A method for preparing a compound spray that improves hemostasis at the wound site

Abstract

This invention discloses a method for preparing a composite spray that improves hemostasis at wound sites. The composite spray includes solution A and solution B, where solution B is an aqueous solution of sodium alginate. The preparation method of solution A includes: 1. Dissolving a lipopeptide conjugate, cholesterol, distearylphosphatidylcholine, distearylphosphatidylserine, and dimethyl sulfoxide in a chloroform-methanol mixed solution to obtain a mixed system; 2. Preparing a precursor solution from the mixed system using a thin-film hydration method; 3. Adding calcium chloride to the precursor solution and reacting it in the dark to obtain solution A. This invention designs the precursor solution based on the function of natural platelets, and its function is equivalent to that of nanoplatelets. Combined with sodium alginate, it can specifically alleviate the adverse effects on the coagulation system caused by blood loss, such as fibrin degradation and reduced platelet activation, at wound sites. It has the characteristics of rapid hemostasis, good bioactivity, and rapid wound healing.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a method for preparing a composite spray that improves hemostasis at wound sites. Background Technology

[0002] It has been reported that massive bleeding caused by tissue / organ damage, surgery, disease, or accidents accounts for 30-40% of trauma mortality, and 50% of these deaths can be prevented with effective hemostasis techniques. Therefore, timely and effective hemostasis is crucial for reducing the risk of postoperative mortality and improving postoperative recovery. Massive blood loss often leads to serious complications, including hemorrhagic shock, hemorrhagic hypothermia, and metabolic acidosis.

[0003] Hemostasis is a biological response involving proteins and cellular components of the wound microenvironment, achieved through multiple interactions. Commercially available hemostatic agents often combat blood loss by improving discrete components of the hemostasis process. For example, commercial gelatin sponges, by simply inducing blood cell aggregation, cannot address complex bleeding, neglecting the changes that occur at the wound site as key components such as platelet function, fibrinogen concentration, and clot stability are adversely affected by blood loss. Furthermore, the traumatic bleeding triad—acute hemorrhage, hypothermia, coagulopathy, and acidosis—exacerbates fibrinogen degradation and reduces platelet activation, which may be difficult to counteract by improving discrete components of hemostasis. These findings illustrate the multifaceted impact of traumatic bleeding on the coagulation system, emphasizing the need for a more comprehensive solution. Ideal hemostatic biomaterials should be able to mimic, scale up, and utilize hemostasis mechanisms. They should also possess storage stability, biocompatibility, biodegradability, and bioactivity to accelerate hemostasis and reduce blood cell loss.

[0004] Providing a targeted approach to alleviate the adverse effects of blood loss on the coagulation system, such as fibrin degradation and reduced platelet activation at the site of injury, is one of the main ways to solve the problem of post-blood loss sequelae in clinical practice. Summary of the Invention

[0005] The technical problem to be solved by this invention is to address the shortcomings of the prior art by providing a method for preparing a composite spray that improves hemostasis at the wound site. This spray is designed with a precursor solution based on the function of natural platelets, functioning similarly to nanoplatelets. Combined with sodium alginate, it can specifically alleviate the adverse effects on the coagulation system caused by blood loss, such as fibrin degradation and reduced platelet activation, resulting in rapid hemostasis, good bioactivity, and rapid wound healing.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: 1. A method for preparing a composite spray that improves the hemostatic ability of a wound site, the composite spray comprising liquid A and liquid B, characterized in that liquid B is an aqueous solution of sodium alginate, and the preparation method of liquid A includes the following steps:

[0007] Step 1: Dissolve the lipopeptide conjugate, cholesterol, distearylphosphatidylcholine, distearylphosphatidylserine, and dimethyl sulfoxide in a chloroform-methanol mixed solution to obtain a mixed system;

[0008] Step 2: Prepare a precursor solution from the mixture described in Step 1 using a thin-film hydration method;

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

[0010] The preparation method of the above-mentioned composite spray for improving hemostasis at the 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 PAP polypeptide, and the PCP-lipopeptide conjugate is a lipopeptide conjugate grafted with PCP polypeptide; the amino acid sequence of the PAP polypeptide is GFOGER, and the molecular weight is 678, as shown in SEQ ID NO:1; the sequence of the PCP polypeptide is GGQQLK, and the molecular weight is 669, as shown in SEQ ID NO:2.

[0011] The preparation method of the above-mentioned compound spray for improving hemostasis at the wound site is characterized in that the preparation method of the lipopeptide conjugate includes the following steps:

[0012] Step 101: Place the distearate phosphatidylethanolamine-polyethylene glycol-carboxyl group in a dimethyl sulfoxide solution and stir to react, to obtain system A;

[0013] Step 102: Place the PAP peptide or PCP peptide in deionized water, sonicate until homogeneous, and then stir to react, to obtain system B;

[0014] Step 103: Add system B to system A, mix, and adjust the pH to 6-8 to obtain system C;

[0015] Step 104: Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to system C, stir and react to obtain system D;

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

[0017] The preparation method of the above-mentioned composite spray for improving hemostasis at the wound site is characterized in that, in step 101, the molecular weight of the distearylphosphatidylethanolamine-polyethylene glycol-carboxyl group is 2000, and the volume of the dimethyl sulfoxide is 0.5 to 3 times the mass of the distearylphosphatidylethanolamine-polyethylene glycol-carboxyl group, wherein the unit of volume is mL and the unit of mass is mg; the stirring reaction in step 101 is carried out at room temperature in the dark for 1 to 4 hours.

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

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

[0020] The preparation method of the above-mentioned composite spray for improving hemostasis at the wound site is characterized in that the preparation method of the mixed system in step one includes: vortexing and ultrasonically mixing a lipopeptide conjugate with a chloroform-methanol mixed solution to obtain a mixed liquid; adding a chloroform-methanol mixed solution containing cholesterol, distearylphosphatidylcholine, and distearylphosphatidylserine to the mixed liquid, and stirring at room temperature in the dark for 0.5 h to 1 h to obtain the mixed system; the chloroform-methanol mixed solution is prepared by mixing chloroform and methanol in a volume ratio of (1~2):1. The mass of the lipopeptide conjugate is 0.8 to 1.2 times the volume of the chloroform-methanol mixed solution, where mass is in mg and volume is in mL; the mass of distearylphosphatidylcholine is 10 to 14 times the mass of cholesterol, and the mass of distearylphosphatidylcholine is 5 to 8 times the mass of distearylphosphatidylserine; the mass of the solute in the chloroform-methanol mixed solution containing cholesterol, distearylphosphatidylcholine, and distearylphosphatidylserine is 0.8 to 1.2 times the volume of the solvent, where mass is in mg and volume is in mL.

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

[0022] The preparation method of the above-mentioned composite spray for improving hemostasis at the wound site is characterized in that the preparation method of the precursor solution in step two includes: placing the mixed system in a single-necked round-bottom flask and ultrasonically mixing it, rotary evaporating it under reduced pressure in a water bath to form a uniform film, vacuum drying to evaporate the organic reagents, adding deionized water for hydration, ultrasonicating with a probe, centrifuging and filtering to obtain the precursor solution; the temperature of the rotary evaporation under reduced pressure in the water bath is 25℃~40℃, and the rotation speed is 90rpm~140rpm; the temperature of the vacuum drying is 25℃~40℃, and the time is 6h~12h; the volume of deionized water added is 0.5~1 times the mass of distearylphosphatidylcholine, the volume unit of deionized water is mL, and the mass unit of distearylphosphatidylcholine is mg; the ultrasonic conditions of the probe are 950W, ultrasonication at 30%~45% power for 5min~10min; the centrifugation conditions are centrifugation at 3000g for 5min~10min.

[0023] The preparation method of the above-mentioned composite spray for improving hemostasis at the wound site is characterized in that, in step three, 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 solution A; the mass ratio of the calcium chloride to the volume of the precursor solution is (1~3):100, where the mass unit is g and the volume unit is mL; the mass ratio of the sodium alginate to the volume of deionized water in the sodium alginate aqueous solution is (1~3):100, where the mass unit is g and the volume unit is mL.

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

[0025] 1. Based on the function of natural platelets, the present invention designs a precursor solution that functions as nanoplatelets. Combined with sodium alginate, it can specifically alleviate the adverse effects on the coagulation system caused by blood loss, such as fibrin degradation and reduced platelet activation capacity. It has the characteristics of rapid hemostasis, good bioactivity, and rapid wound healing.

[0026] 2. In unsanitary environments (e.g., wilderness, battlefield), without professional first aid knowledge and good hygiene conditions, achieving rapid and effective hemostasis remains a significant challenge. The present invention's method for preparing a composite spray to improve hemostasis at wound sites is based on the simple and easy-to-use form of a hemostatic spray. It can achieve rapid and deep hemostasis at the wound site, adapting to irregular wound shapes. By recruiting and activating platelets and counteracting fibrinogen consumption, it improves primary and secondary hemostasis and slows down clot degradation. While achieving rapid and effective hemostasis, it also reduces the adverse effects of blood loss on the body, amplifies its own coagulation cascade reaction, and continuously enhances the hemostatic ability at the wound site.

[0027] 3. The composite spray of the present invention, which improves the hemostatic ability of the wound site, successfully achieves rapid gelation at the wound site, providing a physical barrier for the wound, accelerating tissue repair and promoting wound healing.

[0028] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0029] Figure 1 The image shows the MALDI-TOF diagram of the PCP-lipopeptide conjugate in Example 1-1.

[0030] Figure 2 The image shows the MALDI-TOF diagram of the PAP-lipopeptide conjugate in Example 2-1.

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

[0032] Figure 4 The particle size distribution of the precursor solution prepared in Example 3-1 is shown.

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

[0034] Figure 6 The coagulation parameters of the precursor solution prepared in Example 3-1 are shown in the diagram.

[0035] Figure 7 The image shows the gelation process after spraying of the composite spray prepared in Example 3-1.

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

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

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

[0039] Figure 11 This is a schematic diagram of the CCK8 cytotoxicity analysis results of the composite spray prepared in Example 3-1.

[0040] Figure 12 This is a schematic diagram of the in vitro coagulation of the composite spray prepared in Example 3-1.

[0041] Figure 13 This is a schematic diagram of liver hemostasis using the composite spray prepared in Example 3-1. Detailed Implementation

[0042] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention are described in detail below with reference to examples. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

[0043] Example 1-1

[0044] This embodiment provides a method for preparing PCP-lipopeptide conjugates, specifically including:

[0045] Step 101: Place 20 mg of distearylphosphatidylethanolamine-polyethylene glycol-carboxyl (DSPE-mPEG2000-COOH) in 20 mL of dimethyl sulfoxide (DMSO) solution, stir and react for 2 h at room temperature in the dark to obtain system A. The molecular weight of the distearylphosphatidylethanolamine-polyethylene glycol-carboxyl is 2000.

[0046] Step 102: Place 10 mg of PCP peptide in 20 mL of deionized water, sonicate until homogeneous, and stir at room temperature in the dark for 0.5 h to obtain system B. The molecular weight of PCP is 669, and the PCP peptide sequence is GGQQLK, see SEQ ID NO:2.

[0047] Step 103: Add system B to system A, with a volume ratio of 1:1. After mixing, adjust the pH to 6-8 to obtain system C.

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

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

[0050] Figure 1 The image shows the MALDI-TOF plot of the PCP-lipopeptide conjugate from Example 1-1. According to... Figure 1 It can be seen that the highest peak appears at a mass-to-charge ratio of 2757, indicating that the distearate phosphatidylethanolamine-polyethylene glycol-carboxyl group is linked to the amino group in the PCP peptide, and the PCP-lipopeptide conjugate grafting was successful.

[0051] Examples 1-2

[0052] This embodiment provides a method for preparing PCP-lipopeptide conjugates, specifically including:

[0053] Step 101: Place 20 mg of distearylphosphatidylethanolamine-polyethylene glycol-carboxyl group in 10 mL of dimethyl sulfoxide solution, stir and react for 4 h at room temperature in the dark to obtain system A. The molecular weight of the distearylphosphatidylethanolamine-polyethylene glycol-carboxyl group is 2000.

[0054] Step 102: Place 8 mg of PCP peptide in 8 mL of deionized water, sonicate until homogeneous, and stir at room temperature in the dark for 0.8 h to obtain system B. The molecular weight of PCP is 669 and the sequence of PCP peptide is GGQQLK.

[0055] Step 103: Add system B to system A, with a volume ratio of system A to system B of 2:1. After mixing, adjust the pH to 6-8 to obtain system C.

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

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

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

[0059] Examples 1-3

[0060] This embodiment provides a method for preparing PCP-lipopeptide conjugates, specifically including:

[0061] Step 101: Place 20 mg of distearylphosphatidylethanolamine-polyethylene glycol-carboxyl group in 60 mL of dimethyl sulfoxide solution, stir and react for 1 h at room temperature in the dark to obtain system A. The molecular weight of the distearylphosphatidylethanolamine-polyethylene glycol-carboxyl group is 2000.

[0062] Step 102: Place 8 mg of PCP peptide in 24 mL of deionized water, sonicate until homogeneous, and stir at room temperature in the dark for 1 h to obtain system B. The molecular weight of PCP is 669 and the sequence of PCP peptide is GGQQLK.

[0063] Step 103: Add system B to system A, with a volume ratio of 4:1. After mixing, adjust the pH to 6-8 to obtain system C.

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

[0065] Step 105: Dialyze the system D under ice bath conditions using a 1000 Da dialysis bag for 2 days, then freeze-dry it at -50°C for 3 days to obtain the PCP-lipopeptide conjugate.

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

[0067] Example 2-1

[0068] This embodiment provides a method for preparing PAP-lipopeptide conjugates, specifically including:

[0069] Step 101: Place 20 mg of distearylphosphatidylethanolamine-polyethylene glycol-carboxyl group in 10 mL of dimethyl sulfoxide solution, stir and react for 1 h at room temperature in the dark to obtain system A. The molecular weight of the distearylphosphatidylethanolamine-polyethylene glycol-carboxyl group is 2000.

[0070] Step 102: Place 10 mg of PAP peptide in 5 mL of deionized water, sonicate until homogeneous, and stir at room temperature in the dark for 0.5 h to obtain system B. The PAP molecular weight is 678 and the PAP sequence is GFOGER, see SEQ ID NO:1.

[0071] Step 103: Add system B to system A, with a volume ratio of 1:1. After mixing, adjust the pH to 6-8 to obtain system C.

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

[0073] Step 105: Dialyze the system D under ice bath conditions using a 1000 Da dialysis bag for 1 day, then freeze-dry it at -50℃ for 3 days to obtain the PAP-lipopeptide conjugate.

[0074] Figure 2 The image shows the MALDI-TOF plot of the PCP-lipopeptide conjugate from Example 2-1. According to... Figure 2 It can be seen that the highest peak appears at a mass-to-charge ratio of 2713, indicating that the distearate phosphatidylethanolamine-polyethylene glycol-carboxyl group is linked to the amino group in the PAP peptide, and the grafting of the PAP-lipopeptide conjugate is successful.

[0075] Example 2-2

[0076] This embodiment provides a method for preparing PAP-lipopeptide conjugates, specifically including:

[0077] Step 101: Place 20 mg of distearylphosphatidylethanolamine-polyethylene glycol-carboxyl group in 20 mL of dimethyl sulfoxide solution, stir and react at room temperature in the dark for 2 h to obtain system A. The molecular weight of the distearylphosphatidylethanolamine-polyethylene glycol-carboxyl group is 2000.

[0078] Step 102: Place 15 mg of PAP peptide in 10 mL of deionized water, sonicate until homogeneous, and stir for 1 h at room temperature in the dark to obtain system B. The PAP molecular weight is 678 and the PAP sequence is GFOGER.

[0079] Step 103: Add system B to system A, with a volume ratio of system A to system B of 2:1. After mixing, adjust the pH to 6-8 to obtain system C.

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

[0081] Step 105: Dialyze the system D under ice bath conditions using a 1000 Da dialysis bag for 2 days, then freeze-dry it at -50℃ for 3 days to obtain the PAP-lipopeptide conjugate.

[0082] The PAP-lipopeptide conjugate in this embodiment is the same as that in Example 2-1.

[0083] Example 2-3

[0084] This embodiment provides a method for preparing PAP-lipopeptide conjugates, specifically including:

[0085] Step 1: Place 20 mg of distearylphosphatidylethanolamine-polyethylene glycol-carboxyl group in 60 mL of dimethyl sulfoxide solution and stir for 4 h at room temperature in the dark to obtain system A. The molecular weight of the distearylphosphatidylethanolamine-polyethylene glycol-carboxyl group is 2000.

[0086] Step 2: Place 15 mg of PAP peptide in 30 mL of deionized water, sonicate until homogeneous, and stir at room temperature in the dark for 0.75 h to obtain system B. The PAP molecular weight is 678 and the PAP sequence is GFOGER.

[0087] Step 3: Add system B to system A, with a volume ratio of 4:1. After mixing, adjust the pH to 6-8 to obtain system C.

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

[0089] Step 5: Dialyze the system D under ice bath conditions using a 1000 Da dialysis bag for 2 days, then freeze-dry it at -50℃ for 3 days to obtain the PAP-lipopeptide conjugate.

[0090] The PAP-lipopeptide conjugate in this embodiment is the same as that in Example 2-1.

[0091] Example 3-1

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

[0093] Step 1: Dissolve 2 mg of PAP-lipopeptide conjugate in 2 mL of chloroform-methanol 1:1 mixed solution, and dissolve 2 mg of PCP-lipopeptide conjugate in 2 mL of chloroform-methanol 1:1 mixed solution. Vortex and sonicate the 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 distearylphosphatidylcholine (DSPC) in 20 mL of chloroform-methanol 1:1 mixed solution, and dissolve 5 mg of distearylphosphatidylserine (DSPS) in 4 mL of chloroform-methanol 1:1 mixed solution. Mix the three mixed solutions to obtain a chloroform-methanol mixed solution containing cholesterol, distearylphosphatidylcholine, and distearylphosphatidylserine. Add this solution to the mixed solution and stir at 300 r / min at room temperature in the dark for 0.5 h to obtain a mixed system.

[0094] Step 2: Transfer the mixture to a single-necked round-bottom flask and sonicate to mix. Under 35°C water bath conditions, evaporate the mixture under reduced pressure at 110 rpm to form a uniform thin film. After evaporating the organic reagents by vacuum drying at 25°C for 10 h, add 12.5 mL of deionized water for hydration. Sonicate the mixture with a probe (950W, 35%, 5 min, 2 s / 3 s on / off), centrifuge at 3000g for 8 min, and filter through a 0.22 μm filter membrane to obtain the precursor solution.

[0095] Step 3: Under stirring at 300 r / min, add 0.2 g of calcium chloride to 10 mL of the precursor solution and react in the dark for 10 min to obtain solution A, which is then placed in spray bottle A. Under stirring at 150 r / min, dissolve 0.2 g of sodium alginate in 10 mL of deionized water to obtain solution B, which is then placed in spray bottle B. Apply solutions A and B together to 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, specifically including:

[0098] Step 1: Dissolve 1.2 mg of PAP-lipopeptide conjugate in 1 mL of chloroform-methanol 1:1 mixed solution, and dissolve 1.2 mg of PCP-lipopeptide conjugate in 1 mL of chloroform-methanol 1:1 mixed solution. Vortex the two mixed solutions and sonicate to obtain a mixed solution. At room temperature, dissolve 1 mg of cholesterol in 5 mL of chloroform-methanol 1:1 mixed solution, dissolve 13 mg of distearylphosphatidylcholine (DSPC) in 10 mL of chloroform-methanol 1:1 mixed solution, and dissolve 2 mg of distearylphosphatidylserine (DSPS) in 5 mL of chloroform-methanol 1:1 mixed solution. Mix the three mixed solutions to obtain a chloroform-methanol mixed solution containing cholesterol, distearylphosphatidylcholine, and distearylphosphatidylserine. Then add this solution to the mixed solution and stir at 300 r / min at room temperature in the dark for 1 h to obtain a mixed system.

[0099] Step 2: Transfer the mixture to a single-necked round-bottom flask and sonicate to mix. Under 40°C water bath conditions, evaporate the mixture under reduced pressure at 140 rpm to form a uniform thin film. After vacuum drying at 30°C for 12 h to evaporate the organic reagents, add 13 mL of deionized water for hydration. Sonicate the mixture with a probe (950W, 30%, 10 min, 2 s / 3 s on / off), centrifuge at 3000g for 5 min, and filter through a 0.22 μm filter membrane to obtain the precursor solution.

[0100] Step 3: Under stirring at 200 r / min, add 0.1 g of calcium chloride to 10 mL of the precursor solution and react in the dark for 20 min to obtain solution A, which is then placed in spray bottle A. Under stirring at 200 r / min, dissolve 0.1 g of sodium alginate in 10 mL of deionized water to obtain solution B, which is then placed in spray bottle B. Spray both solution A and solution B onto the wound site to form a composite spray that improves hemostasis at the wound site.

[0101] Example 3-3

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

[0103] Step 1: Dissolve 7.5 mg of PAP-lipopeptide conjugate in 9 mL of chloroform-methanol 2:1 mixed solution, and dissolve 5 mg of PCP-lipopeptide conjugate in 6 mL of chloroform-methanol 2:1 mixed solution. Vortex and sonicate the two mixed solutions to obtain a mixed solution. At room temperature, dissolve 4.3 mg of cholesterol in 5 mL of chloroform-methanol 2:1 mixed solution, dissolve 60 mg of distearylphosphatidylcholine (DSPC) in 50 mL of chloroform-methanol 2:1 mixed solution, and dissolve 7.5 mg of distearylphosphatidylserine (DSPS) in 5 mL of chloroform-methanol 2:1 mixed solution. Mix the three mixed solutions to obtain a chloroform-methanol mixed solution containing cholesterol, distearylphosphatidylcholine, and distearylphosphatidylserine. Add this solution to the mixed solution and stir at 400 r / min at room temperature in the dark for 0.75 h to obtain a mixed system.

[0104] Step 2: Transfer the mixture to a single-necked round-bottom flask and sonicate to mix. Under 25°C water bath conditions, evaporate the mixture under reduced pressure at 90 rpm to form a uniform thin film. After evaporating the organic reagents by vacuum drying at 40°C for 6 hours, add 40 mL of deionized water for hydration. Sonicate the mixture with a probe (950W, 40%, 8 min, 2 s / 3 s on / off). Centrifuge rapidly at 3000g for 10 min. Filter the mixture through a 0.22 μm filter membrane to obtain the precursor solution.

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

[0106] Performance evaluation:

[0107] Figure 3 The image shows a TEM image of the precursor solution prepared in Example 3-1. The precursor solution TEM sample was prepared using a negative staining method. The precursor solution was dropped onto a copper grid specifically designed for TEM, allowed to stand, and then immediately phosphotungstic acid solution was added. After drying, the sample was observed and photographed using a TEM. The TEM analysis results are shown in the figure. Due to the negative staining, the precursor solution appears as a white area, while the background is darker. The image shows that the prepared precursor solution has an approximately circular shape, an intact surface, and no damage. This indicates the successful synthesis of the precursor solution.

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

[0109] Figure 5 This is a SEM image showing the binding of the precursor solution prepared in Example 3-1 to natural platelets. After incubating the natural platelets with the precursor solution, the interaction between the 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 high-affinity peptides enhances its binding with platelets, and the presence of natural platelets with the precursor solution bound to the surface was confirmed by SEM.

[0110] Figure 6 The figure shows the coagulation parameters of the precursor solution prepared in Example 3-1. The precursor solutions were divided into three groups based on different modifications: DSPS, PCP, PAP, and the final precursor solution, APNs. The APTT, PT, TT, and FIB values ​​of the modified precursor solutions were observed. All experimental groups showed a shortened APTT value (activated partial thromboplastin time), indicating successful activation of the intrinsic coagulation pathway. There was no significant change in PT value (prothrombin time) between the experimental groups and the control group, reflecting that the extrinsic coagulation system was not activated. The TT value (activated partial thromboplastin time) was shortened in all experimental groups, indicating a shortened time for fibrinogen to convert to fibrin across all experimental groups. Fibrinogen (FIB) is an important protein involved in coagulation and hemostasis. The figure shows that the FIB content in the group with introduced PCP peptide was higher than in other groups, demonstrating that the cross-linking of platelets and fibrin strengthens the hemostatic barrier and reduces FIB consumption. Figure 6 This indicates that the precursor solution successfully activates the intrinsic coagulation pathway and counteracts fibrinogen depletion.

[0111] Figure 7 This is a schematic diagram of the gelation process after spraying the composite spray prepared in Example 3-1. When liquids A and B of the composite spray are sprayed simultaneously at the same location, they rapidly gel in situ, forming a gel state as shown in the figure. This demonstrates 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 was measured. The test method included immersing the pre-prepared colloid in PBS at 37°C, removing the surface liquid with filter paper at different time points, weighing and recording the weight changes, 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 As can be seen, the 4-hour 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 achieve blood absorption on the basis of rapidly forming a colloidal physical barrier, thus achieving the effect of biological-physical dual coagulation.

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

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

[0118] Where V is the volume of the composite gel and ρ is the density of anhydrous ethanol (0.789 g / cm³). The porosity of the composite spray group was measured to be 39.76% ± 1.04. The porosity of the composite gel helps it to rapidly absorb blood to aggregate blood cells and clotting factors, thereby accelerating hemostasis. In addition, it can effectively absorb wound exudate and reduce the possibility of wound infection.

[0119] Figure 10 The hemolysis rate of the composite spray prepared in Example 3-1 was determined. The test method included: extracting the colloid formed after pre-spraying with physiological saline at 37°C for 72 hours to obtain an extract of 0.1 g / mL. Fresh rat blood was used to separate red blood cells. 5 mL of fresh blood was centrifuged at 2000 rpm for 5 min, and the red blood cells were washed with physiological saline until the supernatant was clear and transparent. This supernatant was then diluted to a 2% (v / v) red blood cell suspension. The extract and red blood cell suspension were mixed in equal proportions in a 1.5 mL EP tube and incubated at 37°C for 1 h. After centrifugation, the absorbance of the supernatant was measured at 545 nm. Triton X-100 and physiological saline were used as positive and negative controls, respectively, followed by quantitative evaluation. The results are as follows: Figure 10 As shown, the hemolysis rate of the composite spray of the present invention is less than 5%, which is considered to be without hemolysis. The results indicate that the composite spray of the present invention does not cause red blood cell rupture and has good blood compatibility.

[0120] Figure 11This is a schematic diagram of the CCK8 assay results for the cytotoxicity of the composite spray prepared in Example 3-1. The test method included: extracting the colloid in RPMI-1640 medium at 37°C for 3 days to obtain an extract with a concentration of 0.1 g / mL. After normal culture for 24 hours, the culture medium for human umbilical vein endothelial cells (HUVEC cells) was replaced with the extracted solution from RPMI-1640 medium. Cells were cultured for 24 hours and 48 hours, respectively. The absorbance was measured using the CCK8 assay, and the survival rate of HUVEC cells was calculated. Each test was repeated in six parallel groups. The results are shown below. Figure 11 As shown. According to Figure 11 As shown, the cell survival rate was over 90% after 24 hours and 48 hours, indicating that the composite spray of the present invention has good biocompatibility and is safe and non-toxic.

[0121] Figure 12 The diagram shows the in vitro coagulation of the composite spray prepared in Example 3-1. The test method includes: placing fresh whole blood in a petri dish, spraying the composite spray of the present invention onto the area where the blood is located, and observing that after the spray forms a gel, the blood is quickly coagulated in situ, indicating that the composite spray of the present invention has a rapid coagulation effect.

[0122] Figure 13 This is a schematic diagram illustrating liver hemostasis using the composite spray prepared in Example 3-1. After anesthetizing the rat, it was fixed on the operating table. The liver was exposed through an abdominal incision, and the serous fluid around the liver was gently removed with gauze. A 0.5 cm long (0.5 cm deep) wound was made on the rat's liver using a scalpel (23# blade). The composite spray of this invention was quickly sprayed onto the bleeding site. Filter paper was placed under the liver, and the bleeding was observed until it stopped. Small areas of blood were observed on the surface of the filter paper under the liver, indicating that the composite spray of this invention has a good hemostatic effect.

[0123] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the present invention. Any simple modifications, alterations, or equivalent structural changes made to the above embodiments based on the technical essence of the invention shall still fall within the protection scope of the present invention.

Claims

1. A method for preparing a composite spray that improves hemostasis at the wound site, the composite spray comprising liquid A and liquid B, characterized in that, Solution B is an aqueous solution of sodium alginate, and the preparation method of solution A includes the following steps: Step 1: Dissolve the lipopeptide conjugate, cholesterol, distearylphosphatidylcholine, distearylphosphatidylserine, and dimethyl sulfoxide in a chloroform-methanol mixed solution to obtain a mixed system; the lipopeptide conjugate includes PAP-lipopeptide conjugate and / or PCP-lipopeptide conjugate; the PAP-lipopeptide conjugate is a lipopeptide conjugate grafted with PAP polypeptide, and the PCP-lipopeptide conjugate is a lipopeptide conjugate grafted with PCP polypeptide; the amino acid sequence of the PAP polypeptide is GFOGER, and the molecular weight is 678; the sequence of the PCP polypeptide is GGQQLK, and the molecular weight is 669; Step 2: Prepare a precursor solution from the mixture described in Step 1 using a thin-film hydration method; Step 3: Add calcium chloride to the precursor solution described in Step 2, and react in the dark to obtain solution A.

2. The method for preparing a composite spray for improving hemostasis at the wound site according to claim 1, characterized in that, The preparation method of the lipopeptide conjugate includes the following steps: Step 101: Place the distearate phosphatidylethanolamine-polyethylene glycol-carboxyl group in a dimethyl sulfoxide solution and stir to react, to obtain system A; Step 102: Place the PAP peptide or PCP peptide in deionized water, sonicate until homogeneous, and then stir to react, to obtain system B; Step 103: Add system B to system A, mix, and adjust the pH to 6-8 to obtain system C; Step 104: Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to system C, stir and react to obtain system D; Step 105: Dialyze the system D and then freeze-dry it to obtain the corresponding lipopeptide conjugate.

3. The method for preparing a composite spray for improving hemostasis at the wound site according to claim 2, characterized in that, The molecular weight of the distearylphosphatidylethanolamine-polyethylene glycol-carboxyl group in step 101 is 2000, and the volume of the dimethyl sulfoxide is 0.5 to 3 times the mass of the distearylphosphatidylethanolamine-polyethylene glycol-carboxyl group, wherein the volume unit is mL and the mass unit is mg; the stirring reaction in step 101 is carried out at room temperature in the dark for 1 to 4 hours.

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

1.

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

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

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

1.

8. The method for preparing a composite spray for improving hemostasis at the wound site according to claim 1, characterized in that, The preparation method of the precursor solution in step two includes: placing the mixed system in a single-necked round-bottom flask and ultrasonically mixing it; rotary evaporating it under reduced pressure in a water bath to form a uniform thin film; vacuum drying to evaporate the organic reagents; adding deionized water for hydration; ultrasonication with a probe; and centrifugation and filtration to obtain the precursor solution; the temperature of the rotary evaporation under reduced pressure is 25℃~40℃, and the rotation speed is 90rpm~140rpm; the temperature of the vacuum drying is 25℃~40℃, and the time is 6h~12h; the volume of deionized water added is 0.5~1 times the mass of distearylphosphatidylcholine, where the volume of deionized water is in mL and the mass of distearylphosphatidylcholine is in mg; the ultrasonication conditions are 950W, 30%~45% power, for 5min~10min; and the centrifugation conditions are 3000g centrifugation for 5min~10min.

9. A method for preparing a composite spray for improving hemostasis at the wound site according to claim 1, characterized in that, In step three, 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 solution A. The mass ratio of calcium chloride to the volume of the precursor solution is (1~3):100, where the mass unit is g and the volume unit is mL. The mass ratio of sodium alginate to deionized water in the sodium alginate aqueous solution is (1~3):100, where the mass unit is g and the volume unit is mL.

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