Preparation method of composite spray for improving hemostatic capacity of wound part

Abstract

The invention discloses a preparation method of a composite spray for improving the hemostatic ability of a wound part, the composite spray comprises a liquid A and a liquid B. The liquid B is a sodium alginate aqueous solution, a preparation method of the solution A comprises the following steps: 1, dissolving a lipopeptide conjugate, cholesterol, distearoyl phosphatidylcholine, distearoyl phosphatidylserine and dimethyl sulfoxide in a chloroform-methanol mixed solution to obtain a mixed system; 2, preparing the mixed system into a precursor solution by utilizing a film hydration method; 3, adding calcium chloride into the precursor solution, and reacting in a dark place to obtain a solution A; a precursor solution is designed according to the function of natural platelets, the function of the precursor solution is equivalent to that of nano platelets, the precursor solution is matched with sodium alginate, adverse effects on a blood coagulation system caused by fibrin degradation, platelet activation capability reduction and the like of a wound part due to blood loss can be relieved in a targeted manner, and the preparation has the advantages of rapid hemostasis, good biological activity, no toxic or side effects and the like. And the wound healing is rapid.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical materials, and in particular relates to a method for preparing a composite spray for improving the hemostasis ability of a wound site. Background Art

[0002] It is reported that death due to massive bleeding caused by tissue / organ damage, surgery, disease or accident accounts for 30-40% of trauma mortality, and 50% of these deaths can be avoided by effective hemostasis techniques. Therefore, timely and effective hemostasis is the key to reducing the risk of postoperative death and improving postoperative recovery. Large amounts of blood loss often lead to serious complications, including hemorrhagic shock, hemorrhagic hypothermia, metabolic acidosis and other complications.

[0003] Hemostasis is a biological response involving protein and cellular components of the wound microenvironment achieved through multiple interactions. Commercially available hemostatic agents often combat blood loss by improving discrete parts of the hemostatic process, such as commercial gelatin sponges that cannot cope with complex bleeding by simply inducing blood cell aggregation, while ignoring the changes that occur at the wound site as blood loss occurs, such as platelet function, fibrinogen concentration, and clot stability, when they are adversely affected. In addition, the trauma-bleeding triad caused by acute bleeding, hypothermia, coagulopathy, and acidosis exacerbates fibrinogen degradation and reduces platelet activation, which may be difficult to combat by improving discrete parts of hemostasis. These illustrate the multifaceted effects of trauma-bleeding on the coagulation system and emphasize the need for more comprehensive solutions. Ideal hemostatic biomaterials should be able to mimic, amplify, and exploit hemostatic mechanisms. They should also be storage stable, biocompatible, biodegradable, and bioactive to accelerate hemostasis and reduce blood cell loss.

[0004] Providing a targeted method to alleviate the adverse effects on the coagulation system caused by blood loss at the trauma site, such as fibrin degradation and reduced platelet activation ability, is one of the main ways to solve the problem of sequelae of blood loss in clinical practice. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method for preparing a composite spray that improves the hemostasis ability of a wound site in view of the above-mentioned deficiencies of the prior art. The spray is designed with a precursor solution according to the function of natural platelets, and its function is equivalent to that of nanoplatelets. In combination with sodium alginate, it can specifically alleviate the adverse effects of fibrin degradation and reduced platelet activation ability caused by blood loss at the wound site, and has the characteristics of rapid hemostasis, good biological activity, and rapid wound healing.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is: 1. A method for preparing a composite spray for improving the hemostatic ability of a wound site, wherein the composite spray comprises liquid A and liquid B, wherein the liquid B is a sodium alginate aqueous solution, and the method for preparing liquid A comprises the following steps:

[0007] Step 1, dissolving the lipopeptide conjugate, cholesterol, distearoylphosphatidylcholine, distearoylphosphatidylserine and dimethyl sulfoxide in a chloroform-methanol mixed solution to obtain a mixed system;

[0008] Step 2: using a thin film hydration method to prepare a precursor solution from the mixed system in step 1;

[0009] Step 3: Add calcium chloride to the precursor solution in step 2, and react in the dark to obtain solution A.

[0010] The above-mentioned method for preparing a composite spray for improving the hemostatic ability of a wound site is characterized in that the lipopeptide conjugate in step one includes a PAP-lipopeptide conjugate and / or a PCP-lipopeptide conjugate; the PAP-lipopeptide conjugate is a lipopeptide conjugate grafted with a PAP polypeptide, and the PCP-lipopeptide conjugate is a lipopeptide conjugate grafted with a PCP polypeptide; the amino acid sequence of the PAP polypeptide is GFOGER, with a molecular weight of 678, see SEQ ID NO: 1; the PCP polypeptide sequence is GGQQLK, with a molecular weight of 669, see SEQ ID NO: 2.

[0011] The method for preparing the composite spray for improving the hemostatic ability of a wound site is characterized in that the method for preparing the lipopeptide conjugate comprises the following steps:

[0012] Step 101, placing distearoyl phosphatidylethanolamine-polyethylene glycol-carboxyl in a dimethyl sulfoxide solution, stirring for reaction, to obtain system A;

[0013] Step 102, placing the PAP polypeptide or PCP polypeptide in deionized water, performing ultrasonic homogenization and stirring for reaction, to obtain system B;

[0014] Step 103, adding system B to the system A, adjusting the pH to 6-8 after mixing, to obtain system C;

[0015] Step 104, adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to the system C, stirring and reacting to obtain the system D;

[0016] Step 105: dialyze the system D and then freeze-dry it to obtain the corresponding lipopeptide conjugate.

[0017] The above-mentioned method for preparing a composite spray for improving the hemostatic ability of a wound site is characterized in that the molecular weight of the distearoyl phosphatidylethanolamine-polyethylene glycol-carboxyl in step 101 is 2000, and the volume of the dimethyl sulfoxide is 0.5 to 3 times the mass of the distearoyl phosphatidylethanolamine-polyethylene glycol-carboxyl, wherein the unit of volume is mL and the unit of mass is mg; the stirring reaction in step 101 is stirred in the dark at room temperature for 1h to 4h.

[0018] The above-mentioned method for preparing a composite spray for improving the hemostatic ability of a wound site is characterized in that the volume of deionized water in step 102 is 0.5 to 2 times the mass of the PAP polypeptide, or the volume of deionized water is 1 to 3 times the mass of the PCP polypeptide, wherein the unit of volume is mL and the unit of mass is mg; the stirring reaction in step 102 is stirring in the dark for 0.5h to 1h at room temperature; and the volume ratio of system A to system B in step 103 is (1 to 4):1.

[0019] The above-mentioned method for preparing a composite spray for improving the hemostatic ability of a wound site is characterized in that the mass of 1-ethyl-(3-dimethylaminopropyl)carbodiimide in step 104 is 0.2 to 0.5 times the mass of distearoylphosphatidylethanolamine-polyethylene glycol-carboxyl; the mass of N-hydroxysuccinimide is 0.3 to 0.8 times the mass of distearoylphosphatidylethanolamine-polyethylene glycol-carboxyl; the stirring reaction in step 104 is stirred at room temperature in the dark for 2h to 6h; the molecular weight cutoff of the dialysis bag used for dialysis in step 105 is 1000Da, the freeze-drying temperature is -50°C, and the time is 3 days.

[0020] The preparation method of the composite spray for improving the hemostatic ability of the wound site is characterized in that the preparation method of the mixed system in step 1 comprises: vortexing and ultrasonically mixing the lipopeptide conjugate and the chloroform-methanol mixed solution to obtain a mixed solution; adding a chloroform-methanol mixed solution containing cholesterol, distearoylphosphatidylcholine and distearoylphosphatidylserine to the mixed solution, stirring for 0.5h to 1h at room temperature and in the dark to obtain a mixed system; the chloroform-methanol mixed solution is prepared by mixing chloroform and methanol in a volume ratio of (1 to 2): 1. The mass of the lipopeptide conjugate is 0.8 to 1.2 times the volume of the chloroform-methanol mixed solution, wherein the unit of mass is mg and the unit of volume is mL; the mass of the distearoylphosphatidylcholine is 10 to 14 times the mass of cholesterol, and the mass of distearoylphosphatidylcholine is 5 to 8 times the mass of distearoylphosphatidylserine; the mass of the solute in the chloroform-methanol mixed solution containing cholesterol, distearoylphosphatidylcholine and distearoylphosphatidylserine is 0.8 to 1.2 times the volume of the solvent, wherein the unit of mass is mg and the unit of volume is mL.

[0021] The method for preparing the composite spray for improving the hemostatic ability of a wound site is characterized in that the mass ratio of the PAP-lipopeptide conjugate to the PCP-lipopeptide conjugate is (1-1.5):1.

[0022] The preparation method of the composite spray for improving the hemostatic ability of the wound site is characterized in that the preparation method of the precursor solution in step 2 comprises: placing the mixed system in a single-mouth round-bottom flask and ultrasonically mixing, vacuum-evaporating in a water bath to form a uniform film, vacuum drying to evaporate the organic reagent, adding deionized water for hydration, ultrasonicating with a probe, and centrifuging to obtain a precursor solution; the temperature of the vacuum-evaporating in a water bath is 25°C to 40°C, and the speed is 90rpm to 140rpm; the temperature of the vacuum drying is 25°C to 40°C, and the time is 6h to 12h; the volume of deionized water added is 0.5 to 1 times the mass of distearoylphosphatidylcholine, the volume unit of deionized water is mL, and the mass unit of distearoylphosphatidylserine is mg; the probe ultrasonic condition is 950W, and the ultrasonic condition is 5min to 10min at 30% to 45% power; the centrifugal condition is 3000g centrifugation for 5min to 10min.

[0023] The preparation method of the composite spray for improving the hemostatic ability of a wound site is characterized in that, in step 3, calcium chloride is added to the precursor solution under stirring conditions of 200 r / min to 400 r / min, and the reaction is carried out in the dark for 10 min to 30 min to obtain liquid A; the ratio of the mass of the calcium chloride to the volume of the precursor solution is (1 to 3):100, wherein the unit of mass is g and the unit of volume is mL; the ratio of the mass of sodium alginate in the sodium alginate aqueous solution to the volume of deionized water is (1 to 3):100, wherein the unit of mass is g and the unit of volume is mL.

[0024] Compared with the prior art, the present invention has the following advantages:

[0025] 1. The present invention designs a precursor solution according to the function of natural platelets, which is equivalent to nanoplatelets. Combined with sodium alginate, it can specifically alleviate the adverse effects on the coagulation system caused by fibrin degradation and reduced platelet activation ability caused by blood loss at the wound site, and has the characteristics of rapid hemostasis, good biological activity, and rapid wound healing.

[0026] 2. In an unsanitary environment (e.g., wilderness, battlefield), without professional rescue knowledge and good sanitary conditions, it is still a huge challenge to stop bleeding quickly and effectively. The preparation method of the composite spray for improving the hemostatic ability of the wound site of the present invention is based on the simple and easy-to-operate form of hemostatic spray, which can achieve rapid and deep hemostasis at the wound site, and can adapt to irregular wound shapes, improve primary and secondary hemostasis and slow down the degradation of blood clots by recruiting activated platelets and counteracting the consumption of fibrinogen, while reducing the adverse effects of blood loss on the body while stopping bleeding quickly and effectively, amplifying the self-coagulation cascade reaction, and continuously enhancing the hemostatic ability of the wound site.

[0027] 3. The composite spray for improving the hemostatic ability of the wound site of the present invention successfully achieves rapid gelation at the wound site, can provide a physical barrier for the wound, accelerate tissue repair and promote wound healing.

[0028] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the MALDI-TOF image of the PCP-lipopeptide conjugate of Example 1-1.

[0030] Figure 2 This is the MALDI-TOF image of the PAP-lipopeptide conjugate of Example 2-1.

[0031] Figure 3 This is the TEM image of the precursor solution prepared in Example 3-1.

[0032] Figure 4 This is a graph showing the particle size of the precursor solution prepared in Example 3-1.

[0033] Figure 5 This is a SEM image of the precursor solution prepared in Example 3-1 binding to platelets.

[0034] Figure 6 This is the four-item graph of coagulation of the precursor solution prepared in Example 3-1.

[0035] Figure 7 This is a gel formation diagram of the composite spray prepared in Example 3-1 after spraying.

[0036] Figure 8 It is the liquid absorption rate of the composite spray prepared in Example 3-1.

[0037] Fig. 9 The porosity of the composite spray prepared in Example 3-1.

[0038] Fig.10 The hemolysis rate of the composite spray prepared in Example 3-1.

[0039] Fig.11 Schematic diagram of the CCK8 analysis results of the cytotoxicity of the composite spray prepared in Example 3-1.

[0040] Fig.12 Schematic diagram of in vitro coagulation of the composite spray prepared in Example 3-1.

[0041] Fig.13 Schematic diagram of liver hemostasis of the composite spray prepared in Example 3-1. DETAILED DESCRIPTION

[0042] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below in conjunction with the embodiments. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments, unless otherwise specified, can be obtained from commercial channels.

[0043] Example 1-1

[0044] This embodiment provides a method for preparing a PCP-lipopeptide conjugate, which specifically comprises:

[0045] Step 101, placing 20 mg of distearoylphosphatidylethanolamine-polyethylene glycol-carboxyl (DSPE-mPEG2000-COOH) in 20 mL of dimethyl sulfoxide (DMSO) solution, stirring and reacting for 2 h at room temperature in the dark, to obtain system A, wherein the molecular weight of the distearoylphosphatidylethanolamine-polyethylene glycol-carboxyl is 2000;

[0046] Step 102, 10 mg of PCP polypeptide was placed in 20 mL of deionized water, and after ultrasonic homogenization, stirred at room temperature in the dark for 0.5 h to obtain system B, the PCP molecular weight was 669, and the PCP polypeptide sequence was GGQQLK, see SEQ ID NO: 2;

[0047] Step 103, adding system B to the system A, with the volume ratio of system A to system B being 1:1, adjusting the pH to 6-8 after mixing, to obtain system C;

[0048] Step 104, adding 5 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 8 mg of N-hydroxysuccinimide (NHS) to the system C, stirring at room temperature in the dark for 2 h, to obtain system D;

[0049] Step 105, dialyzing the system D using a 1000 Da dialysis bag under ice bath conditions for 1 day, and then freeze-drying at -50°C for 3 days to obtain a PCP-lipopeptide conjugate.

[0050] Figure 1 This is the MALDI-TOF image of the PCP-lipopeptide conjugate of Example 1-1. Figure 1 It can be seen that the highest peak appears at a mass-to-charge ratio of 2757, indicating that distearoylphosphatidylethanolamine-polyethylene glycol-carboxyl is connected to the amino group in the PCP polypeptide, and the PCP-lipopeptide conjugate is successfully grafted.

[0051] Example 1-2

[0052] This embodiment provides a method for preparing a PCP-lipopeptide conjugate, which specifically comprises:

[0053] Step 101, placing 20 mg of distearoyl phosphatidylethanolamine-polyethylene glycol-carboxyl in 10 mL of dimethyl sulfoxide solution, stirring and reacting for 4 hours at room temperature in the dark, to obtain system A, wherein the molecular weight of the distearoyl phosphatidylethanolamine-polyethylene glycol-carboxyl is 2000;

[0054] Step 102, 8 mg of PCP polypeptide was placed in 8 mL of deionized water, and after ultrasonic homogenization, stirred at room temperature in the dark for 0.8 h to obtain system B, where the PCP molecular weight was 669 and the PCP polypeptide sequence was GGQQLK;

[0055] Step 103, adding system B to the system A, with the volume ratio of system A to system B being 2:1, and adjusting the pH to 6-8 after mixing to obtain system C;

[0056] Step 104, adding 4 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 6 mg of N-hydroxysuccinimide to the system C, stirring at room temperature in the dark for 4 hours, to obtain system D;

[0057] Step 105, dialyzing the system D using a 1000 Da dialysis bag for 2 days under ice bath conditions and then freeze-drying it at -50°C for 3 days to obtain a PCP-lipopeptide conjugate.

[0058] The PCP-lipopeptide conjugate of this example is consistent with that of Example 1-1.

[0059] Examples 1-3

[0060] This embodiment provides a method for preparing a PCP-lipopeptide conjugate, which specifically comprises:

[0061] Step 101, placing 20 mg of distearoyl phosphatidylethanolamine-polyethylene glycol-carboxyl in 60 mL of dimethyl sulfoxide solution, stirring and reacting for 1 h at room temperature in the dark, to obtain system A, wherein the molecular weight of the distearoyl phosphatidylethanolamine-polyethylene glycol-carboxyl is 2000;

[0062] Step 102, 8 mg of PCP polypeptide was placed in 24 mL of deionized water, and after ultrasonic homogenization, it was stirred in the dark at room temperature for 1 hour to obtain system B, the PCP molecular weight was 669, and the PCP polypeptide sequence was GGQQLK;

[0063] Step 103, adding system B to the system A, with the volume ratio of system A to system B being 4:1, and adjusting the pH to 6-8 after mixing to obtain system C;

[0064] Step 104, adding 10 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 12 mg of N-hydroxysuccinimide to the system C, stirring at room temperature in the dark for 6 hours, to obtain system D;

[0065] Step 105, dialyzing the system D using a 1000Da dialysis bag under ice bath conditions for 2 days and then freeze-drying at -50°C for 3 days to obtain a PCP-lipopeptide conjugate;

[0066] The PCP-lipopeptide conjugate of this example is consistent with that of Example 1-1.

[0067] Example 2-1

[0068] This embodiment provides a method for preparing a PAP-lipopeptide conjugate, which specifically comprises:

[0069] Step 101, placing 20 mg of distearoyl phosphatidylethanolamine-polyethylene glycol-carboxyl in 10 mL of dimethyl sulfoxide solution, stirring and reacting for 1 h at room temperature in the dark, to obtain system A, wherein the molecular weight of the distearoyl phosphatidylethanolamine-polyethylene glycol-carboxyl is 2000;

[0070] Step 102, 10 mg of PAP polypeptide was placed in 5 mL of deionized water, and after homogenization by sonication, stirred at room temperature in the dark for 0.5 h to obtain system B, wherein the molecular weight of PAP is 678, and the sequence of PAP is GFOGER, see SEQ ID NO: 1;

[0071] Step 103, adding system B to the system A, with the volume ratio of system A to system B being 1:1, adjusting the pH to 6-8 after mixing, to obtain system C;

[0072] Step 104, adding 5 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 8 mg of N-hydroxysuccinimide to the system C, stirring at room temperature in the dark for 2 h, to obtain system D;

[0073] Step 105, dialyzing the system D using a 1000Da dialysis bag under ice bath conditions for 1 day, and then freeze-drying at -50°C for 3 days to obtain a PAP-lipopeptide conjugate;

[0074] Figure 2 This is the MALDI-TOF image of the PCP-lipopeptide conjugate of Example 2-1. Figure 2 It can be seen that the highest peak appears at the mass-to-charge ratio of 2713, indicating that distearoylphosphatidylethanolamine-polyethylene glycol-carboxyl group is connected to the amino group in the PAP polypeptide, and the PAP-lipopeptide conjugate is successfully grafted.

[0075] Example 2-2

[0076] This embodiment provides a method for preparing a PAP-lipopeptide conjugate, which specifically comprises:

[0077] Step 101, placing 20 mg of distearoyl phosphatidylethanolamine-polyethylene glycol-carboxyl in 20 mL of dimethyl sulfoxide solution, stirring and reacting for 2 h at room temperature in the dark, to obtain system A, wherein the molecular weight of the distearoyl phosphatidylethanolamine-polyethylene glycol-carboxyl is 2000;

[0078] Step 102, 15 mg of PAP polypeptide was placed in 10 mL of deionized water, and after ultrasonic homogenization, it was stirred at room temperature in the dark for 1 hour to obtain system B, where the molecular weight of PAP was 678 and the sequence of PAP was GFOGER;

[0079] Step 103, adding system B to the system A, with the volume ratio of system A to system B being 2:1, and adjusting the pH to 6-8 after mixing to obtain system C;

[0080] Step 104, adding 6 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 8 mg of N-hydroxysuccinimide to the system C, stirring at room temperature in the dark for 4 hours, to obtain system D;

[0081] Step 105, dialyzing the system D using a 1000Da dialysis bag for 2 days under ice bath conditions and then freeze-drying it at -50°C for 3 days to obtain a PAP-lipopeptide conjugate;

[0082] The PAP-lipopeptide conjugate of this example is consistent with that of Example 2-1.

[0083] Example 2-3

[0084] This embodiment provides a method for preparing a PAP-lipopeptide conjugate, which specifically comprises:

[0085] Step 1, placing 20 mg of distearoyl phosphatidylethanolamine-polyethylene glycol-carboxyl in 60 mL of dimethyl sulfoxide solution, stirring and reacting for 4 hours at room temperature in the dark, to obtain system A, wherein the molecular weight of the distearoyl phosphatidylethanolamine-polyethylene glycol-carboxyl is 2000;

[0086] Step 2: 15 mg of PAP polypeptide was placed in 30 mL of deionized water, and after ultrasonic homogenization, stirred at room temperature in the dark for 0.75 h to obtain system B, where the molecular weight of PAP was 678 and the sequence of PAP was GFOGER;

[0087] Step 3, adding system B to the system A, the volume ratio of system A to system B is 4:1, and adjusting the pH to 6-8 after mixing to obtain system C;

[0088] Step 4: Add 8 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 16 mg of N-hydroxysuccinimide to the system C, and stir at room temperature in the dark for 6 h to obtain system D;

[0089] Step 5, dialyzing the system D using a 1000Da dialysis bag for 2 days under ice bath conditions and then freeze-drying it at -50°C for 3 days to obtain a PAP-lipopeptide conjugate;

[0090] The PAP-lipopeptide conjugate of this example is consistent with that of Example 2-1.

[0091] Example 3-1

[0092] This embodiment provides a method for preparing a composite spray, which specifically includes:

[0093] Step 1, dissolve 2 mg of PAP-lipopeptide conjugate in 2 mL of chloroform-methanol 1:1 mixed solution, dissolve 2 mg of PCP-lipopeptide conjugate in 2 mL of chloroform-methanol 1:1 mixed solution, vortex and ultrasonically mix the above two mixed solutions to obtain a mixed solution; at room temperature, dissolve 2.5 mg of cholesterol in 4 mL of chloroform-methanol 1:1 mixed solution, dissolve 25 mg of distearoylphosphatidylcholine (DSPC) in 20 mL of chloroform-methanol 1:1 mixed solution, and distearoylphosphatidylserine (DSPS) in 4 mL of chloroform-methanol 1:1 mixed solution, mix the above three mixed solutions to obtain a chloroform-methanol mixed solution containing cholesterol, distearoylphosphatidylcholine and distearoylphosphatidylserine, and then add it to the mixed solution, stir at 300 r / min at room temperature and in the dark for 0.5 h to obtain a mixed system;

[0094] Step 2: The mixed system was transferred to a single-mouth round-bottom flask and ultrasonically mixed. Under a 35°C water bath condition, the mixture was decompressed and rotary evaporated at 110 rpm to form a uniform film. After the organic reagent was evaporated under vacuum at 25°C for 10 h, 12.5 mL of deionized water was added for hydration. The mixture was ultrasonically probed (950 W, 35%, 5 min, 2s / 3s on / off), and the mixture was rapidly centrifuged at 3000 g for 8 min. The precursor solution was obtained after filtration with a 0.22 μm filter membrane.

[0095] Step 3: Under stirring conditions of 300 r / min, add 0.2 g of calcium chloride to 10 mL of precursor solution, react in the dark for 10 min to obtain liquid A, which is placed in spray bottle A; under stirring conditions of 150 r / min, dissolve 0.2 g of sodium alginate in 10 mL of deionized water to obtain liquid B, which is placed in spray bottle B, and liquid A and liquid B are sprayed together at the wound site to form a composite spray that improves the hemostatic ability of the wound site.

[0096] Example 3-2

[0097] This embodiment provides a method for preparing a composite spray, which specifically includes:

[0098] Step 1, dissolve 1.2 mg PAP-lipopeptide conjugate in 1 mL chloroform-methanol 1:1 mixed solution, dissolve 1.2 mg PCP-lipopeptide conjugate in 1 mL chloroform-methanol 1:1 mixed solution, vortex and ultrasonically mix the above two mixed solutions to obtain a mixed solution; under room temperature, dissolve 1 mg cholesterol in 5 mL chloroform-methanol 1:1 mixed solution, dissolve 13 mg distearoylphosphatidylcholine (DSPC) in 10 mL chloroform-methanol 1:1 mixed solution, and distearoylphosphatidylserine (DSPS) in 5 mL chloroform-methanol 1:1 mixed solution, mix the above three mixed solutions to obtain a chloroform-methanol mixed solution containing cholesterol, distearoylphosphatidylcholine and distearoylphosphatidylserine, and then add it to the mixed solution, stir at 300 r / min at room temperature and in the dark for 1 hour to obtain a mixed system;

[0099] Step 2: The mixed system was transferred to a single-mouth round-bottom flask and ultrasonically mixed. Under a 40°C water bath condition, the mixture was decompressed and rotary evaporated at 140 rpm to form a uniform film. After the organic reagent was evaporated under vacuum at 30°C for 12 h, 13 mL of deionized water was added for hydration. The mixture was ultrasonically probed (950 W, 30%, 10 min, 2s / 3s on / off), and the mixture was rapidly centrifuged at 3000 g for 5 min. The precursor solution was obtained after filtration with a 0.22 μm filter membrane.

[0100] Step 3: Under stirring conditions of 200 r / min, add 0.1 g of calcium chloride to 10 mL of precursor solution, react in the dark for 20 min to obtain liquid A, which is placed in spray bottle A; under stirring conditions of 200 r / min, dissolve 0.1 g of sodium alginate in 10 mL of deionized water to obtain liquid B, which is placed in spray bottle B, and liquid A and liquid B are sprayed together at the wound site to form a composite spray that improves the hemostatic ability of the wound site.

[0101] Example 3-3

[0102] This embodiment provides a method for preparing a composite spray, which specifically includes:

[0103] Step 1, dissolve 7.5 mg of PAP-lipopeptide conjugate in 9 mL of chloroform-methanol 2:1 mixed solution, dissolve 5 mg of PCP-lipopeptide conjugate in 6 mL of chloroform-methanol 2:1 mixed solution, vortex and ultrasonically mix the above two mixed solutions to obtain a mixed solution; under room temperature, dissolve 4.3 mg of cholesterol in 5 mL of chloroform-methanol 2:1 mixed solution, dissolve 60 mg of distearoylphosphatidylcholine (DSPC) in 50 mL of chloroform-methanol 2:1 mixed solution, and distearoylphosphatidylserine (DSPS) in 5 mL of chloroform-methanol 2:1 mixed solution, mix the above three mixed solutions to obtain a chloroform-methanol mixed solution containing cholesterol, distearoylphosphatidylcholine and distearoylphosphatidylserine, and then add it to the mixed solution, stir at 400 r / min at room temperature and in the dark for 0.75 h to obtain a mixed system;

[0104] Step 2: The mixed system was transferred to a single-mouth round-bottom flask and ultrasonically mixed. Under a 25°C water bath condition, the mixture was decompressed and rotary evaporated at 90 rpm to form a uniform film. The organic reagent was evaporated under vacuum at 40°C for 6 h, and then 40 mL of deionized water was added for hydration. The mixture was ultrasonically probed (950 W, 40%, 8 min, 2s / 3s on / off), and the mixture was rapidly centrifuged at 3000 g for 10 min. The precursor solution was obtained after filtration with a 0.22 μm filter membrane.

[0105] Step 3: Under stirring conditions of 400 r / min, add 0.3 g of calcium chloride to 10 mL of precursor solution, react in the dark for 30 minutes to obtain liquid A, which is placed in spray bottle A; under stirring conditions of 400 r / min, dissolve 0.3 g of sodium alginate in 10 mL of deionized water to obtain liquid B, which is placed in spray bottle B, and liquid A and liquid B are sprayed together at the wound site to form a composite spray that improves the hemostatic ability of the wound site.

[0106] Performance evaluation:

[0107] Figure 3 This is a TEM image of the precursor solution prepared in Example 3-1. The precursor solution TEM sample was prepared by negative staining. The precursor solution was dropped on a copper mesh for transmission electron microscopy and then immediately added with phosphotungstic acid solution after being allowed to stand. After drying, it was observed with a transmission electron microscope and photographed for analysis. The results of the transmission electron microscope analysis are shown in the figure. As a result of negative staining, the precursor solution is a white area, while the background color is darker. The picture shows that the shape of the prepared precursor solution is approximately circular, the surface is intact, and there is no damage. This indicates that the precursor solution was successfully synthesized.

[0108] Figure 4The particle size distribution diagram of the precursor solution prepared in Example 3-1. The hydrated particle size of the precursor solution was measured by a particle size analyzer using a dynamic light scattering method (DLS). As shown in the figure, the average hydrated particle size of the synthesized precursor solution is 135nm, and the polydispersity index (PDI) value is 0.157. The results show that the prepared precursor solution has a stable morphology and good dispersibility.

[0109] Figure 5 This is a SEM image of the precursor solution prepared in Example 3-1 combined with natural platelets. After natural platelets were mixed and incubated with the precursor solution, the interaction between natural platelets and the precursor solution was studied in vitro. Figure 5 It can be seen that the surface of the precursor solution modified with a higher affinity peptide enhances its binding to platelets, and the presence of natural platelets with the precursor solution bound to the surface was confirmed by SEM.

[0110] Figure 6 The four coagulation diagrams of the precursor solution prepared in Example 3-1 are shown in the figure. According to different modified components, they are divided into DSPS, PCP, PAP groups and the final precursor solution, i.e., APNs group. The APTT, PT, TT and FIB values ​​of the differently modified precursor solutions were observed, among which all experimental groups had a shortened APTT value, i.e., the activated partial thromboplastin time, indicating the successful activation of the endogenous coagulation pathway; there was no significant change in the PT value, i.e., the prothrombin time, between different experimental groups and the control group, reflecting that the exogenous coagulation system was not activated; the TT value, i.e., the thromboplastin time, was shortened in all experimental groups, reflecting that the time for fibrinogen to be converted into fibrin between the experimental groups was shortened. Fibrinogen (FIB) is an important protein involved in the coagulation and hemostasis process. It can be seen from the figure that the FIB content of the group introducing the PCP peptide is higher than that of other groups, proving that the cross-linking of platelets and fibrin strengthens the hemostatic barrier and reduces the consumption of FIB. Figure 6 The precursor solution was shown to successfully activate the intrinsic coagulation pathway and antagonize fibrinogen consumption.

[0111] Figure 7 The composite spray prepared in Example 3-1 is a schematic diagram of gelation after spraying. After the A liquid and B liquid of the composite spray are sprayed at the same place at the same time, they are quickly gelled in situ to form a colloidal state as shown in the figure, indicating that the composite spray of the present invention can provide a physical barrier for the wound bleeding site.

[0112] Figure 8 The liquid absorption rate of the composite spray prepared in Example 3-1. The test method includes: soaking the prepared colloid in PBS at 37°C, removing the surface liquid with filter paper at different time points, weighing and recording the weight change, and calculating the liquid absorption rate of the colloid using the following equation:

[0113] Liquid absorption rate (%) = (M1-M0) / M0×100%

[0114] M0 is the mass of the colloid, and M1 is the mass of the composite gel after absorbing the liquid.

[0115] according to Figure 8 It can be seen that the 4h liquid absorption rate of the composite spray prepared in Example 3-1 is 19.40%±0.96, indicating that the composite spray of the present invention can realize the absorption of blood on the basis of quickly forming a colloidal physical barrier, achieving the effect of biological-physical dual coagulation.

[0116] Fig. 9 The porosity of the composite spray prepared in Example 3-1. The test method is as follows: The porosity of the composite gel is tested by the liquid displacement method. The pre-weighed composite gel (M0) is soaked in anhydrous ethanol for 24 hours, and then the ethanol on the surface is removed and weighed (M1). The porosity is calculated according to the following formula:

[0117] Liquid absorption rate (%) = (M1-M0) / ρV×100%

[0118] Where V is the volume of the composite gel, ρ is the density of anhydrous ethanol (0.789 g / cm 3 The porosity of the composite spray group was measured to be 39.76% ± 1.04. The porosity of the composite gel helps it to quickly absorb blood to aggregate blood cells and coagulation factors, thereby accelerating hemostasis. In addition, it can also effectively absorb wound exudate and reduce the possibility of wound infection.

[0119] Fig.10 It is the hemolysis rate of the composite spray prepared in Example 3-1. The testing method includes: extracting the colloid formed after the advance spraying with physiological saline for 72 hours at 37°C to obtain 0.1g / mL of extract. Take fresh rat blood to separate the red blood cells, centrifuge 5mL of fresh blood at 2000rpm for 5min, wash the red blood cells with physiological saline until the supernatant is clear and transparent, and then dilute it to a 2% (v / v) red blood cell suspension. In a 1.5mLep tube, mix the extract and the red blood cell suspension in equal proportions, and incubate at 37°C for 1h. After centrifugation, measure the absorbance of the supernatant at 545nm, use Triton X-100 and physiological saline as positive and negative controls, respectively, and then perform quantitative evaluation. The results are as follows. Fig.10 As shown, it can be seen that the hemolysis rate of the composite spray of the present invention is less than 5%, which is considered to be free of hemolysis. The results show that the composite spray of the present invention does not cause red blood cell rupture and has good blood compatibility.

[0120] Fig.11Schematic diagram of the results of CCK8 analysis of the cytotoxicity of the composite spray prepared in Example 3-1. The test method includes: extracting the colloid in RPMI-1640 medium at 37°C for 3 days to obtain an extract with a concentration of 0.1g / mL. The culture medium of human umbilical vein endothelial cells (HUVEC cells) after normal culture for 24 hours was replaced by the RPMI-1640 medium with the extract, and cultured for 24 hours and 48 hours respectively. The absorbance was measured by the CCK8 method, and the survival rate of the HUVEC cells was calculated. Each test was repeated in 6 parallel groups. The results are shown as follows Fig.11 As shown. Fig.11 As shown, the cell survival rates after 24 hours and 48 hours were both above 90%, indicating that the composite spray of the present invention has good biocompatibility and is safe and non-toxic.

[0121] Fig.12 The figure is a schematic diagram of in vitro coagulation of the composite spray prepared in Example 3-1. The test method comprises: placing fresh whole blood in a culture dish, spraying the composite spray of the present invention on the part where the fresh blood is located, and observing that after spraying into gel, the blood is rapidly coagulated in situ, indicating that the composite spray of the present invention has a rapid coagulation effect.

[0122] Fig.13 The composite spray prepared in Example 3-1 is a schematic diagram of liver hemostasis. After anesthetizing the rat, the rat is fixed on the operating table, the liver of the rat is exposed through an abdominal incision, and the serous fluid around the liver is gently removed with gauze. A 0.5 cm long wound (0.5 cm deep) is cut on the rat liver with a scalpel (23# blade), and the composite spray of the present invention is quickly sprayed on the wound bleeding site. Filter paper is placed under the liver to observe the bleeding of the liver until the bleeding stops. It can be seen that there is a small area of ​​blood on the surface of the filter paper under the liver, indicating that the composite spray of the present invention has a relatively good hemostatic effect.

[0123] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent structural change made to the above embodiment according to the technical essence of the invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A method for preparing a composite spray for improving the hemostasis ability of a wound site, wherein the composite spray comprises liquid A and liquid B, and is characterized in that: The B liquid is a sodium alginate aqueous solution, and the preparation method of the A liquid comprises the following steps: Step 1, dissolving the lipopeptide conjugate, cholesterol, distearoylphosphatidylcholine, distearoylphosphatidylserine and dimethyl sulfoxide in a chloroform-methanol mixed solution to obtain a mixed system; Step 2: using a thin film hydration method to prepare a precursor solution from the mixed system in step 1; Step 3: Add calcium chloride to the precursor solution in step 2, and react in the dark to obtain solution A.

2. The method for preparing a composite spray for improving the hemostasis ability of a wound site according to claim 1, characterized in that: The lipopeptide conjugate in step one includes a PAP-lipopeptide conjugate and / or a PCP-lipopeptide conjugate; the PAP-lipopeptide conjugate is a lipopeptide conjugate grafted with a PAP polypeptide, and the PCP-lipopeptide conjugate is a lipopeptide conjugate grafted with a PCP polypeptide; the amino acid sequence of the PAP polypeptide is GFOGER, and the molecular weight is 678; the PCP polypeptide sequence is GGQQLK, and the molecular weight is 669.

3. The method for preparing a composite spray for improving the hemostasis ability of a wound site according to claim 2, characterized in that: The preparation method of the lipopeptide conjugate comprises the following steps: Step 101, placing distearoyl phosphatidylethanolamine-polyethylene glycol-carboxyl in a dimethyl sulfoxide solution, stirring for reaction, to obtain system A; Step 102, placing the PAP polypeptide or PCP polypeptide in deionized water, performing ultrasonic homogenization and stirring for reaction, to obtain system B; Step 103, adding system B to the system A, adjusting the pH to 6-8 after mixing, to obtain system C; Step 104, adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to the system C, stirring and reacting to obtain the system D; Step 105: dialyze the system D and then freeze-dry it to obtain the corresponding lipopeptide conjugate.

4. The method for preparing a composite spray for improving the hemostasis ability of a wound site according to claim 3, characterized in that: The molecular weight of the distearoylphosphatidylethanolamine-polyethylene glycol-carboxyl in step 101 is 2000, and the volume of the dimethyl sulfoxide is 0.5 to 3 times the mass of the distearoylphosphatidylethanolamine-polyethylene glycol-carboxyl, wherein the unit of volume is mL and the unit of mass is mg; the stirring reaction in step 101 is stirred for 1 h to 4 h in the dark at room temperature.

5. The method for preparing a composite spray for improving the hemostasis ability of a wound site according to claim 3, characterized in that: In step 102, the volume of deionized water is 0.5 to 2 times the mass of the PAP polypeptide, or the volume of deionized water is 1 to 3 times the mass of the PCP polypeptide, wherein the unit of volume is mL and the unit of mass is mg; the stirring reaction in step 102 is stirred for 0.5h to 1h in the dark at room temperature; in step 103, the volume ratio of system A to system B is (1 to 4):

1.

6. The method for preparing a composite spray for improving the hemostasis ability of a wound site according to claim 3, characterized in that: In step 104, the mass of 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 0.2 to 0.5 times the mass of distearoylphosphatidylethanolamine-polyethylene glycol-carboxyl; the mass of N-hydroxysuccinimide is 0.3 to 0.8 times the mass of distearoylphosphatidylethanolamine-polyethylene glycol-carboxyl; the stirring reaction in step 104 is stirred at room temperature in the dark for 2h to 6h; the molecular weight cutoff of the dialysis bag used for dialysis in step 105 is 1000Da, the freeze-drying temperature is -50°C, and the time is 3 days.

7. The method for preparing a composite spray for improving the hemostasis ability of a wound site according to claim 1, characterized in that: The preparation method of the mixed system in step 1 comprises: vortexing and ultrasonically mixing the lipopeptide conjugate and the chloroform-methanol mixed solution to obtain a mixed solution; adding a chloroform-methanol mixed solution containing cholesterol, distearoylphosphatidylcholine and distearoylphosphatidylserine to the mixed solution, stirring for 0.5h to 1h at room temperature and in the dark to obtain a mixed system; the chloroform-methanol mixed solution is prepared by mixing chloroform and methanol in a volume ratio of (1 to 2): 1, and the mass of the lipopeptide conjugate is chloroform-methanol mixed solution. The mass of the distearoylphosphatidylcholine is 10 to 14 times that of the cholesterol, and the mass of the distearoylphosphatidylcholine is 5 to 8 times that of the distearoylphosphatidylserine. The mass of the solute in the chloroform-methanol mixed solution containing cholesterol, distearoylphosphatidylcholine and distearoylphosphatidylserine is 0.8 to 1.2 times the volume of the solvent, wherein the mass unit is mg and the volume unit is mL.

8. The method for preparing a composite spray for improving the hemostasis ability of a wound site according to claim 2, characterized in that: The mass ratio of the PAP-lipopeptide conjugate to the PCP-lipopeptide conjugate is (1-1.5):

1.

9. The method for preparing a composite spray for improving the hemostasis ability of a wound site according to claim 1, characterized in that: The preparation method of the precursor solution in step 2 comprises: placing the mixed system in a single-mouth round-bottom flask and mixing it by ultrasound, performing vacuum rotary evaporation in a water bath to form a uniform film, performing vacuum drying to evaporate the organic reagent, adding deionized water for hydration, performing probe ultrasonication, and obtaining a precursor solution after centrifugal filtration; the temperature of the vacuum rotary evaporation in a water bath is 25°C to 40°C, and the speed is 90rpm to 140rpm; the temperature of the vacuum drying is 25°C to 40°C, and the time is 6h to 12h; the volume of deionized water added is 0.5 to 1 times the mass of distearoylphosphatidylcholine, the volume unit of deionized water is mL, and the mass unit of distearoylphosphatidylserine is mg; the probe ultrasonic condition is 950W, and the ultrasonic condition is 5min to 10min at 30% to 45% power; the centrifugal condition is 3000g centrifugation for 5min to 10min.

10. The method for preparing a composite spray for improving the hemostasis ability of a wound site according to claim 1, characterized in that: In step 3, calcium chloride is added to the precursor solution under stirring conditions of 200 r / min to 400 r / min, and the reaction is carried out in the dark for 10 min to 30 min to obtain liquid A; the ratio of the mass of the calcium chloride to the volume of the precursor solution is (1 to 3):100, wherein the unit of mass is g and the unit of volume is mL; the ratio of the mass of sodium alginate in the sodium alginate aqueous solution to the volume of deionized water is (1 to 3):100, wherein the unit of mass is g and the unit of volume is mL.

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