Preparation method and application of recombinant human serum albumin-arteplase nano-drug

Abstract

The invention discloses a recombinant human serum albumin-arteplase nano-drug (rHSA-HSAbp-rt-PA) as well as a preparation method and an application of the recombinant human serum albumin-arteplase nano-drug (rHSA-HSAbp-rt-PA). The nano-drug comprises arteplase (rt-PA), human serum albumin adhesion peptide (HSAbp) and recombinant human serum albumin (rHSA), and the sequence of the human serum albumin adhesion peptide is VGPLGPHYYYCAADLWRL. According to the present invention, the recombinant human serum albumin-arteplase nano-drug is connected through the human serum albumin adhesion peptide, such that the chemical cross-linking step is avoided, the preparation method is simple, and the recombinant human serum albumin-arteplase nano-drug has the half-life period of as long as 29 min, has good treatment effect, and has wide application prospects in the treatment of thrombotic diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of nano-drugs, and particularly relates to a preparation method and application of recombinant human serum albumin-alteplase. Background Art

[0002] Thromboembolic diseases (such as stroke, acute myocardial infarction, pulmonary embolism, and deep vein thrombosis, etc.) are one of the main causes of disability and death globally. Thrombolysis is an important treatment method for thromboembolic diseases. Even today when interventional surgeries are widely carried out, thrombolysis treatment is still irreplaceable, especially in the treatment of stroke. In recent years, with the emergence of new thrombolytic drugs, clinicians have more choices of drugs to apply, but they may not have in-depth understanding of each drug.

[0003] Currently, common thrombolytic drugs have undergone multiple updates and iterations. The first-generation thrombolytic drug is represented by alteplase. Alteplase (rt-PA) is a recombinant tissue plasminogen activator (t-PA) prepared by genetic engineering technology. It is a single-chain serine protease composed of 527 amino acids, but its half-life is extremely short, only 4 - 5 minutes, and continuous intravenous infusion is required. To solve the problem of its short half-life, many aspects of research have been carried out, mainly including (1) tissue plasminogen activator mutants: represented by reteplase and tenecteplase; (2) tissue plasminogen activator fusion proteins: CN113416724A discloses a tPA 146Y -HSA-FBP fusion protein, which contains a partial sequence of HSA and increases its biocompatibility through the partial sequence of HSA; (3) rtPA modified by a nano-targeted delivery system: coating rtPA with polymers or liposomes to extend its half-life, and at the same time modifying nanoparticles containing rtPA with antibodies, polypeptides, biological membranes or magnetic effects to endow rtPA with thrombus targeting, improve the local drug concentration, and observe more significant therapeutic effects than free rtPA at the animal level.

[0004] Human serum albumin (HSA), as a commonly used drug carrier material, its unique molecular structure endows it with good drug-carrying properties and has been widely used in the preparation of nano-drugs. For example, albumin-bound paclitaxel drugs have pushed the paclitaxel drug market to a new height. HSA has rich amino acid residues and a specific spatial structure, and can bind tightly to active ingredients through a variety of non-covalent interactions, such as hydrogen bonds, hydrophobic interactions, and electrostatic interactions, etc. It can extend the half-life of active ingredients and targeting, providing a new idea for solving the half-life of tissue plasminogen activator. Summary of the Invention

[0005] In view of the deficiencies of the prior art, the present invention discloses a preparation method and application of a recombinant human serum albumin-alteplase nano-drug. The specific content of the present invention is as follows: In the first aspect of the present invention, a recombinant human serum albumin-alteplase nano-drug (rHSA-HSAbp-rt-PA) is provided. The nano-drug comprises alteplase (rt-PA), human serum albumin adhesion peptide (HSAbp), and recombinant human serum albumin (rHSA), wherein the sequence of the human serum albumin adhesion peptide is VGPLGPHYYYCAADLWRL (SEQ ID NO.1).

[0006] Furthermore, in the recombinant human serum albumin-alteplase nano-drug, the molar ratio of alteplase (rt-PA) to recombinant human serum albumin is 0.5:1 to 3:1. Preferably, the molar ratio of alteplase (rt-PA) to recombinant human serum albumin is 1:1.

[0007] Furthermore, the alteplase and the human serum albumin adhesion peptide are connected by a chemical bond or a non-chemical bond.

[0008] Furthermore, the alteplase and the human serum albumin adhesion peptide are connected by a cross-linking method and / or a fusion protein method.

[0009] Furthermore, the cross-linking method includes at least one of a chemical cross-linking method, a physical cross-linking method, an enzymatic cross-linking method, and a photo-cross-linking method.

[0010] Furthermore, the cross-linking agent for the chemical cross-linking method includes at least one of glutaraldehyde, genipin, and carbodiimide (EDC / NHS); the physical cross-linking method includes at least one of ionic cross-linking and thermally induced cross-linking; the enzyme for the enzymatic cross-linking method includes at least one of transglutaminase and horseradish peroxidase (HRP).

[0011] Furthermore, the alteplase and the human serum albumin adhesion peptide are connected by a fusion protein method. Preferably, the human serum albumin adhesion peptide is connected to the N-terminus or C-terminus of the alteplase.

[0012] Furthermore, the human serum albumin adhesion peptide and the recombinant human serum albumin are connected by a chemical bond or a non-chemical bond.

[0013] In a specific embodiment of the present invention, the human serum albumin adhesion peptide and the recombinant human serum albumin are connected by a non-chemical bond.

[0014] In the second aspect of the present invention, a composition is provided, which comprises at least the recombinant human serum albumin-alteplase nano-drug and an additive acceptable in the pharmaceutical field.

[0015] Furthermore, the dosage form of the composition includes at least one of a nano-suspension, a nano-emulsion, and a lyophilized powder.

[0016] Furthermore, the alteplase in the present invention can also be replaced with other protein drugs. Preferably, the protein drugs include at least one of cytokine drugs, enzyme drugs, antibody drugs, hormone drugs, and vaccine protein drugs.

[0017] More preferably, the cytokine drugs include at least one of interleukin (IL), interferon (IFN), tumor necrosis factor (TNF), colony-stimulating factor (CSF), and erythropoietin (EPO); the enzyme drugs include at least one of adenosine deaminase, phenylalanine lyase, pancreatin, and tenecteplase; the hormone drugs include at least one of insulin, growth hormone, and gonadotropin.

[0018] The third aspect of the present invention provides the use of the recombinant human serum albumin-alteplase nano-drug or the composition in the preparation of a drug for preventing and / or treating thromboembolic diseases.

[0019] Furthermore, the thromboembolic diseases include at least one of ischemic stroke, myocardial infarction, peripheral arterial disease, thromboangiitis obliterans, deep vein thrombosis, pulmonary embolism, portal vein thrombosis, cerebral venous sinus thrombosis, disseminated intravascular coagulation, thrombotic microangiopathy, and antiphospholipid antibody syndrome.

[0020] The fourth aspect of the present invention provides a method for preparing the recombinant human serum albumin-alteplase nano-drug, and the method includes the following steps: S1: Linking human serum albumin adhesion peptide and alteplase together by a cross-linking method or recombinant genetic engineering technology; S2: Mixing the HSAbp-rt-PA prepared in step S1 with a recombinant human serum albumin solution to obtain the recombinant human serum albumin-alteplase nano-drug.

[0021] Furthermore, the cross-linking method in step S1 includes at least one of a chemical cross-linking method, a physical cross-linking method, an enzymatic cross-linking method, and a photo-cross-linking method.

[0022] Furthermore, the cross-linking agents for the chemical cross-linking method include at least one of glutaraldehyde, genipin, and carbodiimide (EDC / NHS); the physical cross-linking methods include at least one of ionic cross-linking and thermally induced cross-linking; the enzymes for the enzymatic cross-linking method include at least one of transglutaminase and horseradish peroxidase (HRP).

[0023] Further, the specific operation of connecting human serum albumin adhesion peptide and alteplase by recombinant genetic engineering technology in step S1 is as follows: S1-1: Connect the human serum albumin adhesion peptide to the N-terminus or C-terminus of alteplase to obtain the amino acid sequence of the fusion protein; S1-2: Optimize the nucleotides of the fusion protein according to the codon preference of the recombinant cells; S1-3: Introduce the optimized nucleotides of the fusion protein into the recombinant cells to express the corresponding fusion protein.

[0024] Further, the method further includes the step of purifying the recombinant human serum albumin-alteplase nanomedicine.

[0025] Further, the method further includes the step of freeze-drying and refrigerating the recombinant human serum albumin-alteplase nanomedicine.

[0026] The beneficial effects of the present invention include but are not limited to: The recombinant human serum albumin-alteplase nanomedicine disclosed by the present invention is connected by the human serum albumin adhesion peptide, avoiding the step of chemical cross-linking, and the preparation method is simple. The half-life of the recombinant human serum albumin-alteplase nanomedicine described by the present invention is up to 29 minutes, and the therapeutic effect is better, having a wide application prospect in the treatment of thrombotic diseases. Description of the Drawings

[0027] The drawings described herein are used to provide a further understanding of the present invention, form a part of the present invention, and the schematic embodiments and descriptions thereof are used to explain the present invention and do not constitute an improper limitation to the present invention. In the drawings: Figure 1 is the gel electrophoresis diagram of the nanomedicine (rHSA-HSAbp-rt-PA) in the embodiment of the present invention.

[0028] Figure 2 is the schematic diagram of the cytotoxicity experiment results of rHSA-HSAbp-rt-PA and HSAbp-rt-PA in the embodiment of the present invention.

[0029] Figure 3 is the schematic diagram of the results of the "blood drug activity-time curve" of rHSA-HSAbp-rt-PA and HSAbp-rt-PA in the embodiment of the present invention. Detailed Embodiments

[0030] The present invention will be described in detail below with reference to the embodiments, but the present invention is not limited to these embodiments. Unless otherwise specified, the raw materials and catalysts in the embodiments of the present invention are purchased through commercial channels.

[0031] Recombinant human serum albumin (rHSA): Produced by Tonghua Anruite Biopharmaceutical Co., Ltd., batch number ART103L-240124-01. This rHSA has the same amino acid sequence as human serum albumin; Alteplase: Activase, purchased from Boehringer Ingelheim.

[0032] Example 1: Screening of human serum albumin-binding peptides Using bioinformatics methods, analyze the spatial structure characteristics of monoclonal antibodies or nanobodies against HSA and the surface charge of human serum albumin, etc., and design a series of polypeptides that can bind to HSA. After direct synthesis by chemical synthesis method, use Surface Plasmon Resonance (SPR) technology to measure the affinity between the polypeptide molecule and HSA. The specific steps are as follows: Inject a mixture of EDC / NHS (1:1 ratio) at a flow rate of 10 μL / min to activate the carboxyl groups on the chip surface to form reactive ester groups. Inject the HSA solution (flow rate 10 μL / min) onto the activated chip surface to covalently bind it to the activated carboxyl groups. Inject 1 M ethanolamine (pH 8.5) at a flow rate of 10 μL / min to block the unreacted ester groups and prevent non-specific binding. Use PBS buffer (flow rate 10 μL / min) to wash the chip surface until the baseline is stable. Then inject the 0.1 μM polypeptide solution (flow rate 30 μL / min) onto the activated chip in sequence and monitor the binding signal. Screen out the human serum albumin adhesion peptide sequence with an equilibrium dissociation constant less than 0.01 μM.

[0033] After the above steps, we screened out the human serum albumin adhesion peptide VGPLGPHYYYCAADLWRL (SEQ ID NO.1), named HSAbp.

[0034] Example 2 Preparation of fusion protein HSAbp-rt-PA Connect HSAbp to the N-terminus of recombinant tissue plasminogen activator (the sequence is shown in SEQ ID NO.2) to form the fusion protein HSAbp-rt-PA (the sequence is shown in SEQ ID NO.3).

[0035] Optimize the codons of the nucleotide sequence encoding the fusion protein HSAbp-rt-PA according to the E. coli preference to obtain the corresponding nucleotide sequence (the sequence is shown in SEQ ID NO.4).

[0036] Entrust a biological company to clone the fusion protein HSAbp-rt-PA into the expression vector pET-22b (containing His tag), transform BL21(DE3) Escherichia coli with the expression vector pET-22b, spread it on an LB agar plate containing ampicillin, and culture at 37 °C for 12 hours.

[0037] Pick a single colony and inoculate it into 5 mL of LB liquid medium (containing Amp), and culture it with shaking at 37 °C for 12 hours. Transfer it to 500 mL of LB medium at a ratio of 1:100, and culture at 37 °C until OD 600 = 0.6. Add IPTG to a final concentration of 0.5 mM, induce at low temperature of 25 °C for 16 hours, then collect the bacterial cells and centrifuge at 4 °C and 8000×g for 10 minutes. Discard the supernatant, and wash the bacterial cell pellet with PBS buffer (pH 7.4). Resuspend the bacterial cells in lysis buffer (containing 1 mM PMSF, 10 mM imidazole), and ultrasonically disrupt on ice bath (power 300 W, working for 3 seconds / interval 5 seconds, lasting for 20 minutes). Centrifuge at 4 °C and 12000×g for 30 minutes, and collect the supernatant.

[0038] Equilibrate the Ni-NTA column with binding buffer (20 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole, pH 8.0). Load the crude extract onto the column at a flow rate of 1 mL / min to bind the His-tagged protein. Wash away the non-specifically bound proteins with buffers containing 20 mM and 50 mM imidazole in sequence. Elute the nanobody with a buffer containing 250 mM imidazole, and collect the elution peak. Load the eluate into a dialysis bag (cut-off molecular weight 25 kDa), and dialyze with PBS for 24 hours (4 °C, changing the solution 3 times) to remove imidazole and obtain the purified fusion protein HSAbp-rt-PA. Detect it by SDS-PAGE, and the molecular weight of the fusion protein HSAbp-rt-PA is about 61 kDa.

[0039] Example 3 Construction of a nanodrug (rHSA-HSAbp-rt-PA) Weigh 100 mg of rHSA, dissolve it in 10 mL of PBS (pH 7.4) to prepare a 10 mg / mL rHSA solution. Filter and sterilize it with a 0.22 μm filter membrane, and store it at 4 °C for later use.

[0040] Weigh 100 mg of the fusion protein HSAbp-rt-PA, dissolve it in 10 mL of PBS (pH 7.4) to prepare a 10 mg / mL HSAbp-rt-PA solution. Filter and sterilize it with a 0.22 μm filter membrane, and store it at 4 °C for later use.

[0041] Mix HSAbp-rt-PA and rHSA at a molar ratio of 0.5 - 3:1 and react for 3 - 5 min. Load the reaction solution into a dialysis bag (MWCO 100 kDa) and store it to obtain the stock solution of (rHSA-HSAbp-rt-PA). Dialyze it in 1 L of PBS for 24 hours (change the solution 3 times). Detect the purified nanoparticle suspension by gel electrophoresis, then freeze it (-80 °C, 2 hours), and then lyophilize it for 24 hours to obtain a solid powder.

[0042] The results are as Figure 1 shown.

[0043] It can be Figure 1 seen that HSAbp-rt-PA and rHSA are completely combined when the molar ratio of HSAbp-rt-PA:rHSA is 1:1. When the molar ratio of HSAbp-rt-PA:HSA is 2:1 and 3:1, a light-colored band of about 190 kDa appears. Therefore, it is speculated that HSA contains two binding sites for the fusion protein HSAbp-rt-PA, but the binding ability of one of them is poor. Select rHSA-HSAbp-rt-PA with a molar ratio of HSAbp-rt-PA:rHSA of 1:1 for the subsequent experiments.

[0044] Example 4 Cytotoxicity experiment of rHSA-HSAbp-rt-PA and HSAbp-rt-PA (CCK-8 method) MTT / CCK-8 method: Seed HUVEC cells (10 4 cells / well). After they adhere to the wall, divide them into groups. The blank group is blank medium (without cells), the cell control group is (HUVEC cells + complete medium), the alteplase group is (1, 2.5, 5, 10, 20, 50 μg / mL), the HSAbp-rt-PA fusion protein group is (equivalent rt-PA concentration 1, 2.5, 5, 10, 20, 50 μg / mL), the rHSA-HSAbp-rt-PA nanoparticle group is (equivalent rt-PA concentration 1, 2.5, 5, 10, 20, 50 μg / mL), and the rHSA group is (rHSANPs without drugs, excluding the toxicity of the carrier). After incubation for 24 h, add 10 μL of CCK-8 working solution (10% CCK-8 + 90% serum-free medium) to each well. Measure the absorbance at 450 nm after 1 h. Conduct independent repeated experiments ≥3 times to exclude the influence of edge effects. Use SPSS to statistically analyze the experimental results. The results are shown in Table 1 and Figure 2 shown.

[0045] Calculate the cell survival rate:

[0046] Table 1

[0047] Among them, * indicates P < 0.05 compared with the cell control group, ** indicates P < 0.01 compared with the cell control group, # indicates P < 0.05 compared with the rt-PA group, and ## indicates P < 0.01 compared with the rt-PA group.

[0048] According to Table 1 and Figure 2 it can be seen that the cytotoxicity of the HSAbp-rt-PA group and the rHSA-HSAbp-rt-PA group is much less than that of the rt-PA group.

[0049] Example 5 Activity Experiments of rHSA-HSAbp-rt-PA and HSAbp-rt-PA The activities of rHSA-HSAbp-rt-PA and HSAbp-rt-PA solutions were detected using a human t-PA ELISA kit (purchased from Shanghai Huabang Biotech Co., Ltd.). The equivalent rt-PA concentration was 1 μg / mL, and rt-PA was used as a reference. The results are shown in Table 2.

[0050] Table 2

[0051] The results prove that the activities of rHSA-HSAbp-rt-PA and HSAbp-rt-PA are not much different from that of rt-PA.

[0052] Example 6 In Vitro Thrombolysis Detection of rHSA-HSAbp-rt-PA and HSAbp-rt-PA Fresh blood from healthy individuals was placed in an anticoagulant tube, and the anticoagulated whole blood was added with thrombin (1 - 5 U / mL) and CaCl2 (10 - 20 mM). After mixing, it was injected into a silicone tube or a 96-well plate and left to stand at 37°C for 1 - 2 hours to form a thrombus. The thrombus was gently taken out, rinsed 3 times with PBS, and the surface moisture was blotted dry with filter paper and weighed (denoted as W0). The thrombus was placed in a PBS solution containing a thrombolytic drug, and the control group used an equal volume of PBS. It was incubated at 37°C with constant shaking (50 - 100 rpm) for 2 h, and the reaction was terminated: the remaining thrombus was taken out, rinsed with PBS, blotted dry, and weighed (denoted as W1). The thrombus dissolution rate was calculated. Among them, the experimental groups were rHSA-HSAbp-rt-PA and HSAbp-rt-PA, the positive control was alteplase (rt-PA), the concentrations of the active ingredient rt-PA in rHSA-HSAbp-rt-PA, HSAbp-rt-PA, and alteplase were the same (10 μg / mL), and the negative control was an equal volume of PBS solution. Formula: Thrombus dissolution rate (%) = , and the results are shown in Table 3.

[0053] Table 3

[0054] **Indicates that P value ≤ 0.01 compared with the PBS group.

[0055] As can be seen from Table 3, through statistical analysis, there is no significant difference in the in vitro thrombolysis effect among rHSA-HSAbp-rt-PA, HSAbp-rt-PA and alteplase.

[0056] Example 7 Detection of the half-life of rHSA-HSAbp-rt-PA and HSAbp-rt-PA Twenty adult SD rats were selected, with 10 males and 10 females in each group, divided into 2 groups; each group was intravenously injected with rHSA-HSAbp-rt-PA and HSAbp-rt-PA respectively (where the clinical equivalent dose of rt-PA: 0.9 mg / kg); blood was taken from the tail vein of the rats at 0 (baseline), 2, 5, 10, 15, 20, 25, 30, 40, 50, 60, 80, 100, 120 min respectively, and the serum was collected. The biological activity of rt-PA retained in the serum was detected using a human t-PA ELISA kit (purchased from Shanghai Huabang Biotechnology Co., Ltd.). The "blood drug activity-time curve" was plotted, and data fitting was performed using software (as shown in the figure), and the pharmacokinetic parameters were analyzed. The results are shown in Table 4 and Figure 3 as follows. Table 4

[0057] As can be seen from Table 4 and Figure 3 it can be known that the half-lives of rHSA-HSAbp-rt-PA and HSAbp-rt-PA are significantly longer than that of rt-PA (half-life 4 - 5 min), and the half-life of rHSA-HSAbp-rt-PA is increased compared with the half-life of HSAbp-rt-PA. It is speculated that part of HSAbp-rt-PA is cleared before binding to HSA in plasma after entering the blood.

[0058] Example 7 Animal model experiments of rHSA-HSAbp-rt-PA and HSAbp-rt-PA Middle cerebral artery occlusion model (MCAO): Before the experiment, adult mice were fasted for 12 h without water deprivation. The mice were anesthetized by intraperitoneal injection of 5% chloral hydrate (0.1 mL / 10 g body weight). After the anesthesia took effect, the mice were fixed supine on the operating table. A midline incision was made in the neck, and the right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were dissected. The proximal end of the ECA was ligated, and threads were passed under the CCA and ICA for standby. A small incision was made at about 3 - 4 mm from the bifurcation of the ECA, and a pre-treated thread embolism (diameter about 0.26 - 0.28 mm) was inserted into the ICA through the ECA incision. The depth of the thread insertion was about 18 - 20 mm to block the blood flow of the middle cerebral artery and cause cerebral ischemia. The ischemia time was controlled for 60 min, and then the thread embolism was slowly withdrawn to achieve blood reperfusion.

[0059] Drug administration: The experimental group was administered rHSA-HSAbp-rt-PA and HSAbp-rt-PA, the positive control group was administered alteplase (rt-PA), and the concentration of the active ingredient rt-PA in rHSA-HSAbp-rt-PA, HSAbp-rt-PA, and alteplase was the same. The negative control group was administered an equal volume of PBS solution. During cerebral ischemia-reperfusion, each group of drugs was injected through the tail vein at a relative dose of 0.9 mg / kg body weight. After 24 h of reperfusion, the mice were anesthetized with 5% chloral hydrate, decapitated, and the brains were removed. The mouse brains were sliced in a brain trough and placed in 2% TTC staining solution, and stained in the dark at 37°C in an incubator for 8 min. Photos were taken and the infarct volume was calculated.

[0060] Infarct volume percentage = (contralateral hemisphere volume - ipsilateral hemisphere non-infarcted area volume) / contralateral infarcted hemisphere volume × 100%.

[0061] The results are shown in Table 5.

[0062] Table 5

[0063] Among them, * indicates P < 0.05 compared with the PBS group, ** indicates P < 0.01 compared with the PBS group, # indicates P < 0.05 compared with the rt-PA group, and ## indicates P < 0.01 compared with the rt-PA group.

[0064] As can be seen from Table 5, the effects of rHSA-HSAbp-rt-PA and HSAbp-rt-PA in treating cerebral infarction are significantly better than those of the rt-PA group.

[0065] As described above, these are only embodiments of the present invention. The protection scope of the present invention is not limited by these specific embodiments, but is determined by the claims of the present invention. For those skilled in the art, various modifications and variations can be made to the present invention. Any modification, equivalent replacement, improvement, etc. made within the technical idea and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A recombinant human serum albumin-tenecteplase nanomedicine, characterized in that, The nano-drug includes alteplase (rt-PA), human serum albumin adhesion peptide (HSAbp) and recombinant human serum albumin (rHSA), wherein the sequence of the human serum albumin adhesion peptide is VGPLGPHYYYCAADLWRL (SEQ ID NO. 1).

2. The recombinant human serum albumin-tenecteplase nanomedicine according to claim 1, wherein The alteplase and the human serum albumin adhesion peptide are connected by a chemical bond or a non-chemical bond.

3. The recombinant human serum albumin-tenecteplase nanomedicine according to claim 2, wherein The alteplase and the human serum albumin adhesion peptide are connected by a cross-linking method and / or in the form of a fusion protein.

4. The recombinant human serum albumin-alteplase nanomedicine according to claim 1, wherein The human serum albumin adhesion peptide and the recombinant human serum albumin are connected by a chemical bond or a non-chemical bond.

5. A composition, characterized in that, The composition at least includes the recombinant human serum albumin-alteplase nano-drug according to any one of claims 1-4 and an additive acceptable in the pharmaceutical field.

6. The composition according to claim 5, characterized in that, The dosage form of the composition includes at least one of a nano-suspension, a nano-emulsion and a freeze-dried powder.

7. Use of the recombinant human serum albumin-alteplase nano-drug according to any one of claims 1-4 or the composition according to claim 5 or 6 in the preparation of a drug for preventing and / or treating thrombotic diseases.

8. The application according to claim 7, wherein The thrombotic diseases include at least one of ischemic stroke, myocardial infarction, peripheral arterial disease, thromboangiitis obliterans, deep vein thrombosis, pulmonary embolism, portal vein thrombosis, cerebral venous sinus thrombosis, disseminated intravascular coagulation, thrombotic microangiopathy, antiphospholipid antibody syndrome.

9. A method for preparing the recombinant human serum albumin-tPA nanomedicine according to any one of claims 1-4, characterized in that, The method includes the following steps: S1: Connect the human serum albumin adhesion peptide and alteplase together by a cross-linking method or recombinant genetic engineering technology; S2: Mix the HSAbp-rt-PA prepared in step S1 with a recombinant human serum albumin solution to obtain a recombinant human serum albumin-alteplase nano-drug.

10. The method according to claim 9, characterized in that, The method further includes a step of purifying the recombinant human serum albumin-alteplase nano-drug.

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