Copper ion mediated bivalirudin self-assembled nano-drug as well as preparation method and application thereof

Abstract

The invention discloses a copper ion mediated bivalirudin self-assembled nano-drug and a preparation method and application thereof, and belongs to the field of biological medicines.The preparation method comprises the following steps that bivalirudin and targeting peptide cRGD are dissolved in an organic solvent, copper salt is dissolved in deionized water, the organic solution and an aqueous solution are mixed, the pH is adjusted to be faintly acid or neutral, and the copper ion mediated bivalirudin self-assembled nano-drug is obtained; and fully stirring and reacting to obtain a suspension, centrifuging to remove supernate, and freeze-drying to obtain the freeze-dried powder. On the basis of a metal coordination polypeptide self-assembly strategy, metal ions are introduced on the basis of traditional polypeptide self-assembly, special reaction performance is introduced through coordination of polypeptide functional groups and the metal ions to participate in regulation and control of the polypeptide self-assembly process, and thrombus targeting peptide is introduced on the basis to achieve precise drug delivery at the thrombus part.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to a copper ion-mediated bivalirudin self-assembled nanodrug and a preparation method and application thereof. BACKGROUND

[0002] Bivalirudin (BIV) is a 20-peptide anticoagulant based on natural hirudin synthesized artificially, which was approved for marketing by FDA in 2000. As a short-acting direct thrombin inhibitor, it inhibits the coagulation process by reversibly combining with thrombin, thereby effectively preventing thrombus formation in a short time. However, its clinical application is limited by two major defects: (1) the 20-peptide structure is easily hydrolyzed by plasma proteases and rapidly filtered through the glomerulus (half-life about 25 min), resulting in short-term drug efficacy maintenance; (2) non-specific inhibition of thrombin may cause severe bleeding. These shortcomings seriously limit its application in antithrombotic therapy. Existing modification techniques such as PEGylation reduce drug activity, and carrier delivery systems have the defect of complex process. SUMMARY

[0003] To solve the above problems, the present application provides a bivalirudin self-assembled nanodrug based on metal coordination polypeptide self-assembly strategy and a preparation method and application thereof. Metal coordination polypeptide self-assembly is based on traditional polypeptide self-assembly and introduces metal ions such as copper, zinc, and iron ions. Through coordination of polypeptide functional groups and metal ions, special reaction performance is introduced to participate in the regulation of polypeptide self-assembly process, such as forming a core-shell nanostructure that can shield protease attack and delay kidney clearance. On this basis, thrombus-targeting peptides are introduced to achieve precise drug delivery at the thrombus site. This strategy is the first in the field of BIV modification and provides a new path to solve its clinical pain points.

[0004] The object of the present application is achieved by the following technical solution: A preparation method of a copper ion-mediated bivalirudin self-assembled nanodrug, comprising the following steps: (1) dissolving bivalirudin and targeting peptide cRGD in an organic solvent to obtain solution A, and dissolving copper salt in deionized water to obtain solution B; (2) mixing the solution A and the solution B, adjusting the pH to weakly acidic or neutral, fully stirring and reacting to obtain a suspension, removing the supernatant by centrifugation, and freeze-drying to obtain the product.

[0005] In some preferred embodiments, the molar ratio of bivalirudin to copper ions in the copper salt is 1: (2-6).

[0006] In some preferred embodiments, the mass ratio of bivalirudin to targeting peptide cRGD is 1: (0.3-3).

[0007] In some preferred embodiments, the organic solvent is methanol.

[0008] In some preferred embodiments, the volume ratio of the solution A to the solution B is 4:1.

[0009] In some preferred embodiments, the pH is 5-7.

[0010] In some preferred embodiments, the reaction temperature of the stirring reaction is 30-50℃, the stirring rate is 800-1000rpm, and the reaction time is 50-80min.

[0011] The second aspect of the present application is to provide a copper ion-mediated bivalirudin self-assembled nanodrug, which is prepared by the aforementioned preparation method.

[0012] The third aspect of the present application is to provide an application of the copper ion-mediated bivalirudin self-assembled nanodrug, in particular, in the preparation of a drug for treating anti-thrombosis.

[0013] The present application has the following advantages: (1) The present application uses a metal coordination polypeptide self-assembly method to synthesize bivalirudin nanodrug, which belongs to a nanodrug delivery system, has a convenient preparation method, high drug loading efficiency, and high biological safety.

[0014] (2) The introduction of cRGD targeting membrane penetrating peptide further improves the targeting effect of the nanodrug on the thrombus site.

[0015] (3) Compared with bivalirudin monomers, the bivalirudin self-assembled nanodrug can more effectively inhibit thrombus formation and has no toxic side effects on normal cells. (4) In vitro experiments, bivalirudin nanodrug can effectively inhibit thrombin activity, which is no different from monomer inhibition activity, and has good blood compatibility and no cytotoxicity, proving that the nanodrug has good anticoagulant effect and biocompatibility. (5) Mouse carotid thrombosis modeling experiments prove that bivalirudin nanodrug can effectively prolong the thrombus formation time, which greatly promotes the further optimization and clinical application of bivalirudin. BRIEF DESCRIPTION OF DRAWINGS

[0016] The present application is further illustrated by the accompanying drawings, but the embodiments in the drawings do not constitute any limitation on the present application. For ordinary skilled in the art, other drawings can be obtained without creative labor on the basis of the following drawings.

[0017] Figure 1 Different BIV and Cu 2+The amount of substance ratio of BIV@Cu synthesized by different pH values of the reaction; Figure 2 The thrombin inhibition rate of BIV@Cu@cRGD synthesized by different pH values of the reaction; Figure 3 The thrombin inhibition rate of BIV@Cu@cRGD synthesized by different molar ratios of cRGD; Figure 4 In a and b are FESEM images of the bivalirudin nanomedicine BIV@Cu@cRGD and EDS point scanning images of BIV@Cu@cRGD, respectively, and the percentage of each atom (C, N, O, Cu) of BIV@Cu@cRGD EDS; Figure 5 The particle size and surface charge of the bivalirudin nanomedicine BIV@Cu and BIV@Cu@cRGD; Figure 6 The XPS full spectrum (a) of the bivalirudin nanomedicine BIV@Cu@cRGD and its (b) C 1s, (c) N 1s, (d) O 1s and (e) Zn 2p binding energy spectrum; Figure 7 In a and b are the in vitro fluorescence signals in the artificial blood clots treated with BIV@Cu and BIV@Cu@cRGD synthesized by different molar ratios of cRGD, respectively, and the quantitative fluorescence intensity of each group of blood clots; Figure 8 The in vitro drug release curve of the bivalirudin nanomedicine BIV@Cu and BIV@Cu@cRGD; Figure 9 The survival rate of HUVEC cells co-incubated with the bivalirudin nanomedicine BIV@Cu and BIV@Cu@cRGD for 48 h; Figure 10 In a, b, c, d, respectively, represent the changes in blood flow perfusion in the right carotid artery of mice after administration of PBS, BIV@Cu, BIV, and BIV@Cu@cRGD; Figure 11 The tail bleeding time of the thrombus model mice in each treatment group; Figure 12 The 7-day weight changes of mice in each group after injection of drugs; Figure 13 H&E sections of the main organs (heart, liver, spleen, lung, kidney) of mice in each treatment group; Figure 14 In a and b, respectively, represent the liver function indicators: AST, serum glutathione transaminase; ALP, alkaline phosphatase; ALT, serum glutathione transaminase; and the kidney function indicators: UA, uric acid; CRE, creatinine; UREA, urea. DETAILED DESCRIPTION

[0018] The present application is further described in connection with the following examples.

[0019] Embodiments of the present application relate to a copper ion-mediated bivalirudin self-assembly nanodrug, and a preparation method thereof, comprising the following steps: (1) dissolving bivalirudin and targeting peptide cRGD in an organic solvent to obtain solution A, and dissolving a copper salt in deionized water to obtain solution B; (2) mixing the solution A and the solution B, adjusting the pH to weakly acidic or neutral, fully stirring to react, obtaining a suspension, removing the supernatant by centrifugation, and freeze-drying to obtain; The targeting peptide cRGD is a specially designed short peptide for targeting membrane penetration, which is a cyclic arginine-glycine-aspartic acid polypeptide sequence. It can specifically recognize and tightly bind to the integrin receptor on the cell surface, especially the integrin αvβ3 and αvβ5 highly expressed on the cell surface of tumor cells, neovascular endothelial cells and the like. It also has the ability to penetrate the cell membrane and carry the coupled substances into the cell interior; In some embodiments, the molar ratio of the bivalirudin to the copper ion in the copper salt is 1: (2-6), such as 1:2, 1:4 or 1:6; In some embodiments, the mass ratio of the bivalirudin to the targeting peptide cRGD is 1: (0.3-3), such as 3:1, 1:1, 1:2 or 1:3; In some embodiments, the organic solvent is methanol; In some embodiments, the copper salt is copper chloride; In some embodiments, the volume ratio of the solution A to the solution B is 4:1; In some embodiments, the pH is 5-7, such as 5, 6 or 7; In some embodiments, the reaction temperature of the stirring reaction is 30-50℃, such as 30℃, 40℃, 50℃, the stirring rate is 800-1000 rpm, such as 800 rpm, 900 rpm, 1000 rpm, and the reaction time is 50-80 min, such as 50 min, 60 min, 70 min, 80 min.

[0020] Example 1 This embodiment relates to a bivalirudin nanodrug, and a preparation method thereof, comprising the following steps: Accurately weigh 10 mg of bivalirudin (BIV) and fully dissolve it in 4 mL of methanol, dissolve 1.23 mg of CuCl2 in 1 mL of ddH2O, and mix well in a reaction bottle, the molar ratio of BIV to Cu 2+The molar ratio was 1:2. The pH was adjusted to 6.0 with 1 M NaOH solution. The reaction system was magnetically stirred at 900 rpm for 60 min at a constant temperature of 37 ℃. The solution was then transferred to centrifuge tubes and centrifuged at 12000 rpm for 10 min to remove the supernatant. The precipitate was resuspended in 1 mL of ultrapure water, and the washing and centrifugation were repeated three times. Finally, the purified sample was pre-frozen at -80 ℃ for 30 min, followed by freeze-drying for 14-18 h to obtain dried BIV@Cu powder. Following the above synthesis steps, the total reaction volume was 5 mL, and the volume ratio of MeOH to water was 4:1. The amounts of BIV and Cu were varied. 2+ The molar ratios of the substances were 1:4 or 1:6 to synthesize bivalirudin nanomedicines (BIV@Cu) in different proportions.

[0021] The inhibitory effects of BIV@Cu and BIV on thrombin were verified using a chromogenic substrate method. The specific method is as follows: BIV and BIV@Cu were dissolved separately in prepared Tris-HCl (pH 7.4), with an equivalent BIV drug concentration of 0.8 mg / mL. First, 100 µL of Tris-HCl buffer was added to each well of a 96-well plate, followed by 40 µL of bivalirudin nanoparticle solution, and then 10 U of thrombin solution. The absorbance of the solution was measured at 405 nm at 37 ℃ as a blank. Then, 2 mM of S-2238 was added to each well to initiate the enzyme-catalyzed reaction. The absorbance was measured every 1 min, using the drug solvent as a negative control. The thrombin inhibition rate of the drugs was calculated using the absorbance of the sample after 5 min. Results are as follows: Figure 1 This indicates that BIV and Cu 2+ The BIV@Cu synthesized under a molar ratio of 1:4 exhibited stronger thrombin inhibitory activity than those synthesized under ratios of 1:2 and 1:6. Therefore, BIV and Cu... 2+ The optimal molar ratio is 1:4.

[0022] Example 2 This embodiment relates to a copper ion-mediated bivalirudin self-assembled nanomedicine, the preparation method of which includes the following steps: 10 mg BIV and 8.53 mg cRGD were accurately weighed and dissolved together in 4 mL of methanol, with a molar ratio of BIV to cRGD of 1:3. 2.46 mg CuCl2 was accurately weighed and dissolved in 1 mL ddH2O. The methanol solution and copper chloride solution were placed together in a reaction flask and mixed thoroughly. The pH was adjusted to 6.0 with 1 M NaOH solution. The reaction system was magnetically stirred at 900 rpm for 60 min at a constant temperature of 37 ℃. The solution was then transferred to a centrifuge tube and centrifuged at 12000 rpm for 10 min to remove the supernatant. The precipitate was resuspended in 1 mL of ultrapure water and washed and centrifuged three times. Finally, the purified sample was pre-frozen at -80 ℃ for 30 min and then freeze-dried for 14–18 h to obtain the copper ion-mediated bivalirudin self-assembled nanomedicine, denoted as BIV@Cu@cRGD.

[0023] Example 3 This embodiment relates to a copper ion-mediated bivalirudin self-assembled nanomedicine, which is prepared in the same way as in Example 2, except that the pH value is adjusted to 5.0.

[0024] Example 4 This embodiment relates to a copper ion-mediated bivalirudin self-assembled nanomedicine, which is prepared in the same way as in Example 2, except that the pH value is adjusted to 7.0.

[0025] The inhibitory effect of the nanomedicines prepared in Examples 2-4 on thrombin was verified using the chromogenic substrate method. The specific method was the same as in Example 1, and the results are as follows: Figure 2 The results showed that at pH 5, the inhibitory effect of BIV@Cu@cRGD on thrombin was minimized to only about 12%. At pH 7, the thrombin inhibition rate of BIV@Cu@cRGD was 38.4%, lower than that of BIV monomer (43.7%). However, at pH 6, the thrombin inhibition rate of BIV@Cu@cRGD was as high as 44.6%. Therefore, the final reaction pH was determined to be 6.

[0026] Example 5 This embodiment relates to a copper ion-mediated bivalirudin self-assembled nanomedicine, which is prepared in the same way as in Example 2, except that the molar ratio of BIV to cRGD is 3:1.

[0027] Example 6 This embodiment relates to a copper ion-mediated bivalirudin self-assembled nanomedicine, which is prepared in the same way as in Example 2, except that the molar ratio of BIV to cRGD is 1:1.

[0028] Example 7 This embodiment relates to a copper ion-mediated bivalirudin self-assembled nanomedicine, which is prepared in the same way as in Example 2, except that the molar ratio of BIV to cRGD is 1:2.

[0029] The inhibitory effect of the nanomedicines prepared in Examples 2 and 5-7 on thrombin was verified using the chromogenic substrate method. The specific method was the same as in Example 1, and the results are as follows: Figure 3 The results showed that there was no significant difference in thrombin inhibition rate between each group and the control group (BIV), indicating that BIV@Cu@cRGD with different cRGD ratios all exhibited good anticoagulant effects.

[0030] Experimental Example (1) Morphology and elemental analysis Morphology and elemental analysis of the BIV@Cu@cRGD nanomedicine prepared in Example 2 were performed using field emission scanning electron microscopy. The BIV@Cu@cRGD nanomedicine prepared in Example 2 was resuspended in ultrapure water to a concentration of 0.01 mg / mL, sonicated for 5 min to ensure uniform dispersion, and 10 μL was added dropwise onto a silicon wafer. The wafer was dried at room temperature for 12 h, and gold was sputtered onto the sample. During electron microscopy observation, the morphological characteristics of the BIV nanomedicine were acquired at an appropriate magnification, and elemental mapping and image acquisition were performed using energy dispersive spectroscopy (EDS).

[0031] Experimental results are as follows Figure 4 As shown in 4a, BIV@Cu@cRGD mainly exhibits a network morphology composed of multiple stacked nanoparticles. The nanoparticles are relatively uniform in size, with a main size of about 100 nm. Five random points were selected from the BIV@Cu@cRGD sample for elemental energy dispersive spectroscopy (EDS), and the results are as follows: Figure 4 As shown in b, the molar percentage of Cu ions in BIV@Cu@cRGD is approximately 2.96%.

[0032] (2) Characterization of particle size and surface charge The particle size and surface charge of the nanomedicines prepared in Examples 1 and 2 were characterized. A Malvern zeta potential analyzer was used to analyze the particle size and zeta potential of the BIVAR nanomedicines (BIV@Cu and BIV@Cu@cRGD). The particle size results are shown below. Figure 5 As shown in Figure a, in the ultrasonically treated aqueous solution, the main particle size distributions of BIV@Cu and BIV@Cu@cRGD were 315 nm and 476 nm, respectively, indicating a very uniform particle size distribution in the polydispersity coefficient surface solution; the zeta potential was as follows. Figure 5As shown in b, the zeta potential of BIV@Cu is -13.89 mV, while that of BIV@Cu@cRGD is -11.00 mV. Since cRGD also carries a positive charge under pH 7 conditions, the higher surface charge of BIV@Cu@cRGD compared to BIV@Cu also proves the successful introduction of cRGD.

[0033] (3) XPS characterization The nanomedicine BIV@Cu@cRGD prepared in Example 2 was characterized by X-ray photoelectron spectroscopy. The results are as follows: Figure 6 As shown, peaks for C1s, N1s, O1s, and Cu2p were detected, indicating that Cu was successfully incorporated into BIV@Cu@cRGD.

[0034] (4) Targeting effect The targeting effects of FITC-labeled bivalirudin monomer, bivalirudin nanodrug BIV@Cu prepared in Example 1, and nanodrugs BIV@Cu@cRGD prepared under different BIV and cRGD molar ratios in Examples 2, 5-7 on in vitro thrombi were determined.

[0035] Artificial blood clots were incubated with BIV@Cu and BIV@Cu@cRGD (BIV:cRGD molar ratios of 3:1, 1:1, 1:2, and 1:3), respectively. Fluorescence imaging results are shown below. Figure 7 As shown in Figure a, the fluorescence intensity of the BIV@Cu@cRGD (1:3) group was significantly higher than that of the other drug groups. The quantitative statistical data of fluorescence intensity for each group are shown in Figure a. Figure 7 As shown in b, the fluorescence intensity of the thrombus in the BIV@Cu@cRGD (1:3) group was the strongest and showed a significant difference compared with BIV@Cu and other molar ratio groups. The above results indicate that BIV@Cu@cRGD synthesized at a BIV:cRGD molar ratio of 1:3 exhibits superior in vitro thrombus targeting performance compared with BIV@Cu.

[0036] (5) In vitro drug release In vitro drug release studies of the nanomedicines prepared in Examples 1 and 2.

[0037] Weigh 1 mg of lyophilized BIV@Cu and BIV@Cu@cRGD, resuspend them in 1 mL of PBS (pH 7.4), and transfer the sample solutions to dialysis bags with a molecular weight cutoff of 3500 Da. After sealing, completely immerse the bags in 40 mL of PBS release medium. Place the release system in a 37°C constant temperature water bath and magnetically stir at 200 rpm. Take 1 mL samples at predetermined time points (0.5, 1, 2, 4, 8, and 12 h), and simultaneously add an equal amount of fresh PBS to keep the total volume of the system constant. Measure the absorbance of BIV@Cu and BIV@Cu@cRGD at 276 nm, and calculate the cumulative drug release percentage based on the standard curve.

[0038] Experimental results are as follows Figure 8 As shown, both nanomedicines exhibited significant sustained-release characteristics. The cumulative release rates of BIV@Cu at 0.5 h, 2 h, and 8 h were 15.36%, 36.91%, and 71.87%, respectively, while the release rate of BIV@Cu@cRGD was slightly faster, with release rates of 24.66%, 58.92%, and 80.46% at the corresponding time points. Within 4 h of administration, both nanomedicines continued to release rapidly, but the faster release mode of BIV@Cu@cRGD is more beneficial for anticoagulation therapy in acute thrombosis.

[0039] (6) Cytotoxicity The toxicity of the nanomedicines prepared in Examples 1 and 2 to HUVECs was determined. The cytotoxicity of BIV@Cu and BIV@Cu@cRGD to HUVECs was detected by the CCK-8 assay. Different concentrations of BIV@Cu and BIV@Cu@cRGD solutions were co-incubated with HUVECs for 48 h, and cell viability was measured. Results are as follows: Figure 9 As shown, the cell survival rate of all drug treatment groups exceeded 90%, and no significant differences were observed; these results indicate that BIV@Cu and BIV@Cu@cRGD did not have significant cytotoxicity on HUVEC cells.

[0040] (7) Mouse model validation The therapeutic application and parameter changes of the nanomedicines prepared in Examples 1 and 2 in a mouse carotid artery thrombosis model were studied.

[0041] A FeCl3-induced carotid artery thrombosis model was established using 10-week-old male C57BL / 6J mice. Healthy male C57BL / 6J mice were anesthetized, fixed on a surgical board, and the skin and tissues of the neck were cut open. The fascia was bluntly dissected, and the vagus nerve was gently dissected to avoid rupture, exposing a segment of the right carotid artery. The therapeutic drug was injected into the tail vein of the mice using a tail vein injection imaging device. Immediately afterwards, saturated filter paper (2*2 mm) soaked in 10% FeCl3 was placed on the exposed right carotid artery for 5 minutes to induce thrombus formation. The filter paper was then removed, and the surface of the right carotid artery was rinsed with physiological saline to remove residual FeCl3. The changes in blood perfusion in the right carotid artery of each group of mice were recorded in real time using a laser speckle blood flow imaging system until thrombus formation and complete cessation of blood flow. The animals were divided into four groups, with four animals in each group: PBS control group; BIV group; BIV@Cu group; BIV@Cu@cRGD group. All doses of the drug were dissolved in PBS to a volume of 100 μL. The control group was injected with 100 μL of sterile PBS via tail vein. All experimental doses were 1 µmol / kg.

[0042] The results showed that complete coagulation and vascular embolism occurred in the control group (sterile PBS) approximately 6 minutes after the removal of FeCl3-saturated filter paper. Figure 10 a) By injecting 1 μmol / kg of free bivalirudin into model mice, the clotting time ( Figure 10 c) Delaying clotting time to 9 minutes, while injecting an equal volume of bivalirudin BIV@Cu can delay clotting time to 11 minutes. Figure 10 (b) BIV@Cu@cRGD showed significantly higher efficacy than the same dose of free bivalirudin and BIV@Cu in inhibiting FeCl3-induced carotid artery thrombosis, with a clotting time as long as 16 min (b). Figure 10 d) The main reason for the prolonged anticoagulation time of BIV@Cu and BIV@Cu@cRGD is that the nano-drugs formed by BIV and copper ions through metal coordination self-assembly allow for sustained drug release in vivo, thus prolonging the drug circulation time. The coagulation time trends of bivalirudin monomer and nano-drug show that the nano-drugs formed by bivalirudin through metal coordination self-assembly have a significant sustained-release effect compared to the monomer drug. Most importantly, the difference between BIV@Cu@cRGD and BIV@Cu reflects the contribution of cRGD to platelet activation at the thrombus site, which greatly increases the accumulation of drug in the thrombus area and produces a high local concentration of bivalirudin.

[0043] Under the above-described model, the tail bleeding time was measured after administration of the drug via the tail vein in mice. Specifically, the tail was transversely cut with scissors 2 mm from the end of the mouse tail, and the tail was immediately immersed in 37 °C physiological saline. The bleeding time was observed, and the time to initial hemostasis (30 seconds) was recorded. The tail was then wiped with filter paper to verify hemostasis. This experiment was performed in parallel with thrombosis induction.

[0044] Experimental results are as follows Figure 11 The tail bleeding time in the PBS group was 325 s, while the tail bleeding time in the BIV@Cu@cRGD group (416 s) was slightly longer. However, compared with the BIV group (544 s), the bleeding time was significantly shorter, indicating that cRGD enriches the nanomedicine at the thrombus site and reduces the uncontrollable systemic circulation of the drug with venous blood. At the same time, we found that the tail bleeding time in the BIV@Cu group was 488 s, which also effectively reduced the bleeding time compared with the BIV group. This is because the sustained release effect of the self-assembled drug in vivo reduces the accumulation of the drug at non-target sites. The above results show that BIV@Cu@cRGD can significantly shorten the bleeding adverse reaction time.

[0045] Ten-week-old C57BL / 6J mice were randomly divided into four groups: PBS control group; BIV group; BIV@Cu group; and BIV@Cu@cRGD group, with five mice in each group. After administration of 1 µmol / kg, the body weight was recorded daily. Seven days later, whole blood was collected from the mice to detect liver and kidney indicators, and the mice were dissected and the five major organs (heart, liver, spleen, lung, and kidney) were removed for pathological sections. Figure 12 The curves showing the change in mouse body weight over time within 7 days after a single dose reveal a slight upward trend in body weight across all groups after one week, with no significant difference in weight gain among the groups. Figure 13 As shown, no significant pathological changes were found in the tissue sections of the major organs in either the PBS control group or the single BIV, BIV@Cu, or BIV@Cu@cRGD treatment groups; Figure 14 As shown, compared with the control group (PBS) and the BIV group, the screening liver and kidney function markers in the BIV@Cu or BIV@Cu@cRGD groups showed no statistically significant changes, indicating that these effects were transient and benign. These results indicate that treatment with BIV@Cu@cRGD does not cause liver or kidney damage. All of these results demonstrate that the bivalirudin self-assembled nanomedicine has good biocompatibility, further highlighting the safety of this nanomedicine.

[0046] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for preparing copper ion-mediated bivalirudin self-assembled nanomedicine, characterized in that, Includes the following steps: (1) Dissolve bivalirudin and the targeting peptide cRGD in an organic solvent to obtain solution A, and dissolve the copper salt in deionized water to obtain solution B; (2) Mix the solution A and solution B, adjust the pH to weakly acidic or neutral, stir the reaction thoroughly to obtain a suspension, centrifuge to remove the supernatant, and freeze dry to obtain the product.

2. The method for preparing a copper ion-mediated bivalirudin self-assembled nanomedicine according to claim 1, characterized in that, The molar ratio of bivalirudin to copper ions in the copper salt is 1:(2-6).

3. The method for preparing a copper ion-mediated bivalirudin self-assembled nanomedicine according to claim 1, characterized in that, The mass ratio of bivalirudin to the target peptide cRGD is 1:(0.3-3).

4. The method for preparing a copper ion-mediated bivalirudin self-assembled nanomedicine according to claim 1, characterized in that, The organic solvent is methanol.

5. The method for preparing a copper ion-mediated bivalirudin self-assembled nanomedicine according to claim 1, characterized in that, The volume ratio of solution A to solution B is 4:

1.

6. The method for preparing a copper ion-mediated bivalirudin self-assembled nanomedicine according to claim 1, characterized in that, The pH is 5-7.

7. The method for preparing a copper ion-mediated bivalirudin self-assembled nanomedicine according to claim 1, characterized in that, The reaction temperature of the stirring reaction is 30-50℃, the stirring rate is 800-1000rpm, and the reaction time is 50-80min.

8. A copper ion-mediated bivalirudin self-assembled nanomedicine, characterized in that, It is prepared by the preparation method according to any one of claims 1-7.

9. The application of the copper ion-mediated bivalirudin self-assembled nanomedicine according to claim 8 in the preparation of a drug for treating antithrombotic diseases.

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