Anticoagulant coating based on novel tick-derived anticoagulant protein Qinghaenin and preparation method thereof

Abstract

The invention relates to the technical field of coating surface preparation, in particular to an anticoagulant coating based on novel tick-derived anticoagulant protein Qinghaenin and a preparation method of the anticoagulant coating. The functional layer of the anticoagulant coating comprises the following components: anticoagulant protein Qinghaenin or a functional peptide fragment Qinghaenin 167-192 of the anticoagulant protein Qinghaenin, wherein the amino acid sequence of the functional peptide fragment is as shown in SEQ ID NO. 2. The preparation method of the anticoagulant coating comprises the following steps: by using a polydopamine coating as a substrate layer, covalently grafting through an amido bond, immobilizing anticoagulant protein Qinghaenin capable of inhibiting FXII factor activation or a functional peptide fragment Qinghaenin 167-192 thereof on the surface of a medical polymer material, and successfully constructing a novel anticoagulant coating. Compared with an unmodified medical polymer material, the modified coating can significantly enhance the hydrophilic performance of the material and inhibit platelet adhesion and activation, finally effectively prolongs the in-vitro blood coagulation time, and shows excellent anticoagulation performance.

Description

Technical Field

[0001] This invention relates to the field of coating surface preparation technology, specifically to an anticoagulant coating based on a novel tick-derived anticoagulant protein Qinghaienin and its preparation method. Background Technology

[0002] In clinical medical practice, biomaterials that come into contact with blood, such as cannulas, artificial valves, vascular grafts, stents, central venous catheters, dialyzers, and blood collection tubes, are frequently used. Upon contact with blood, these biomaterials undergo a series of complex physiological reactions, including protein adsorption, activation and aggregation of blood cells, activation of the intrinsic coagulation pathway, and activation of complement, ultimately leading to thrombosis. Despite continuous efforts to improve the blood compatibility of these materials, thrombosis and thrombosis-related complications continue to hinder the long-term use of these blood-contact devices.

[0003] Currently, the most commonly used biomedical anticoagulant coating is heparin coating. However, the biological activity of heparin coating decreases after it is fixed on the surface of polymer materials, and it can also reduce platelet activity, leading to complications such as abnormal bleeding. Qinghaienin is derived from the Qinghai haematomarginatus, a species unique to my country. Haemaphysalis qinghaiensis A novel anticoagulant protein was discovered in vivo. Recombinant Qinghaienin prolongs plasma APTT in vitro by inhibiting FXII activation through competition for binding sites on the anion surface, thereby affecting the activation of the intrinsic coagulation pathway. Studies have shown that inhibiting the activation of key coagulation factors such as FXI, FXII, and prokallikrein in the intrinsic coagulation pathway can reduce thrombus formation while retaining some hemostatic potential to cope with bleeding in the event of vascular injury. Therefore, anticoagulants developed targeting coagulation factors in the intrinsic coagulation pathway have the advantage of minimizing bleeding complications. The full-length amino acid sequence of the anticoagulant protein Qinghaienin involved in this invention is disclosed in patent (ZL202310665559.5.), as shown in SEQ ID NO:1. Summary of the Invention

[0004] To overcome the shortcomings of the prior art, the present invention provides a novel active ingredient and preparation method for an anticoating coating.

[0005] The first aspect of this invention discloses a functional peptide segment of the anticoagulant protein Qinghaienin. 167-192 The amino acid sequence of the anticoagulant protein Qinghaienin is shown in SEQ ID NO: 1, and the amino acid sequence of the functional peptide is shown in SEQ ID NO: 2; the sequences correspond to amino acids 167 to 192 of the Qinghaienin protein.

[0006] The second aspect of the present invention discloses an anticoagulant coating on the surface of a biomaterial based on a novel tick-derived anticoagulant protein, Qinghaienin, wherein the active ingredient of the anticoagulant coating is selected from the complete anticoagulant protein Qinghaienin or the above-mentioned functional peptides.

[0007] The third aspect of this invention discloses a method for preparing the above-mentioned anticoagulant coating on the surface of biomaterials, wherein the active components of the anticoagulant coating are fixed to the material surface by covalent grafting, comprising the following steps: (1) Material surface pretreatment; (2) Preparation of the base layer; (3) Preparation of functional layers.

[0008] Preferably, the substrate is a medical polymer material; Base layer: Dopamine hydrochloride (DA) + polyethyleneimine (PEI); Functional layer: Full-length anticoagulant protein Qinghaienin / Functional peptide segment of anticoagulant protein Qinghaienin 167-192 .

[0009] As an embodiment of the present invention, the material surface pretreatment includes the following steps: ① Prepare material samples from medical polymer materials; ②The prepared material samples are completely immersed in ethanol for 4-6 hours, and then ultrasonically treated with deionized water for 30-50 minutes. ③ After cleaning, place the materials in an oven to dry for later use.

[0010] As an embodiment of the present invention, the substrate preparation includes the following steps: ① Dissolve 2 mg / mL dopamine hydrochloride (DA) and 0.5 mg / mL polyethyleneimine (PEI) in phosphate buffer solution (PBS) at pH = 8.5 to obtain the basal layer solution; ② Immerse the surface-pretreated material completely into the base layer solution; ③ Stir or shake slowly at 30 ℃ for 6 h to form the PDA-PEI substrate layer; ④ Sonicate in deionized water for 5 minutes and rinse to remove loosely bound dopamine hydrochloride and polyethyleneimine on the PDA-PEI substrate.

[0011] As an embodiment of the present invention, the preparation of the functional layer includes the following steps: ① Express and purify the anticoagulant protein Qinghaienin to obtain a protein sample; ②Qinghaienin, a chemically synthesized bioactive peptide167-192 The powder was dissolved in deionized water to obtain a functional peptide solution; ③ Dissolve 16 mg / mL 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and 12 mg / mL N-hydroxysuccinimide (NHS) in PBS at pH = 5.6 to obtain a solution for activating carboxyl groups; ④ Mix the protein samples and functional peptide solutions obtained in steps ① and ② with the solution obtained in step ③ at a volume ratio of 4:1 to obtain the corresponding functional layer solutions; ⑤ Immerse the base layer in the functional layer solution and react at 41°C for 12 hours; ⑥ After removal, wash the poorly bound bioactive substances on the substrate surface after step ⑤ with deionized water, and then air dry at room temperature to obtain the anticoagulant coating PDA-PEI / Qinghaienin. 167-192 .

[0012] The fourth aspect of this invention discloses the application of the above-mentioned anticoagulant protein Qinghaienin or functional peptides on the surface of medical polymer materials or medical devices.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. Compared to existing anticoagulant coatings, the anticoagulant coating based on the novel tick-derived anticoagulant protein Qinghaienin proposed in this invention exhibits significant advantages. Through the self-polymerization of polydopamine, the adhesion between the coating and the substrate material is greatly improved, ensuring the stability and durability of the coating. Simultaneously, Qinghaienin is an anticoagulant agent developed targeting coagulation factors in the intrinsic coagulation pathway, and its addition has the advantage of minimizing bleeding complications. Furthermore, the preparation method is simple, environmentally friendly, and easy to promote. In summary, the anticoagulant coating of this invention has broad application prospects in the biomedical field, providing strong support for improving the blood compatibility of medical devices.

[0014] 2. The anticoagulant coating of this invention uses Qinghaienin, a novel anticoagulant protein extracted from Qinghai ticks, and further proposes its functional peptide segment Qinghaienin. 167-192 Furthermore, its independent anticoagulant activity was verified, broadening the range of active ingredients to be selected.

[0015] 3. The embodiments of the present invention validate the functional peptide Qinghaienin. 167-192 The effect is close to that of full-length protein, indicating that peptides have the potential to replace full-length protein. Attached Figure Description

[0016] Figure 1This is a flowchart of the anti-coating coating preparation method in this invention (taking PDA-PEI / Qinghaienin as an example).

[0017] Figure 2 This is a schematic diagram of the coating structure during the preparation process of the anti-coating coating in this invention (taking PDA-PEI / Qinghaienin as an example).

[0018] Figure 3 This is a schematic diagram showing the results of water contact angles for different test samples in this invention.

[0019] Figure 4 This is a schematic diagram showing the results of hemolysis rates for different test samples in this invention.

[0020] Figure 5 This is a schematic diagram showing the results of platelet adhesion experiments on different test samples in this invention.

[0021] Figure 6 This is a schematic diagram showing the anticoagulant effect of different test samples in this invention. Detailed Implementation

[0022] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0023] Example 1

[0024] This embodiment is used to illustrate the preparation of the anticoagulant coating PDA-PEI / Qinghaienin. Figure 1 ).

[0025] (1) Material surface pretreatment: PVC material samples of uniform size are made, completely soaked in ethanol for 5 hours, and then ultrasonically treated with deionized water for 40 minutes. After cleaning, the PVC material samples are placed in an oven at 50°C for drying.

[0026] (2) Preparation of the base layer: Dissolve 2 mg / mL DA and 0.5 mg / mL PEI in PBS at pH = 8.5 to obtain the base layer solution. Then, completely immerse the pretreated PVC material in the base layer solution, and slowly stir or shake at 30 °C for 6 h. After that, sonicate with deionized water for 5 min and rinse to remove the loosely bound DA and PEI, and finally form the PDA-PEI base layer.

[0027] (3) Functional layer preparation: Recombinant expression and purification of the anticoagulant protein Qinghaienin were performed to obtain the protein Qinghaienin. 16 mg / mL EDC and 12 mg / mL NHS were dissolved in PBS at pH = 5.6, and then the protein Qinghaienin was mixed with them at a volume ratio of 4:1. Finally, the PVC material after preparing the PDA-PEI base layer was immersed in the mixed solution and reacted at 41℃ for 12 h. After removal, the surface of the poorly bound protein Qinghaienin was washed with deionized water, and then dried at room temperature to obtain the anticoagulant coating PDA-PEI / Qinghaienin ( Figure 2 ).

[0028] Example 2

[0029] This example illustrates the anticoagulant coating PDA-PEI / Qinghaienin. 167-192 Preparation of .

[0030] (1) Material surface pretreatment: PVC material samples of uniform size are made, completely soaked in ethanol for 5 hours, and then ultrasonically treated with deionized water for 40 minutes. After cleaning, the PVC material samples are placed in an oven at 50°C for drying.

[0031] (2) Preparation of the base layer: Dissolve 2 mg / mL DA and 0.5 mg / mL PEI in PBS at pH = 8.5 to obtain the base layer solution. Then, completely immerse the pretreated PVC material in the base layer solution, and slowly stir or shake at 30 °C for 6 h. After that, sonicate with deionized water for 5 min and rinse to remove the loosely bound DA and PEI, and finally form the PDA-PEI base layer.

[0032] (3) Preparation of functional layer: The chemically synthesized functional peptide Qinghaienin 167-192 The powder was dissolved in deionized water to obtain a functional peptide solution. 16 mg / mL EDC and 12 mg / mL NHS were dissolved in PBS at pH 5.6, and the functional peptide solution was then mixed with PBS at a volume ratio of 4:1. Finally, the PVC material with the prepared PDA-PEI base layer was immersed in the mixed solution and reacted at 41°C for 12 hours. After removal, the surface of the material was washed with deionized water to remove any loosely bound functional peptides, and then air-dried at room temperature to obtain the anticoagulant coating PDA-PEI / Qinghaienin. 167-192 .

[0033] The above embodiments of the present invention were measured and evaluated in the following manner.

[0034] 1. Hydrophilic properties Untreated PVC material, PVC material grafted with PDA-PEI primer, PVC material coated with PDA-PEI / Qinghaienin anti-gel coating (Example 1), and PVC material coated with PDA-PEI / Qinghaienin were tested at room temperature. 167-192 The contact angle of the PVC material with anti-condensation coating (Example 2) surface is shown in the following results. Figure 3 As shown, the water contact angle of the grafted functional layer is significantly reduced, indicating that the prepared anti-condensation coating improves the hydrophilicity of PVC materials.

[0035] 2. Hemolysis rate 8 mL of fresh human whole blood, anticoagulated with sodium citrate, was diluted with 10 mL of physiological saline. PVC material coated with a PDA-PEI / Qinghaienin anticoagulant coating (Example 1) and PDA-PEI / Qinghaienin-coated materials were then compared. 167-192 The PVC material with the anticoagulant coating (Example 2) was immersed in diluted blood and incubated at 37°C for 1 hour. After incubation, 9 mL of physiological saline was added to the blood, and the diluted blood was centrifuged at 2000 rpm for 5 minutes. The absorbance of the supernatant at 545 nm was measured using a UV spectrophotometer and recorded as X1. 9 mL of deionized water was added to 0.5 mL of diluted blood, and the mixture was centrifuged at 2000 rpm for 5 minutes. The supernatant was used as a positive reference, and its absorbance was measured at 545 nm and recorded as X2. 9 mL of physiological saline was added to 0.5 mL of diluted blood, and the mixture was centrifuged at 2000 rpm for 5 minutes. The supernatant was used as a negative reference, and its absorbance was measured at 545 nm and recorded as X3. The hemolysis rate (%) was calculated as follows: (X1-X3) / (X2-X3) × 100%. The results are as follows: Figure 4 As shown, the hemolysis rate of each embodiment is less than 1%, which does not cause hemolysis and has good blood compatibility.

[0036] 3. Platelet adhesion test Fresh human whole blood, anticoagulated with sodium citrate, was centrifuged at 1500 rpm for 10 minutes to obtain platelet-rich plasma (PRP). Untreated PVC material, PVC material grafted with PDA-PEI primer, PVC material coated with a PDA-PEI / Qinghaienin anticoagulant coating (Example 1), and PVC material coated with PDA-PEI / Qinghaienin... 167-192The PVC material with the anti-coating coating (Example 2) was incubated with 500 μL PRP at 37°C for 1 h. After rinsing with PBS at pH 7.4, the sample was fixed in 2.5% glutaraldehyde solution for 12 h. After dehydration in aqueous ethanol solution (40, 50, 60, 70, 80, 90, and 100% v / v) for 30 min, followed by isoamyl acetate replacement, critical point drying, and ion sputtering coating, the morphology of the samples was observed using a scanning electron microscope (GeminiSEM 300). The results are as follows: Figure 5 As shown, Examples 1 and 2 can inhibit the adhesion and activation of platelets by PVC materials to varying degrees, and the effect of inhibiting platelet adhesion and activation is relatively good.

[0037] 4. Anti-coagulation performance Fresh anticoagulated human whole blood was mixed with CaCl2 solution (final concentration 5mM) and immediately then mixed with untreated PVC material, PVC material grafted with PDA-PEI primer, PVC material coated with PDA-PEI / Qinghaienin anticoagulant coating (Example 1), and PVC material coated with PDA-PEI / Qinghaienin. 167-192 The PVC material with an anticoagulant coating (Example 2) was immersed to ensure full contact between the sample and blood, and allowed to stand for 1 hour. The sample was then slowly and vertically removed, rinsed in deionized water for 20 seconds, and the adhesion of thrombi to the sample surface was observed and photographed. Results are as follows: Figure 6 As shown, Examples 1 and 2 can prevent thrombus formation and adhesion on the surface of PVC materials to varying degrees, and have significant anticoagulant effects.

[0038] This invention utilizes a polydopamine coating as a functionalized substrate and covalently grafts Qinghaienin, an anticoagulant protein that inhibits factor FXII activation, or its synthetic peptide, onto the surface of a medical polymer material, successfully constructing a novel anticoagulant coating. Compared to the unmodified material, the modified coating significantly improves the surface hydrophilicity, inhibits platelet adhesion and activation, and effectively prolongs in vitro blood clotting time, exhibiting excellent anticoagulant properties.

[0039] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A functional peptide segment of the anticoagulant protein Qinghaienin. 167-192 The amino acid sequence of the anticoagulant protein Qinghaienin is shown in SEQ ID NO: 1, characterized in that, The amino acid sequence of the functional peptide is shown in SEQ ID NO:

2.

2. An anticoagulant coating on the surface of a biomaterial based on the tick-derived anticoagulant protein Qinghaienin, characterized in that, The active ingredient of the anticoagulant coating is selected from the complete anticoagulant protein Qinghaienin or the functional peptides of claim 1.

3. The method for preparing the anti-condensation coating according to claim 2, characterized in that, The active components of the anti-coating coating are fixed to the material surface via covalent grafting, including the following steps: (1) Material surface pretreatment; (2) Preparation of the base layer; (3) Preparation of functional layers.

4. The method for preparing the anti-condensation coating according to claim 3, characterized in that, The material surface pretreatment includes the following steps: ① Prepare material samples from medical polymer materials; ②The prepared material samples are completely immersed in ethanol for 4-6 hours, and then ultrasonically treated with deionized water for 30-50 minutes. ③ After cleaning, place the materials in an oven to dry for later use.

5. The method for preparing the anti-condensation coating according to claim 3, characterized in that, The preparation of the substrate layer includes the following steps: ① Dissolve 2 mg / mL dopamine hydrochloride and 0.5 mg / mL polyethyleneimine in a phosphate buffer solution with pH = 8.5 to obtain a base layer solution; ② Immerse the surface-pretreated material completely into the base layer solution; ③ Stir or shake slowly at 30 ℃ for 6 h to form the PDA-PEI substrate layer; ④ Sonicate in deionized water for 5 minutes and rinse to remove loosely bound dopamine hydrochloride and polyethyleneimine on the PDA-PEI substrate.

6. The method for preparing the anti-condensation coating according to claim 3, characterized in that, The preparation of the functional layer includes the following steps: ① Express and purify the anticoagulant protein Qinghaienin to obtain a protein sample; ②Qinghaienin, a chemically synthesized bioactive peptide 167-192 The powder was dissolved in deionized water to obtain a functional peptide solution; ③ Dissolve 16 mg / mL 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 12 mg / mL N-hydroxysuccinimide in PBS at pH = 5.6 to obtain a solution for activating carboxyl groups; ④ Mix the protein samples and functional peptide solutions obtained in steps ① and ② with the solution obtained in step ③ at a volume ratio of 4:1 to obtain the corresponding functional layer solutions; ⑤ Immerse the base layer in the functional layer solution and react at 41°C for 12 hours; ⑥ After removal, wash the poorly bound bioactive substances on the substrate surface after step ⑤ with deionized water, and then air dry at room temperature to obtain the anticoagulant coating PDA-PEI / Qinghaienin. 167-192 .

7. The application of the anticoagulant protein Qinghaienin or functional peptide as described in claim 1 on the surface of medical polymer materials or medical devices.

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