Injectable polypeptide hemostatic material based on co-targeting of endothelial cells and platelets

Abstract

The invention discloses an injectable polypeptide hemostatic material that forms a thrombus at a wound and blocks the wound by targeting wound endothelial cells and platelets, comprising the following steps: (1) synthesizing endothelial cell binding peptide and platelet binding peptide sequences respectively; (2) adding a carboxyl activator and a stabilizer to an alginate solution, and stirring at room temperature for 1 hour; (3) adding the endothelial cell binding peptide and the platelet binding peptide to the alginate solution in step (2), stirring for 1 hour, and then standing overnight; (4) the polymer obtained in step (3) can be placed in a dialysis bag for 48 hours. The endothelial cell binding peptide and the platelet binding peptide can effectively enrich the activated platelets at the wound to the vicinity of the exposed endothelial cells, forming a thrombus to block the wound. The material has low cost, simple preparation method, easy operation, easy large-scale production and storage; the material has excellent biocompatibility, is degradable, non-toxic and non-immunogenic, and has high hemostatic efficiency; the injectable material breaks through the limitations of traditional materials and has outstanding ability to deal with complex wounds.

Description

Technical Field

[0001] The invention relates to an injectable polypeptide hemostatic material which can form a thrombus at a wound and block the wound by targeting wound endothelial cells and platelets. Background Art

[0002] In the military, hospitals and other emergency situations, about 40% of traumatic deaths are caused by uncontrolled bleeding. Due to the high time sensitivity of traumatic bleeding, the control of bleeding caused by trauma is challenging, and achieving rapid and thorough hemostasis within the first few hours is essential to ensure the survival of patients with massive bleeding. At present, injectable hemostatic materials, such as patent CN202210938659, disclose a foam hemostatic material, a hemostatic injection, and its preparation method and application. At present, there are few injectable materials, and the existing ones are injected into the wound by foam, using the multi-parallel channels of the material to quickly absorb blood, promote the aggregation of blood cells and platelets, and then achieve the purpose of hemostasis. At present, peptide hemostatic materials, such as patent CN201710284205, disclose a method for preparing a controllable degradable RGD phage / silk fibroin composite rapid hemostatic material; patent CN201310248965 discloses a method for preparing a silk fibroin peptide powder hemostatic material. These materials attract negatively charged platelets and red blood cells in the blood through charge action, increase platelet adhesion and promote thrombosis. Compared with the above patents, endothelial cell binding peptides and platelet binding peptides are materials that the body itself possesses, and their good biocompatibility is unquestionable. They are biodegradable and can locally amplify the aggregation of activated platelets on the endothelial cells of damaged blood vessels, which can quickly achieve thrombus formation and improve hemostasis efficiency. Summary of the invention

[0003] The purpose of the present invention is to construct an injectable polypeptide hemostatic material, which is obtained by using alginate as a carrier and connecting endothelial cell binding peptide and platelet binding peptide to the carboxyl group of alginate through covalent bonds. The present invention provides a hemostatic method that can be injected at the site of damaged blood vessels.

[0004] In order to realize the above technical solution, the injectable polypeptide hemostatic material based on endothelial cell and platelet co-targeting according to the present invention specifically comprises the following steps:

[0005] (1) synthesizing endothelial cell binding peptide and platelet binding peptide sequences respectively;

[0006] (2) adding a carboxyl activator and a stabilizer to the alginate solution and stirring at room temperature for 1 h;

[0007] (3) adding the endothelial cell binding peptide and the platelet binding peptide to the alginate solution in step (2), stirring for 1 hour, and then standing overnight;

[0008] (4) The polymer obtained in step (3) can be placed in a dialysis bag and dialyzed for 48 hours.

[0009] The endothelial cell binding peptide sequence involved in the present invention is SSTSYNRGDSTFESK (Seq_1), and the platelet binding peptide sequence is HHLGGAKQAGDV (Seq_2).

[0010] In the step (2) of the present invention, the carboxyl activating and stabilizing agents are 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide, and the stirring time is 30-60 minutes.

[0011] The concentration of the endothelial cell binding peptide and the platelet binding peptide in step (3) of the present invention is 5 mg / mL, 10 mg / mL or 15 mg / mL.

[0012] The molecular weight of the dialysis bag in step (4) of the present invention is 50-100 KDa.

[0013] Compared with the prior art, the present invention has the following advantages: (1) low material cost, simple preparation method, easy operation, and easy large-scale production and storage; (2) excellent material biocompatibility, degradability, non-toxicity and non-immunogenicity; (3) two polypeptides can directly target platelets to endothelial cells, so that the formed thrombus is more stable and has high hemostasis efficiency; (4) the injectable material breaks through the limitations of traditional materials and has outstanding ability to deal with complex wounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Attached Figure 1 The molecular structure of the polypeptide of Example 1 of the present invention;

[0015] Attached Figure 2 The degree of polypeptide modification on alginate in Example 1 of the present invention;

[0016] Attached Figure 3 This is a schematic diagram of the polymer hemostasis principle of Example 1 of the present invention;

[0017] Attached Figure 4 The coagulation test results of Example 1 of the present invention;

[0018] Attached Figure 5 This is the cytotoxicity result of Example 1 of the present invention.

[0019] Specific embodiments of the invention

[0020] The present invention will be further described below through specific embodiments and accompanying drawings.

[0021] Embodiment 1:

[0022] (1) Synthesis of endothelial cell-binding peptide and platelet-binding peptide

[0023] The Fmoc solid phase synthesis method is used to swell the resin, then the first amino acid is attached and then deprotected, and then the color reaction is detected and then washed and condensed, and then the color detection is performed again. After it is colorless, it is washed and purified, and finally the sample is freeze-dried for use. Its molecular structure is shown in the attached Figure 1 .

[0024] (2) Activation and stabilization of alginate carboxyl groups

[0025] Dissolve 6 mg of alginate in 1 mL of ultrapure water and stir at room temperature for 1 h. After the alginate is completely dissolved, add 13 uL of 2 mg / mL 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and 3 mg / mL N-hydroxysuccinimide, and stir at room temperature for 1 h.

[0026] (3) Alginate and peptide grafting

[0027] 200uL of 5mg / mL endothelial cell binding peptide and platelet binding peptide were added to the carboxyl activated alginate, stirred at room temperature for 1h, and then placed at room temperature overnight after being evenly dispersed. The degree of peptide modification on alginate was verified by NMR. The results are shown in the attached figure. Figure 2 shown.

[0028] (4) Purification of alginate-peptide polymers

[0029] The alginate-peptide polymer synthesized overnight was placed in a dialysis bag with a molecular weight of 50KDa and dialyzed with pure water for 48 hours, with the dialysate replaced every 8 hours. After purification, it was stored at 4°C. The schematic diagram of the polymer hemostasis principle is shown in the attached figure. Figure 3 Shown

[0030] (5) Coagulation test

[0031] In vitro coagulation: 200uL of purified polymer was mixed with 800uL of antagonized blood, incubated at 37°C, and the blood coagulation time was measured.

[0032] In vivo experiment: 200uL of purified polymer was injected into a wound with a length × width of 0.5*0.5cm. The time when the wound stopped bleeding was used as the coagulation index and the coagulation time was recorded. The liver of SD rats was used as the in vivo sample. The coagulation experiment results are shown in the attached figure. Figure 4 shown.

[0033] (6) Cell experiments

[0034] NIH-3T3 cells were cultured with Dulbecco's modified Eagle's medium (supplemented with 10% bovine serum) at 37°C in a 5% CO2 humidity incubator. The cell culture medium pre-incubated with the injectable material overnight was used to culture NIH-3T3 cells for 24 hours. The cells were fluorescently photographed by the live-dead double staining method to record the cell status. The results are shown in the attached figure. Figure 5 shown.

[0035] Embodiment 2:

[0036] This example is the same as Example 1 except that the polypeptide concentration in step (3) is 10 mg / mL; the dialysis bag in step (4) is 80 KDa, and the other steps are the same.

[0037] Embodiment 3:

[0038] This example is the same as Example 1 except that the polypeptide concentration in step (3) is 15 mg / mL; the dialysis bag in step (4) is 100 KDa, and the other steps are the same.

[0039] The test shows that the injectable polypeptide hemostatic materials prepared in Examples 2 and 3 are similar to those in Example 1 except for the differences in liquid viscosity and hemostatic effect.

Claims

1. An injectable polypeptide hemostatic material based on co-targeting of endothelial cells and platelets, characterized in that: The specific steps include: (1) synthesizing an endothelial cell binding peptide and a platelet binding peptide respectively, wherein the endothelial cell binding peptide sequence is SSTSYNRGDSTFESK and the platelet binding peptide sequence is HHLGGAKQAGDV; (2) adding a carboxyl activator and a stabilizer to the alginate solution and stirring at room temperature for 1 h; (3) adding the endothelial cell binding peptide and the platelet binding peptide to the alginate solution in step (2), stirring for 1 hour, and then standing overnight; (4) The polymer obtained in step (3) was placed in a dialysis bag and dialyzed for 48 hours.

2. The injectable polypeptide hemostatic material based on endothelial cell and platelet co-targeting according to claim 1, characterized in that: In step (2), the carboxyl activator and stabilizer are 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, and the stirring time is 1 hour.

3. The injectable polypeptide hemostatic material based on endothelial cell and platelet co-targeting according to claim 2, characterized in that: The concentrations of the endothelial cell binding peptide and the platelet binding peptide in step (3) are 5 mg / mL, 10 mg / mL or 15 mg / mL.

4. The injectable polypeptide hemostatic material based on endothelial cell and platelet co-targeting according to claim 3, characterized in that: The molecular weight of the dialysis bag in step (4) is 50-100 kDa.

5. The injectable polypeptide hemostatic material based on endothelial cell and platelet co-targeting according to any one of claims 1 to 4, characterized in that: An injectable polypeptide hemostatic material is constructed by using endothelial cell binding peptide as a target and platelet binding peptide to enrich activated platelets, which can effectively deal with complex wound types.

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