional hemostatic adhesive and preparation method and application thereof

Abstract

This invention discloses a multifunctional hemostatic adhesive, its preparation method, and its application. The steps are as follows: A pale yellow zein powder is dispersed in an SDS aqueous solution under ultrasonication to form a stable zein / SDS solution, and the pH is adjusted; polylysine is dissolved in water to form a polylysine solution; the polylysine solution is added dropwise to the zein / SDS solution to form a wet adhesive for the multifunctional hemostatic adhesive; the wet adhesive is freeze-dried to obtain a powdered multifunctional hemostatic adhesive. The hemostatic adhesive prepared by this invention is suitable for hemostasis of venous and arterial bleeding in live animals; it can also achieve hemostasis in different physical states and can be used in combination. The hemostatic adhesive of this invention has both blood-absorbing and hemostatic functions. The product of this invention has good biocompatibility at the cellular level, has no significant effect on the activity of fibroblasts and blood cells, and matches the self-repair speed of arteries and veins.

Description

Technical Field

[0001] This invention relates to the field of medical devices, and more specifically to a multifunctional hemostatic adhesive, its preparation method, and its application. Background Technology

[0002] Uncontrolled bleeding following trauma or during surgery is a leading cause of patient death. In addition to traditional electrocoagulation and suturing, there is an urgent clinical need to develop effective hemostatic materials, especially for bleeding from irregularly shaped, fragile internal organs and arterial / venous bleeding in cases of high blood pressure. An ideal hemostatic material should meet the following requirements: rapid and robust blood absorption to concentrate clotting factors; strong adhesion and good mechanical properties to provide a physical barrier to maintain blood pressure at the wound site; and good blood compatibility and biocompatibility. Fibrin glue is a commonly used biomaterial for in vivo hemostasis in clinical practice. It is mainly composed of adhesive proteins (containing fibrinogen and clotting factor X), thrombin, and calcium ions, exhibiting good tissue compatibility and no toxic side effects. However, its disadvantages include slow fibrinogen dissolution, low gel strength, and rapid degradation, which can lead to difficulties in clinical application, incomplete hemostasis, and rebleeding. Furthermore, blood often weakens the adhesion between the hydrogel and tissue, resulting in decreased hemostatic efficacy. Existing polymer hemostatic materials often have a clear hemostatic effect, but their efficacy is limited, they produce toxic byproducts, and can potentially cause tissue necrosis and local irritation, thus restricting their widespread application. Hemostatic powders can be directly deposited on irregularly shaped, incompressible bleeding wounds, promoting hemostasis by absorbing blood. However, traditional hemostatic powders cannot form stable scabs and may even dissolve in the blood, failing to reliably stop bleeding. Furthermore, bone wax used in orthopedic hemostasis has poor biocompatibility, leading to foreign body granulomas, nonunion, and reduced tissue resistance to infection.

[0003] Therefore, providing a hemostatic adhesive that is biocompatible, has a moderate degradation rate, is unaffected by blood, can be applied in various scenarios, can adhere clearly, and has multiple usage forms is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0004] In view of this, the present invention provides a multifunctional hemostatic adhesive and its preparation method that can be used for life-threatening arterial bleeding, hemostasis of smooth organ wounds that are difficult to operate, and bleeding from irregularly shaped bones.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A method for preparing a multifunctional hemostatic adhesive includes the following steps:

[0007] (1) Disperse the pale yellow zein powder into an SDS aqueous solution under ultrasonication to form a stable zein / SDS solution, and then adjust the pH of the zein / SDS solution for later use;

[0008] (2) Dissolve polylysine in water to form a transparent polylysine solution, and then adjust the pH of the polylysine solution to be the same as that of the zein / SDS solution.

[0009] (3) Add polylysine solution dropwise to zein / SDS solution to form a wet adhesive of water-insoluble multifunctional hemostatic adhesive;

[0010] (4) Freeze-dry the wet adhesive of the multifunctional hemostatic adhesive to obtain a powdered multifunctional hemostatic adhesive.

[0011] Furthermore, the zein concentration in the zein / SDS solution is 10-100 mg mL⁻¹, and the concentration of the SDS aqueous solution is 10-200 mM;

[0012] The ultrasonic frequency is 20000-40000Hz, and the ultrasonic time is 1-2min.

[0013] Furthermore, the pH of the zein / SDS solution described in step (1) is adjusted to 1-8.

[0014] Furthermore, the concentration of the polylysine solution in step (2) is 200-500 mM.

[0015] Furthermore, in step (3), the final molar ratio of polylysine to SDS in the polylysine solution and the zein / SDS solution is 1:1-5:1.

[0016] Furthermore, the freeze-drying temperature in step (4) is -80℃, and the freeze-drying time is 8-16h.

[0017] This invention also provides the application of the above-mentioned multifunctional hemostatic adhesive in the preparation of hemostatic drugs for treating arterial and venous bleeding in vivo.

[0018] The beneficial effects of this invention are as follows: the hemostatic adhesive prepared by this invention is applicable to hemostasis of venous and arterial bleeding in live animals; it can also achieve hemostasis in different physical states and can be used in combination. The hemostatic adhesive of this invention has both blood absorption and hemostasis functions, and its burst pressure can reach 99.5-122.3 mmHg. The product of this invention has good biocompatibility at the cellular level, with no significant impact on the activity of fibroblasts and blood cells, and degrades in approximately 21 days, matching the self-repair rate of arteries and veins. The hemostatic adhesive prepared by this invention effectively meets various clinical hemostasis needs that are difficult to perform. Attached Figure Description

[0019] Figure 1 The diagram shows three application forms of the multifunctional hemostatic adhesive of the present invention.

[0020] Figure 2 This is a cell compatibility diagram of the multifunctional hemostatic adhesive of the present invention;

[0021] Figure 3 This is a graph showing the hemolysis rate of the multifunctional hemostatic adhesive of the present invention after incubation with red blood cells for 3 hours.

[0022] Figure 4 The graph shows the degradation effect of the multifunctional hemostatic adhesive of the present invention in rats at different time points after implantation.

[0023] Figure 5 This is a schematic diagram of the in vitro burst pressure testing device for the multifunctional hemostatic adhesive and fibrin glue of the present invention.

[0024] Figure 6 The image shows the hemostatic effect of the fibrin glue and multifunctional hemostatic adhesive group of the present invention on a rabbit abdominal aortic bleeding model.

[0025] Figure 7 The image shows the effect of fibrin glue and multifunctional hemostatic adhesive of the present invention on vascular patency after hemostasis in a rabbit abdominal aortic bleeding model.

[0026] Figure 8 The image shows the hemostatic effect of the fibrin glue and multifunctional hemostatic adhesive of the present invention on a rat femoral artery bleeding model.

[0027] Figure 9 The image shows a section of the heart, liver, and kidney after three weeks of hemostasis in a rat femoral artery bleeding model using the multifunctional hemostatic adhesive of the present invention.

[0028] Figure 10 The image shows the hemostatic effect of fibrin glue and the multifunctional hemostatic adhesive of the present invention on a rat liver bleeding model.

[0029] Figure 11 The image shows the hemostatic effect of the multifunctional hemostatic agent of the present invention on a rabbit tibial hemorrhage model.

[0030] Figure 12 This is a diagram illustrating the blood-absorbing effect of the powdered multifunctional hemostatic adhesive of the present invention. Detailed Implementation

[0031] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] Example 1

[0033] To achieve the above objectives, the present invention adopts the following technical solution:

[0034] A method for preparing a multifunctional hemostatic adhesive includes the following steps:

[0035] (1) 75 mg mL -1 The pale yellow zein powder was dispersed in 150 mL of MSDS aqueous solution under ultrasonic treatment (30000 Hz, 1.5 min) to form a stable zein / SDS solution (pH 4.5). The pH of the zein / SDS solution was then adjusted to 7.4 with HCl and NaOH for later use.

[0036] (2) Dissolve polylysine in water to form a 200mM transparent polylysine solution, and then adjust the pH of the polylysine solution to be the same as that of the zein / SDS solution using HCl and NaOH.

[0037] (3) Add polylysine solution dropwise to zein / SDS solution and make the final molar ratio of polylysine to SDS 2:1 to form a water-insoluble multifunctional hemostatic adhesive wet adhesive.

[0038] (4) The wet adhesive of the multifunctional hemostatic adhesive was freeze-dried at -80℃ for 12h to obtain powdered multifunctional hemostatic adhesive.

[0039] Experimental Example 1: Biocompatibility Testing

[0040] (1) Cytotoxicity

[0041] To determine the cytotoxicity of the multifunctional hemostatic adhesive, the CCK-8 assay was used. The multifunctional hemostatic adhesive from Example 1 was immersed in DMEM medium at a concentration of 200 mg / mL according to national medical device standards and incubated at 37°C and 5% CO2 for 24 h to obtain the extract. The obtained extract was serially diluted to obtain 100%, 50%, 25%, 12.5%, and 6.25% dilutions for later use. After resuscitating mouse fibroblast 3T3 cells, they were stably passaged twice and then seeded in 96-well plates at a cell density of approximately 5000 cells / well. After culturing at 37°C and 5% CO2 for 24 h to confirm good cell growth, the medium was replaced with the extract dilution for further cell culture. Cell viability was observed at 24 h, 48 h, and 72 h. Results are as follows: Figure 2 As shown in the figure. The extract dilution prepared according to national medical device standards is non-cytotoxic, and live / dead staining shows no significant difference between the multifunctional hemostatic adhesive group and the blank group. The multifunctional hemostatic adhesive exhibits good biocompatibility at the cellular level.

[0042] (2) Hemolysis test

[0043] First, the multifunctional hemostatic adhesive from Example 1 was soaked in physiological saline at a concentration of 200 mg / mL according to national medical device standards and incubated at 37°C for 24 hours to obtain an extract. The obtained extract was serially diluted to obtain 100%, 50%, 25%, 12.5%, and 6.25% extract dilutions for later use. Fresh venous blood was collected from Japanese white rabbits using anticoagulant tubes, centrifuged at 1500 rpm for 15 minutes, and the supernatant was removed. After washing with 0.9% NaCl, the mixture was centrifuged repeatedly until the supernatant was clear. The collected red blood cells were then added to 0.9% NaCl to prepare a 2% red blood cell suspension. Seven groups were established: 100%, 50%, 25%, 12.5%, and 6.25% extract groups, a physiological saline group (negative control group), and a deionized water group (positive control group). Three replicates were set up for each group. 1 mL of extract / physiological saline / deionized water and erythrocyte suspension were added to each 1.5 mL centrifuge tube at a 1:1 volume ratio, totaling 1 mL. The centrifuge tubes were incubated at 37°C in a 5% CO2 incubator for 3 hours. After the incubation period, the tubes were removed and the hemolysis was observed. The appearance and absorbance test results are as follows: Figure 3 As shown, the extracts at all concentrations were similar in appearance to the negative control group, with roughly the same absorbance, all significantly lower than the positive control group. Microscopic observation revealed that the cell morphology of the multifunctional hemostatic adhesive group was similar to that of the saline group, and no ruptured red blood cells were observed as in the deionized water group, demonstrating the good blood compatibility of the multifunctional hemostatic adhesive. This ensures its applicability to the blood system.

[0044] Degradation performance of test example 2

[0045] Male SD rats, weighing approximately 200-250g, were used. After successful anesthesia, a subcutaneous space was created on each side of the rat's back, and 50mg of the multifunctional hemostatic adhesive from Example 1 was implanted into each side. The skin incisions were then sutured. Animals were sacrificed at 3, 6, 9, 12, 15, 18, and 21 days post-surgery to observe the degradation of the multifunctional hemostatic adhesive. The results are as follows: Figure 3 As shown in the figure. The tissue specimens were then subjected to HE staining to assess the inflammatory response of the materials, and the results are as follows. Figure 4 As shown, the multifunctional hemostatic adhesive showed no significant inflammatory response during degradation, and it completely degraded after approximately 21 days, matching the self-repair rate of arteries and veins.

[0046] Test Example 3: Burst Pressure Test of Multifunctional Hemostatic Adhesive

[0047] The burst pressure (e.g., pressure of the multifunctional hemostatic adhesive of Example 1) was measured based on ASTM F2392-04 (Standard Test Method for Burst Strength of Surgical Sealant). Figure 5 (As shown in the image). The tissue used was bovine aortic tissue. A 1.6mm diameter needle was used to puncture the aorta, creating a hole. Physiological saline was then injected into the pressure chamber at a rate of 0.8mL / min, slightly wetting the aortic surface (simulating blood leakage due to vascular rupture during surgery). Afterward, multifunctional hemostatic adhesive and fibrin glue were applied for sealing. The extracorporeal burst pressure of the multifunctional hemostatic adhesive was 122.3mmHg, while that of the fibrin glue was 34.79mmHg, showing a significant difference. Furthermore, the burst pressure of the multifunctional hemostatic adhesive was sufficient to meet the clinical requirements for arterial hemostasis during surgery.

[0048] Experiment 4: Hemostasis and patency testing of abdominal aortic bleeding in rabbits

[0049] The abdominal aorta is one of the largest arteries in the animal body, and its rapid blood flow and pulsation pose a significant challenge to the adhesion of hemostatic materials. Male Japanese White rabbits, weighing approximately 2.5 kg, were selected. After anesthesia with antacids, the abdominal skin was prepared and draped, and a longitudinal incision of approximately 5 cm was made in the midline of the abdomen. After the abdominal wall was opened, the abdominal aorta was bluntly dissected, and an 18G indwelling needle was used to puncture the abdominal aorta. The blood flow rate of the abdominal aorta was calculated over 1 minute by collecting distal blood loss. The indwelling needle was then closed, and a defect and bleeding in the abdominal aorta were created using a 50 ml syringe needle. After confirming bleeding, the arterial clamp was closed. The experimental group used 1 ml of powdered multifunctional hemostatic adhesive, while the control group used 1 ml of fibrin glue to seal the arterial defect for hemostasis. After opening the arterial clamp, gauze was used to absorb the bleeding to observe and compare the hemostatic effects of the two groups. The patency of the blood vessels was then observed after the indwelling needle was removed.

[0050] Depend on Figure 6 It can be seen that after using powdered multifunctional hemostatic adhesive to stop bleeding, it quickly absorbs blood and forms a gel, adhering firmly to the bleeding site to form a good seal, thus stopping the bleeding immediately. No rebleeding occurred at 2, 4, and 6 minutes, demonstrating the good hemostatic effect of the multifunctional hemostatic adhesive. With the addition of fibrin glue, due to the presence of a small amount of blood on the wound surface, the fibrin glue could not adhere firmly to the wound, resulting in some continued bleeding. Furthermore, the clotting factors within the fibrin glue require time to take effect, so there was still a small amount of blood loss at 10 and 20 seconds, although the bleeding rate gradually slowed down. At 40 seconds, the bleeding in the fibrin glue group completely stopped, and the clotting process was complete, with some blood clots visible in the surgical field. In summary, the multifunctional hemostatic adhesive has a better hemostatic effect than fibrin glue. This is because its good blood absorption properties lead to good adhesion, allowing the multifunctional hemostatic adhesive to remain stably attached to the bleeding site and achieve sealing and hemostasis even under significant mechanical disturbances. In contrast, fibrin glue's adhesion is poor under the interference of bleeding and arterial pulsation. Figure 7The amount of blood loss in the control group was consistent with that in the control group. The multifunctional hemostatic adhesive had a clear hemostatic effect, and the fibrin glue had a certain hemostatic effect. Its blood loss was significantly less than that of the control group but more than that of the multifunctional hemostatic adhesive group. Figure 7 The blood flow results confirmed that the abdominal aorta remained patent after treatment with the multifunctional hemostatic adhesive, with no vascular obstruction. This is largely due to the adhesive's ability to rapidly absorb blood and gel in situ, ensuring that the powder does not enter the blood vessels and form emboli. This ensures that the multifunctional hemostatic adhesive can achieve hemostasis without affecting blood supply.

[0051] Experimental Case 5: Femoral Artery Hemorrhage and Vascular Repair in Rats

[0052] The femoral artery is located deep within the body, surrounded by complex muscle structures, thus often requiring various hemostatic materials for auxiliary hemostasis. Male SD rats weighing approximately 250g were anesthetized with 20mg / kg sodium pentobarbital. After anesthesia took effect, the skin was prepared, disinfected, and draped. A surgical incision of approximately 3cm was made below the groin on the right lower limb, and the surrounding muscles and vascular sheath were bluntly dissected to fully expose the femoral artery. A bleeding model was created by puncturing the femoral artery using a 5ml syringe. The experimental group used 50mg of powdered fibrin glue, while the control group used 50mg of fibrin glue for hemostasis. The hemostatic effect was as follows: Figure 8 As shown. Blood samples were collected 3 days post-surgery for routine blood tests and blood biochemistry examinations. Heart, liver, and kidney tissues from rats were collected for corresponding toxicity assays.

[0053] A large amount of blood was observed to gush out after the puncture. Multifunctional hemostatic adhesive and fibrin glue were applied to the incision for hemostasis. After the femoral artery was repositioned, observation and timing were performed. At 0 seconds, the multifunctional hemostatic adhesive rapidly absorbed blood at the wound site, forming a gel and adhering well, thus acting as a physical barrier to seal the bleeding. The fibrin glue, however, had poor adhesion to moist surfaces and did not seal the bleeding site well after repositioning, resulting in continued bleeding. At 30 seconds, the multifunctional hemostatic adhesive group showed complete hemostasis, while the fibrin glue group still showed slight bleeding. At 60 seconds, both the multifunctional hemostatic adhesive and fibrin glue groups showed complete hemostasis. Due to the good initial sealing effect of the multifunctional hemostatic adhesive, there was no significant blood accumulation in the surgical field, making it clearer and providing better conditions for subsequent debridement and closure. The fibrin glue contained clotting factors, thus also achieving hemostasis and completely coagulating the blood in the surgical field. However, the presence of a large amount of blood clots was detrimental to subsequent debridement, suturing, and wound healing. All animals survived normally post-surgery, and no abnormalities were found in blood routine and blood biochemistry one week later, proving that the multifunctional hemostatic adhesive had no significant effect on the blood system. Three weeks later, after euthanasia, tissue sections of the heart, liver, and kidneys were examined, and no abnormalities were found. This demonstrates that both the multifunctional hemostatic adhesive and fibrin glue can be normally degraded in vivo, and their metabolites are non-toxic to internal organs. Example 6: Hemostasis after hepatic vein bleeding in rats.

[0054] As a highly vascularized organ, hemostasis of liver bleeding presents a significant challenge in clinical practice. Furthermore, the liver's fragility makes hemostasis difficult through pressure alone. Male SD rats weighing approximately 250g were anesthetized with 20mg / kg sodium pentobarbital. After anesthesia, the skin was prepared, disinfected, and draped. Heparin was applied at 100u / kg. A 1cm incision was made in the right hypochondrium to expose the middle lobe of the liver. A 5mm long and 5mm deep incision was made in the lower third of the liver. After confirming bleeding, the experimental group used 50mg of powdered multifunctional hemostatic adhesive, while the control group used 50mg of fibrin glue for hemostasis. Bleeding was collected using filter paper, and the hemostatic effects of the two groups were compared. Figure 10 The multifunctional hemostatic adhesive rapidly absorbs blood at the wound site, forming a sealant that immediately stops blood loss and generally achieves complete hemostasis. Simultaneously, the adhesive's absorption of blood promotes the concentration of red blood cells and platelets, further facilitating hemostasis. During absorption, the adhesive eliminates the liquid barrier between itself and the organ surface, resulting in rapid adhesion to wet surfaces and better bonding to the bleeding site for sealing and hemostasis. Therefore, the bleeding area on the filter paper and the bar graph show that the multifunctional hemostatic adhesive has a significantly better hemostatic effect compared to the control group. Fibrin, unable to absorb blood, cannot adhere well to the wound, thus its sealing effect is slightly inferior to the multifunctional hemostatic adhesive, and it also results in greater blood loss.

[0055] Experimental Example 7: Hemostasis after femoral bleeding in rabbits

[0056] Although the amount of bleeding from the bone is not large, the irregular shape and deep wounds of the bone surface defects and medullary cavity necessitate the combined use of liquid and gel-based multifunctional hemostatic adhesives. Male Japanese White rabbits, weighing approximately 2.5 kg, were selected. After anesthesia with Anatel, the skin of the right lower limb was prepared and draped. A longitudinal incision of approximately 5 cm was made on the front of the tibia to fully expose the bone. The periosteum was dissected, and a 3 mm diameter bone defect was created using Kirschner wires. Successful modeling was indicated by significant bleeding from the medullary cavity and bone surface. After 30 seconds of bleeding, liquid multifunctional hemostatic adhesive was injected into the medullary cavity defect, and gel-based multifunctional hemostatic adhesive was used to seal the bone surface. Bleeding cessation was clearly observed upon completion of the procedure.

[0057] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. The application of a multifunctional hemostatic adhesive in the preparation of hemostatic drugs for treating arterial and venous bleeding in vivo, characterized in that, The preparation method of the multifunctional hemostatic adhesive is as follows: (1) Disperse the pale yellow zein powder into an SDS aqueous solution under ultrasonication to form a stable zein / SDS solution. Then adjust the pH of the zein / SDS solution to 7.4 for later use. The zein concentration in the zein / SDS solution is 10-100 mg / mL. -1 The concentration of SDS aqueous solution is 10-200 mM; the ultrasonic frequency is 20000-40000 Hz; and the ultrasonic time is 1-2 min. (2) Dissolve polylysine in water to form a transparent polylysine solution, and then adjust the pH of the polylysine solution to be the same as that of the zein / SDS solution; the concentration of the polylysine solution is 200-500 mM; (3) Add polylysine solution dropwise to zein / SDS solution to form a water-insoluble multifunctional hemostatic adhesive wet adhesive; the final molar ratio of polylysine to SDS in polylysine solution and zein / SDS solution is 1:1-5:1; (4) The wet adhesive of the multifunctional hemostatic adhesive is freeze-dried at a temperature of -80℃ for 8-16 hours to obtain a powdered multifunctional hemostatic adhesive.

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