High-liquid-absorption, tissue-adhesion and antibacterial composite powder and application thereof

Abstract

The invention relates to high-liquid-absorption, tissue-adhesion and antibacterial composite powder and application thereof. The composite powder comprises a first component, a second component and a third component, the first component is a porous microsphere, the second component is selected from a polyethylene glycol derivative or a natural polymer derivative, and the third component is polylysine. The composite powder can rapidly absorb a large amount of liquid to promote coagulation and can be gelatinized to form high-adhesion hydrogel, so that effective hemostasis and sealing of a massive hemorrhage wound surface are realized. And the inherent excellent antibacterial property is realized through the positive ion and positive charge characteristics of the material. The composite powder solves the problems that existing styptic powder is low in liquid absorption rate, low in tissue adhesive force, incapable of effectively blocking massive hemorrhage, poor in antibacterial performance and the like. The composite powder is excellent in biocompatibility and definite in component, facilitates clinical transformation, has wide application prospects, can be used for emergency hemostasis nursing and can be used as an antibacterial product and a wound dressing.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a composite powder with high liquid absorption, tissue adhesion, and antibacterial properties and its applications. Background Art

[0002] Uncontrolled acute massive hemorrhage caused by severe trauma is one of the main causes of death in military and civilian settings. Although the human body has a complex blood coagulation cascade mechanism, it cannot effectively function in the face of extreme bleeding situations. Using materials for on-site intervention can save lives and prevent complications. Powders / granules, as common materials, have the advantages of simple operation and the ability to cover large wound areas. However, currently commercially available hemostatic powders are mainly based on inorganic mineral materials such as kaolin and montmorillonite, which promote blood coagulation by adsorbing coagulation factors in blood. However, inorganic minerals generally have poor biocompatibility, and residues in the body can cause tissue rejection, inflammation, and risks of embolism and systemic thrombosis. Polysaccharide-based powders such as starch (e.g., Arista™) and chitosan (e.g., Celox®) have relatively good safety, but such powders have weak abilities to promptly seal and adhere to wound surfaces and are easily washed away under high-pressure blood flow conditions, and are not suitable for sealing massive hemorrhage. Therefore, there is a need to develop high-performance powders with both biosecurity and strong sealing capabilities.

[0003] Self-adhesive powders can transform into adhesive hydrogels after contacting blood, thereby combining the strong liquid absorption ability of the powder and the adhesion and sealing advantages of the hydrogel to enhance performance. Currently developed self-adhesive powders include polyethyleneimine / polyacrylic acid / quaternized chitosan, giant salamander skin secretions, and freeze-dried okra mucus, etc., which have certain effects. However, the liquid absorption abilities of these self-gelling powders are limited, making it impossible for them to quickly absorb blood when dealing with massive bleeding and achieve effective adhesion to wet tissue interfaces. In addition, such powders also have problems such as being difficult to degrade and unclear compositions, which may cause safety hazards. In addition, in a complex open wound environment, infections by microorganisms such as bacteria and viruses will seriously affect wound healing and patient recovery. The above-mentioned commercial powders generally do not have antibacterial properties. Local use of antibiotics easily leads to bacterial drug resistance, making subsequent treatment more difficult; silver ions added to materials have blood and cell toxicity and have biocompatibility problems.

[0004] In summary, it is necessary to rationally design self-gelling powders with high liquid absorption, strong tissue adhesion, and inherent antibacterial abilities to effectively seal massive wound hemorrhage. Summary of the Invention

[0005] The object of the present invention is to disclose a composite powder with high liquid absorption, tissue adhesion, and antibacterial properties and its applications, so as to solve one or more technical problems existing in the prior art and provide at least one beneficial choice or create conditions.

[0006] A first aspect of the present invention is to provide a composite powder with self-gelling properties.

[0007] A second aspect of the present invention is to provide an application direction of the composite powder described in the first aspect of the present invention.

[0008] The composite powder described in the first aspect of the present invention includes a first component, a second component, and a third component. The first component is a porous microsphere, the second component is selected from polyethylene glycol derivatives or natural polymer derivatives, and the third component is polylysine. The porous microsphere can rapidly absorb a large amount of blood to enrich blood cells and concentrate coagulation factors, while removing the liquid at the wound tissue. The polyethylene glycol or natural polymer derivative can in-situ chemically crosslink with polylysine to form a hydrogel in the hydrated state. Polylysine is rich in cationic amino groups and has the advantages of antibacterial, immune regulation, and reducing inflammation at the wound site. The above three components work synergistically to endow the composite powder with effective adhesion and plugging to the wound, achieving hemostasis, antibacterial and anti-infection, and promoting tissue healing. Through experiments, it is proved that after the composite powder obtained by mixing the three components contacts with blood (or other liquids), it can rapidly absorb a large amount of liquid and spontaneously form a tissue-adhesive hydrogel, thereby effectively exerting the hemostatic plugging function. At the same time, the composite powder has good cell compatibility and antibacterial ability against Gram-negative bacteria and Gram-positive bacteria, avoiding infection and reducing wound inflammation, thus promoting healing.

[0009] In a further application embodiment, the porous microsphere is selected from chitosan porous microspheres, sodium alginate porous microspheres, and / or gelatin porous microspheres.

[0010] In a further application embodiment, the polyethylene glycol derivative includes one or more compounds shown in Formulas I, II, III, and IV.

[0011]

[0012] Among them, n is 20 - 120, and R1 is succinimidyl butanedioate, succinimidyl glutarate, succinimidyl carbonate, or aldehyde group. The second component containing the active ester or aldehyde group can chemically bond with the amino group on the tissue surface, thereby forming strong tissue adhesion.

[0013] In a further application embodiment, the molecular weight of the polyethylene glycol derivative is 1.0 - 50.0 kDa; preferably, the molecular weight of the polyethylene glycol derivative is 5.0 - 20.0 kDa.

[0014] In a further application embodiment, the natural polymer derivative is selected from oxidized hyaluronic acid, oxidized cellulose, oxidized dextran, and / or oxidized sodium alginate.

[0015] In a further application embodiment, the molecular weight of the natural polymer derivative is 2.0 to 500.0 kDa; preferably, the molecular weight of the natural polymer derivative is 10.0 to 200.0 kDa.

[0016] In a further application embodiment, the polylysine comprises one or more compounds represented by Formulas V, VI, VII, and VIII,

[0017] wherein n is 20 to 1000.

[0018] In a further application embodiment, the mass ratio of the first component, the second component, and the third component is (0.1 to 100.0):(0.1 to 100):(0.1 to 100.0).

[0019] In a further application embodiment, the first component, the second component, and the third component are all dry powders with a particle size range of 0.1 to 1000.0 µm.

[0020] The second aspect of the present invention is to apply the composite powder to the preparation of hemostatic drugs, biological scaffolds, or carrier materials. For example, to prepare hemostatic products, wound dressings, antibacterial products, or bioadhesives. The hemostatic products can be used for any form of bleeding, including but not limited to bleeding from surface wounds, internal organ wounds, and bleeding points at any location accompanied by bacterial infections. The wound dressings can deal with, including but not limited to, acute traumatic wounds, chronic wounds, and infected wounds. The antibacterial products refer to inhibiting or killing pathogens such as bacteria, and the bacteria include but are not limited to various pathogenic bacteria, and the bacteria can be Escherichia coli or Staphylococcus aureus. The bioadhesives can deal with, including but not limited to, various wound closures, precise anastomosis between biological tissue sections, and other adhesions between biological tissues.

[0021] Taking the above-provided hemostatic product as an example, the method for its application in hemostasis is: applying the hemostatic product containing the composite powder to the bleeding point of the test subject for hemostasis, and the test subject includes but is not limited to humans or non-human animals.

[0022] The beneficial effects of the present invention are: The present invention provides a high-absorbing, tissue-adhesive, antibacterial self-gelling composite powder suitable for emergency bleeding control. When the composite powder acts on a bleeding wound surface by mixing porous microspheres, polyethylene glycol or natural polymer derivatives, and polylysine components, it can rapidly absorb a large amount of liquid to promote blood coagulation while gelling itself to form a strongly adhesive hydrogel, achieving effective hemostatic plugging of a large bleeding wound surface. And through the cationic positive charge characteristics of the material itself, inherent excellent antibacterial properties are realized. The composite powder solves the problems of low liquid absorption rate, low tissue adhesion force, inability to effectively plug large bleeding, and poor antibacterial performance of existing hemostatic powders. The composite powder has excellent biocompatibility, clear components, is conducive to clinical transformation, has broad application prospects, and can be used for emergency hemostasis care and as an antibacterial product and wound dressing. Description of the Drawings

[0023] Figure 1 Scanning electron microscope image of the CPE-derived hydrogel prepared in Example 1; Figure 2 Scanning electron microscope image of the CPD-derived hydrogel prepared in Example 2; Figure 3 Scanning electron microscope image of the CPH-derived hydrogel prepared in Example 3; Figure 4 Scanning electron microscope image of the GPL-derived hydrogel prepared in Example 4; Figure 5 Scanning electron microscope image of the GDH-derived hydrogel prepared in Example 5; Figure 6 Scanning electron microscope image of the GPE-derived hydrogel prepared in Example 6; Figure 7 Scanning electron microscope image of the APD-derived hydrogel prepared in Example 7; Figure 8 Scanning electron microscope image of the AHH-derived hydrogel prepared in Example 8; Figure 9 Scanning electron microscope image of the APE-derived hydrogel prepared in Example 9; Figure 10 Comparison chart of the liquid absorption rates of CPE, GPL, AHH, commercial hemostatic powder products, and PEG / EPL powder for water, PBS, and blood; Figure 11 Schematic diagram and b) quantitative burst pressure of the a) burst pressure test of CPE, GPL, AHH-derived hydrogels and commercial fibrin glue; Figure 12 Coagulation index of CPE and PEG / EPL powder after incubation with 2, 5, and 10 times the mass of blood for 30 seconds Figure 13 In vivo biocompatibility test of CPE; a) Schematic diagram of in vivo subcutaneous implantation test in rats; b) H&E and Masson staining images of CPE and surrounding tissues in subcutaneous implantation test (the areas within the asterisk, triangle, and dotted line represent hydrogel, normal tissue cells, and inflammatory cells, respectively); c) Complete blood cell count and d) blood biochemical analysis results of SD rats subcutaneously implanted with CPE; e) H&E section images of the heart, liver, spleen, lung, and kidney of rats implanted with CPE and healthy rats 28 days after implantation. Figure 14 Evaluation of the hemostatic effect of APD in a rat bleeding model; a) Representative bleeding and hemostasis processes of different experimental groups and b) cumulative blood loss and c) hemostasis time in a partial liver resection model of rats. Figure 15 H&E section of the liver wound site after hemostasis of CPE in a rat bleeding model. Figure 16 Evaluation of the hemostatic effect of CPE in a rat femoral artery transection resection model; a) Representative bleeding and hemostasis processes of different experimental groups and b) cumulative blood loss within three minutes. Figure 17 Hemostatic process of GDH in a spleen linear bleeding in a pig model. Figure 18 Evaluation of the hemostatic effect of CPE in a pig bleeding model; a) Hemostatic process of a liver defect model; b) Hemostatic process of a pig femoral artery bleeding model. Figure 19 Promoting wound healing effect of CPE on rat skin trauma; a) Photos of full-thickness skin defect models of rats treated with different methods; b) Healing superimposed images of corresponding wounds of skin defects; c) H&E staining and Masson staining of skin defects on the 7th and 11th days; d) Quantitative analysis of wound healing rate; e) CD31 staining of skin wounds on the 7th and 11th days; f) Calculation of the relative area of CD31 in immunohistochemistry; g) TNF-α staining of skin wounds on the 7th and 11th days; h) Calculation of the relative area of TNF-α in immunohistochemistry. Detailed implementation manners

[0024] The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Modifications and substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the present invention all fall within the scope of the present invention.

[0025] Unless otherwise specified, the technical means used in the examples are conventional means well-known to those skilled in the art.

[0026] Preparation and testing methods Preparation method of composite powder: First, dissolve the third component in pure water or PBS, then add sodium hydroxide to adjust the pH of polylysine to 7.00 - 9.00. After drying or freeze-drying, thoroughly grind the material into powder particles. Add the first component, the second component, and the third component to the stirring device in proportion, and use vortex to mix at 2000 rpm for 3 minutes to prepare the composite powder.

[0027] Gelling ability test: Add 200 μL of water dyed with red food dye to the powder sample. After a predetermined time interval (5, 10, 15, 30, and 60 seconds), add 2 mL of deionized water again to determine the gelling ability of the sample. Samples in which the particulate powder does not disperse in water but forms a stable gel indicate in-situ gelling ability, and the gel time is defined as the time when the stable hydrogel is formed.

[0028] Shear stripping test: First, cut the pigskin into a 35×10 mm rectangle and moisten the surface. Sprinkle approximately 10 mg of composite powder evenly on the surface of one piece of pigskin, then quickly attach another piece of pigskin of the same size on its surface, with a contact area of 10×15 mm. Press under a 200 g weight for 20 seconds and let it stand for 5 minutes. Use an Instron universal tensile machine to perform a tensile test on the pigskin specimen lap-bonded with the hemostatic powder at a speed of 50 mm / minute to obtain the adhesion strength. Each group of tests is repeated five times to ensure the accuracy of the results.

[0029] Liquid absorption rate test: Weigh different composite hemostatic powders ( W 0) and place them on a 120-mesh nylon screen, then immerse the screen in distilled water, whole blood, or PBS for 30 seconds and then take it out. After blotting the excess liquid with absorbent paper, weigh ( W t ). The formula for calculating the water absorption rate of the material is: Liquid absorption (%) = W t / W 0× 100% (g / g).

[0030] Burst pressure test: Fill the aorta of an ex vivo pig heart with PBS and connect it to an injection pump and a pressure monitor. Make a circular hole with a diameter of 2 mm on the surface, and add 80 mg of composite powder to the circular hole to form a hydrogel layer. After 5 minutes, gradually inject PBS into the device and apply a force to the sealed incision at a speed of 10 mL / minute. The critical pressure at which the seal fails is recorded as the burst pressure. All experiments are carried out at room temperature (25°C) and 40% humidity and repeated three times.

[0031] In vitro coagulation test: Take 360 µL of freshly collected rat whole blood treated with sodium citrate, and add 40 µL of calcium chloride with a concentration of 0.1 mol / L to restore its coagulation ability. Take 10 mg of the composite powder and add it to a 5 mL EP tube. Drop 50 µL of rat blood into the tube and start timing. Immediately after reaching the preset time, add 2 mL of deionized water to the tube and use a 1 mL pipette to blow and disperse the uncoagulated blood. Take 100 µL of the liquid and add it to a 96-well plate, and read the absorbance at 540 nm under an enzyme-linked immunosorbent assay (ELISA) reader to calculate the blood coagulation index (BCI).

[0032] Blood compatibility test: Add the composite powder sample to 500 µL of RBC suspension (5%, v / v in PBS), with final concentrations of 10 mg / mL and 30 mg / mL. After incubating at 37 °C for 1 hour, centrifuge the sample at 2000 rpm for 10 minutes. 0.1% Triton X-100 and PBS are used as positive and negative controls, respectively. Read the absorbance of the resulting supernatant at 540 nm using an ELISA reader and calculate the hemolysis rate.

[0033] Erythrocyte adhesion test: Freshly anticoagulated rat whole blood is centrifuged at 2000 rpm for 10 minutes to obtain an erythrocyte suspension (RBC), which is diluted with PBS (10% v / v ).) and platelet-rich plasma (PRP). Drop 100 µL of the RBCs suspension onto 10 mg of the sample and incubate at 37 °C for 30 minutes. Wash the sample with PBS to remove the unadhered RBCs, then add deionized water (4 mL) and place it at 37 °C for 1 hour to lyse the adhered erythrocytes. Measure the absorbance value of the supernatant at 540 nm. Suspend 100 µL of RBCs in 4 mL of deionized water as a reference value to calculate the erythrocyte adhesion rate.

[0034] Antibacterial performance test: Take 10 mg of the composite powder sample. Drop 100 µL of the suspension with a concentration of 10 7 CFU / mL of E.coli and S.aureus onto the sample, then add 900 µL of PBS and mix well, and incubate at 37 °C for 12 hours. Take out 10 µL of the solution and spread it on a plate and incubate for another 12 hours. Similarly, drop 100 µL of the suspension with a concentration of 10 7 CFU / mL of E.coli and S.aureus onto the sample, then add 900 µL of PBS and mix well and incubate for 12 hours. Then add 900 µL of LB broth and incubate for another 12 hours. Finally, take 200 µL of the suspension into a 96-well plate and read the absorbance at 600 nm using an ELISA reader.

[0035] SEM test: The hydrogel prepared from the obtained composite powder was freeze-dried at -50 °C for 48 hours. The freeze-dried sample was adhered to the conductive adhesive. A layer of Pt was sprayed on the sample surface before testing (for 90 seconds). The hydrogel derived from the hemostatic powder was observed using a scanning electron microscope. During the test, the acceleration voltage was set to 10 kV.

[0036] Example 1 In a dry environment, chitosan microspheres (CM), four-armed polyethylene glycol succinimidyl succinate (4-PEG-NHS), and ε-polylysine (EPL) were mixed at a mass ratio of 1:2:1 using a vortex at 2000 rpm for 3 minutes to obtain the CPE hemostatic powder of the present invention.

[0037] Gelation time: 30 s; Adhesion strength: 56 kPa; Liquid absorption rate: 12 times; Bursting pressure: 192 mmHg; BCI (30 s): 1.4%; Hemolysis rate: 2.3%; Red blood cell adhesion rate: 79%; Bacteriostatic efficiency: 99%; Figure 1 It is the SEM morphology diagram of the hydrogel derived from the CPE hemostatic powder.

[0038] Example 2 In a dry environment, chitosan microspheres (CM), octa-armed polyethylene glycol succinimidyl aldehyde (8-PEG-CHO), and dendritic polylysine (DPL) were mixed at a mass ratio of 2:2:1 using a vortex at 2000 rpm for 3 minutes to obtain the CPD hemostatic powder of the present invention.

[0039] Gelation time: 170 s; Adhesion strength: 21 kPa; Liquid absorption rate: 16 times; Bursting pressure: 85 mmHg; BCI (30 s): 3.1%; Hemolysis rate: 2.1%; Red blood cell adhesion rate: 59%; Bacteriostatic efficiency: 98%; Figure 2 It is the SEM morphology diagram of the hydrogel derived from the CPD hemostatic powder.

[0040] Example 3 In a dry environment, chitosan microspheres (CM), six-armed polyethylene glycol succinimidyl carbonate (6-PEG-NHS), and hyperbranched polylysine (HPL) were mixed at a mass ratio of 4:2:1 using a vortex at 2000 rpm for 3 minutes to obtain the CPH hemostatic powder of the present invention.

[0041] Gelation time: 60 s; Adhesion strength: 17 kPa; Liquid absorption rate: 19 times; Bursting pressure: 57 mmHg; BCI (30 s): 5.6%; Hemolysis rate: 3.4%; Red blood cell adhesion rate: 51%; Bacteriostatic efficiency: 88%; Figure 3 It is the SEM morphology diagram of the hydrogel derived from the CPH hemostatic powder.

[0042] Example 4 Under a dry environment, gelatin microspheres (GM), two-arm polyethylene glycol succinimidyl glutarate (2-PEG-NHS), and poly(L-lysine) (PLL) were mixed at a mass ratio of 1:2:1 using a vortex at a rotation speed of 2000 rpm for 3 minutes to obtain the GPL hemostatic powder of the present invention.

[0043] Gelation time: 30 s; Adhesion strength: 38 kPa; Liquid absorption rate: 10 times; Bursting pressure: 155 mmHg; BCI (30 s): 1.5%; Hemolysis rate: 2.0%; Red blood cell adhesion rate: 76%; Bacteriostatic efficiency: 94%; Figure 4 SEM morphology diagram of the hydrogel derived from the GPL hemostatic powder.

[0044] Example 5 Under a dry environment, gelatin microspheres (GM), oxidized dextran (O-DEX), and hyperbranched polylysine (HPL) were mixed at a mass ratio of 2:2:1 using a vortex at a rotation speed of 2000 rpm for 3 minutes to obtain the GDH hemostatic powder of the present invention.

[0045] Gelation time: 60 s; Adhesion strength: 29 kPa; Liquid absorption rate: 14 times; Bursting pressure: 137 mmHg; BCI (30 s): 4.5%; Hemolysis rate: 3.9%; Red blood cell adhesion rate: 78%; Bacteriostatic efficiency: 93%; Figure 5 SEM morphology diagram of the hydrogel derived from the GDH hemostatic powder.

[0046] Example 6 Under a dry environment, gelatin microspheres (GM), six-arm polyethylene glycol succinimidyl succinate (6-PEG-NHS), and ε-polylysine (EPL) were mixed at a mass ratio of 1:2:1 using a vortex at a rotation speed of 2000 rpm for 3 minutes to obtain the GPE hemostatic powder of the present invention.

[0047] Gelation time: 30 s; Adhesion strength: 48 kPa; Liquid absorption rate: 10 times; Bursting pressure: 177 mmHg; BCI (30 s): 1.2%; Hemolysis rate: 1.7%; Red blood cell adhesion rate: 78%; Bacteriostatic efficiency: 97%; Figure 6 SEM morphology diagram of the hydrogel derived from the GPE hemostatic powder.

[0048] Example 7 Under a dry environment, sodium alginate microspheres (AM), four-arm polyethylene glycol succinimidyl carbonate (4-PEG-NHS), and dendritic polylysine (DPL) were mixed at a mass ratio of 2:2:1 using a vortex at a rotation speed of 2000 rpm for 3 minutes to obtain the APD hemostatic powder of the present invention.

[0049] Gelation time: 45 s; Adhesion strength: 24 kPa; Liquid absorption rate: 16 times; Bursting pressure: 103 mmHg; BCI (30 s): 2.8%; Hemolysis rate: 2.6%; Red blood cell adhesion rate: 67%; Bacteriostatic efficiency: 88%; Figure 7 SEM morphology diagram of the hydrogel derived from APD hemostatic powder.

[0050] Example 8 Under a dry environment, sodium alginate microspheres (AM), oxidized hyaluronic acid (OHA) and hyperbranched polylysine (HPL) were mixed at a mass ratio of 2:2:1 using a vortex at a rotational speed of 2000 rpm for 3 minutes to obtain the AHH hemostatic powder of the present invention.

[0051] Gelation time: 140 s; Adhesion strength: 20 kPa; Liquid absorption rate: 14 times; Bursting pressure: 91 mmHg; BCI (30 s): 2.8%; Hemolysis rate: 3.0%; Red blood cell adhesion rate: 63%; Bacteriostatic efficiency: 73%; Figure 8 SEM morphology diagram of the hydrogel derived from AHH hemostatic powder.

[0052] Example 9 Under a dry environment, sodium alginate microspheres (AM), octa-arm polyethylene glycol succinimidyl glutarate (8-PEG-NHS) and ε-polylysine (EPL) were mixed at a mass ratio of 2:2:1 using a vortex at a rotational speed of 2000 rpm for 3 minutes to obtain the APE hemostatic powder of the present invention.

[0053] Gelation time: 45 s; Adhesion strength: 25 kPa; Liquid absorption rate: 15 times; Bursting pressure: 102 mmHg; BCI (30 s): 1.1%; Hemolysis rate: 1.3%; Red blood cell adhesion rate: 65%; Bacteriostatic efficiency: 82%; Figure 9 SEM morphology diagram of the hydrogel derived from APE hemostatic powder.

[0054] Example 10 Taking the CPE, GPL, and AHH composite powders obtained in Examples 1, 4, and 8 as examples, their liquid absorption performance was tested and compared with commercial hemostatic powders Yunnan Baiyao YB (purchased from Yunnan Baiyao Group Co., Ltd., China), Celox (purchased from Medtrade Products Limited, UK), and a powder obtained by mixing tetra-arm polyethylene glycol active ester and polylysine in a mass ratio of 2:1 (denoted as PEG / EPL). YB has only an absorption rate of about 2.5 times, Celox has an absorption rate of about 4.8 times, and the absorption rate of PEG / EPL is also only about 3 times. However, the absorption rates of the CPE, GPL, and AHH hemostatic powders prepared by the present invention can reach 12, 10, and 14 times their own weights respectively, which are much higher than those of commercial products and PEG / EPL ( Figure 10 ). It can be seen that the addition of porous microspheres significantly improves the liquid absorption rate of the composite powder.

[0055] Example 11 A self-made device was used to test the bursting pressure to evaluate the adhesion and plugging abilities of the CPE, GPL, and AHH powders and commercial fibrin glue (purchased from Guangzhou Beixiu Biotechnology Co., Ltd., China). The method flow is as shown in Figure 11 at a.

[0056] The results are as shown in Figure 11 at b. The commercial fibrin glue has only a bursting pressure of about 53 mmHg, while the measured rupture pressures of the adhesive hydrogels induced by CPE, GPL, and AHH after hydration are 192 mmHg, 155 mmHg, and 91 mmHg respectively. Among the tested powders, the bursting pressures of CPE and GPL are significantly higher than the normal human blood pressure (120 mmHg).

[0057] Example 12 Equal masses of CPE prepared in Example 1 and PEG / EPL powder in Example 10 were taken and incubated with fresh whole blood at 2, 5, and 10 times their masses for 30 seconds respectively, and the blood coagulation index BCI was measured. The results are as shown in Figure 12 It can be seen that due to its excellent rapid liquid absorption ability, the blood coagulation index BCI of CPE is significantly higher than that of PEG / EPL, showing significant advantages in dealing with massive bleeding.

[0058] Example 13 SD rats were implanted subcutaneously on the back to evaluate the in vivo biocompatibility of the hemostatic powder, and samples were taken at different time points (7, 14, 21, and 28 days) for histopathological and blood analyses ( Figure 13a). Taking the CPE hemostatic powder as an example, H&E and Masson staining showed that obvious acute inflammatory reactions were induced 7 days after the implantation of the hemostatic powder. During the subsequent experiment period, the inflammation was observed to significantly weaken. Only very slight inflammatory reactions could be detected on the 21st day, and the inflammatory cell layer basically disappeared on the 28th day ( Figure 13 b). In addition, the blood analysis results indicated that there were no significant differences in blood biochemical and blood cell parameters between the experimental group and healthy rats during the implantation of the hemostatic powder, suggesting that the in vivo biodegradation of the powder had no systemic toxicity to animals ( Figure 13 c, 13d). In addition, after 28 days of implantation, the hearts, livers, spleens, lungs, and kidneys of the implanted rats and healthy rats were taken for sectioning and H&E staining analysis. The results showed that there were no significant differences between the internal organs of the implanted rats and healthy rats, and no signs of any potential organ toxicity were found ( Figure 13 e). These results proved that the CPE powder had excellent biocompatibility.

[0059] Example 14 The performance of the hemostatic powder was tested in detail using a liver incision defect model of SD rats.

[0060] Construction of the rat liver lobectomy wound model: Rats aged 14 - 15 weeks were selected and anesthetized with isoflurane. The rat liver was exposed through a transverse abdominal incision, and a pre-weighed filter paper was placed under the viscera. A complete transection incision about 20 mm long was made in the lower part of the liver lobe. After free bleeding for about 5 seconds, 100 mg of the composite powder was immediately applied to the bleeding wound, while no treatment was performed after bleeding as the control group. The blood loss was calculated by weighing the weight of the filter paper after absorbing blood, and the bleeding time was recorded simultaneously. After the experiment, all animals were euthanized in accordance with animal welfare and animal ethics standards.

[0061] Taking the APD obtained in Example 7 as an example for comparison with commercial products. The experimental results of the Celox group showed that during a bleeding time of about 120 seconds, the average blood loss was about 570 mg. In contrast, the blood loss of the APD group was only about 251 mg, and hemostasis was successfully achieved within about 75 seconds ( Figure 14 a, b, c).

[0062] Example 15 The CPE obtained in Example 1 was used for hemostasis in a rat liver incision defect model. After hemostasis was completed, the CPE-derived hydrogel adhered to the liver was fixed together with the liver using paraformaldehyde fixative and then sectioned and stained with H&E. The section results showed that the CPE powder formed a tight adhesion with the liver tissue, and a large number of red blood cells were enriched on the surface of the CM in the CPE powder, proving that CM played a function of rapidly absorbing blood and enriching red blood cells at the initial stage of the application of the CPE hemostatic powder ( Figure 15 )

[0063] Example 16 A rat femoral artery bleeding model was used to further test the hemostatic ability of CPE powder.

[0064] Method for the hemostasis test of rat femoral artery: After anesthetizing the rat with isoflurane, the femoral artery of the rat was transversely incised with surgical scissors to induce bleeding. After free bleeding for about 3 seconds, a gauze or 250 mg of the composite powder was applied to the wound, and the blood loss in the first 3 minutes was recorded.

[0065] For the control group, after applying the gauze to the wound, it failed to stop bleeding effectively. For Celox, although hemostasis was achieved in 3 minutes, relatively severe leakage was observed during hemostasis. While the CPE adhesive powder showed excellent hemostatic effect: CPE quickly absorbed the blood and formed a firm sealing layer, successfully stopping the bleeding within 3 minutes, with the least blood loss among the three groups, and no secondary bleeding was found during the 30-minute observation period ( Figure 16 a, b).

[0066] Example 17 The preclinical functional evaluation of the GDH powder obtained in Example 5 was carried out using a porcine spleen linear wound bleeding model. A linear wound with a length of 20 mm and a depth of 10 mm was created on the porcine spleen. After applying the GDH hemostatic powder (about 2 g) to the wound and gently pressing, the initial massive bleeding could be controlled within 3 min. The resulting hydrogel and blood clot complex remained stable and no secondary bleeding occurred after flushing the spleen with water ( Figure 17 )

[0067] Example 18 The preclinical functional evaluation of the CPE obtained in Example 1 was carried out using a porcine liver circular defect and porcine femoral artery bleeding.

[0068] Construction of the Bama pig bleeding model: A mixture of xylazine hydrochloride and midazolam was used to anesthetize the Bama pig (female, about 30 kg) by intramuscular injection, and then inhalation of sevoflurane was used to maintain anesthesia. The pig was placed in the supine position and the abdominal cavity was opened. Subsequently, a linear wound with a length of 20 mm and a depth of 10 mm was created on the spleen, and a circular defect wound with a length of 20 mm and a depth of 5 mm was created on the liver. The composite powder was immediately applied to the bleeding wound. The hemostatic effect was examined and photos of the wound site were taken. For femoral artery hemostasis, the femoral artery of the pig was transversely incised with a scalpel to induce bleeding, and then the composite hemostatic powder was immediately applied to the bleeding site, and hemostasis was achieved by gently pressing with the hand as well.

[0069] After applying CPE (about 2 g) to the wound and gently pressing, the initial massive bleeding could be controlled within 3 min ( Figure 18a). For severe bleeding caused by femoral artery injury, it was effectively controlled after applying CPE hemostatic powder (about 3 g) and supplemented with slight pressure, and complete hemostasis was achieved at the 3-minute time point as well ( Figure 18 b). The research results confirmed that the powder provided by this invention can provide strong adhesion and effective hemostatic sealing for fatal and inoppressible injuries of porcine visceral organs, showing great potential for practical applications.

[0070] Example 19 A favorable wound-healing effect is also a key factor for an ideal hemostatic material.

[0071] Wound healing model construction: Rats were anesthetized using a small animal anesthesia machine with inhaled isoflurane. A full-thickness circular skin defect (diameter: 15 mm) was created on the back of the rats using a puncher. Then, the wounds were treated with PBS, Celox, and the composite hemostatic powder, and the wounds were photographed to record the healing process on the 0th, 3rd, 5th, 7th, 9th, and 11th days after surgery and calculate the healing rate.

[0072] During the 11 days of the experimental process, the wounds in the three groups treated with PBS, Celox, and CPE-derived hydrogel healed gradually, and the area gradually decreased. Gross observation showed that compared with the control group and the control group, the wounds in the CPE group healed better ( Figure 19 a, b). H&E and Masson staining of the repaired tissue on the 7th and 11th days showed that compared with the PBS group and the Celox group, the width of the dermal space in the CPE gel group was significantly reduced, and the degree of wound healing was higher ( Figure 19 c). Quantitative analysis of the wound area showed that at all time points, the average value of the wound closure area in the CPE-derived hydrogel group was higher than that in the PBS and Celox groups, and there were significant differences from the PBS group at 3, 5, 7, and 11 days; there were significant differences from the Celox group at 3 and 11 days. It is worth noting that the wound closure area in the CPE-derived hydrogel group reached 93% after 11 days, higher than 85% and 83% in the PBS and Celox groups ( Figure 19 d).

[0073] Further immunohistochemical staining analysis showed that the inflammatory response induced by the CPE-derived hydrogel was significantly lower and the microvessel density was higher: compared with PBS, the expression of TNF-α in the CPE gel group was significantly less and the CD31 expression was higher on the 7th and 11th days; compared with Celox, the CPE-derived hydrogel group had significantly less TNF-α expression and higher CD31 expression on the 11th day, indicating that the CPE-derived hydrogel can inhibit the inflammatory response and promote new blood vessel formation. Figure 19e, f, g, h). The results showed that the CPE powder-derived hydrogel had better effects than PBS and Celox in repairing full-thickness excisional wounds, which was mainly attributed to the combined effects of its inherent antibacterial activity, inflammation inhibition, angiogenesis enhancement, collagen deposition increase, and creation of a moist wound healing environment.

[0074] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above-described exemplary embodiments, and that the present invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the present invention. Therefore, in any regard, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be embraced within the present invention.

Claims

1. A composite powder, characterized in that, It includes a first component, a second component and a third component. The first component is a porous microsphere, the second component is selected from polyethylene glycol derivatives or natural polymer derivatives, and the third component is polylysine.

2. The composite powder according to claim 1, wherein The porous microsphere is selected from chitosan porous microspheres, sodium alginate porous microspheres and / or gelatin porous microspheres.

3. The composite powder according to claim 1, wherein The polyethylene glycol derivatives include one or more compounds shown in Formulas Ⅰ, Ⅱ, Ⅲ and Ⅳ, wherein, n is 20 - 120, and R1 is succinimidyl succinate, succinimidyl glutarate, succinimidyl carbonate or aldehyde group.

4. The composite powder according to claim 3, characterized in that, The molecular weight of the polyethylene glycol derivative is 1.0 - 50.0 kDa; preferably, the molecular weight of the polyethylene glycol derivative is 5.0 - 20.0 kDa.

5. The composite powder according to claim 1, wherein The natural polymer derivative is selected from oxidized hyaluronic acid, oxidized cellulose, oxidized dextran and / or oxidized sodium alginate.

6. The composite powder according to claim 5, characterized in that, The molecular weight of the natural polymer derivative is 2.0 - 500.0 kDa; preferably, the molecular weight of the natural polymer derivative is 10.0 - 200.0 kDa.

7. The composite powder according to claim 1, wherein The polylysine includes one or more compounds shown in Formulas Ⅴ, Ⅵ, Ⅶ and Ⅷ, wherein, n is 20 - 1000.

8. The composite powder according to any one of claims 1 to 7, characterized in that, The mass ratio of the first component, the second component and the third component is (0.1 - 100.0):(0.1 - 100):(0.1 - 100.0).

9. The composite powder according to claim 8, wherein The first component, the second component and the third component are all dry powders, and the particle size range is 0.1 - 1000.0 µm.

10. Use of the composite powder according to any one of claims 1 to 9 in the preparation of a hemostatic drug, a biological scaffold or a carrier material.

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