Preparation method and application of recombinant human serum albumin-alteplase nanomedicine

Abstract

The present invention discloses a recombinant human serum albumin-alteplase nanodrug (rHSA-HSAbp-rt-PA), its preparation method, and application. The nanodrug comprises alteplase (rt-PA), a human serum albumin adhesion peptide (HSAbp), and recombinant human serum albumin (rHSA), wherein the sequence of the HSA adhesion peptide is VGPLGPHYYYCAADLWRL. The rHSA-alteplase nanodrug is linked via the HSA adhesion peptide, avoiding the chemical cross-linking step and simplifying the preparation method. The rHSA-alteplase nanodrug has a half-life of up to 29 minutes, exhibits improved therapeutic efficacy, and has broad application prospects in treating thrombotic diseases.

Description

Technical Field

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

[0002] Thrombotic diseases (such as stroke, acute myocardial infarction, pulmonary embolism, and deep vein thrombosis) are among the leading causes of disability and death worldwide. Thrombolysis is an important treatment for thrombotic diseases. Even with the widespread use of interventional procedures, thrombolytic therapy remains irreplaceable, particularly in stroke treatment. The emergence of new thrombolytic drugs in recent years has expanded the range of drug options available to clinicians, but they may not have a thorough understanding of each drug.

[0003] Currently, common thrombolytic drugs have undergone multiple iterations. The first generation of thrombolytic drugs 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. However, its half-life is extremely short, only 4-5 minutes, and it requires continuous intravenous infusion. 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 protein: 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 nano-targeted delivery system: rtPA is encapsulated by polymers or liposomes to extend its half-life, and nanoparticles containing rtPA are modified by antibodies, peptides, biofilms or magnetic effects to give rtPA thrombus targeting, increase local drug concentration, and observe more significant therapeutic effects than free rtPA at the animal level.

[0004] Human serum albumin (HSA) is a commonly used drug delivery material. Its unique molecular structure endows it with excellent drug-carrying properties and has been widely used in the preparation of nanomedicines. For example, albumin-bound paclitaxel has propelled the paclitaxel market to new heights. HSA, with its rich amino acid residues and specific spatial structure, can tightly bind to active ingredients through various non-covalent interactions, such as hydrogen bonding, hydrophobic interactions, and electrostatic interactions. Its ability to extend the half-life of active ingredients and its targeted properties provides new insights into addressing the half-life of tissue plasminogen activator. Summary of the Invention

[0005] In response to the shortcomings of the existing technology, the present invention discloses a method for preparing and using a recombinant human serum albumin-alteplase nanoparticle drug. The present invention is specifically described as follows:

[0006] In a first aspect, the present invention provides a recombinant human serum albumin-alteplase nanoparticle drug (rHSA-HSAbp-rt-PA). The nanoparticle drug comprises alteplase (rt-PA), a human serum albumin adhesion peptide (HSAbp), and recombinant human serum albumin (rHSA). The sequence of the human serum albumin adhesion peptide is VGPLGPHYYYCAADLWRL (SEQ ID NO. 1).

[0007] Furthermore, the molar ratio of alteplase (rt-PA) to recombinant human serum albumin in the recombinant human serum albumin-alteplase nanodrug is 0.5:1~3:1. Preferably, the molar ratio of alteplase (rt-PA) to recombinant human serum albumin is 1:1.

[0008] Furthermore, the alteplase and human serum albumin adhesion peptide are connected via a chemical bond or a non-chemical bond.

[0009] Furthermore, the alteplase and human serum albumin adhesion peptide are connected by cross-linking and / or fusion protein.

[0010] 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 photocross-linking method.

[0011] Furthermore, the crosslinking agent of the chemical crosslinking method includes at least one of glutaraldehyde, genipin, and carbodiimide (EDC / NHS); the physical crosslinking method includes at least one of ionic crosslinking and thermal crosslinking; and the enzyme of the enzymatic crosslinking method includes at least one of transglutaminase and horseradish peroxidase (HRP).

[0012] Furthermore, the alteplase and human serum albumin adhesion peptide are connected via a fusion protein. Preferably, the human serum albumin adhesion peptide is connected to the N-terminus or C-terminus of alteplase.

[0013] Furthermore, the human serum albumin adhesion peptide is connected to the recombinant human serum albumin via a chemical bond or a non-chemical bond.

[0014] In a specific embodiment of the present invention, the human serum albumin adhesion peptide is connected to the recombinant human serum albumin via a non-chemical bond.

[0015] In a second aspect, the present invention provides a composition comprising at least the recombinant human serum albumin-alteplase nanomedicine and additives acceptable in the medical field.

[0016] Furthermore, the dosage form of the composition includes at least one of a nanosuspension, a nanoemulsion, and a lyophilized powder.

[0017] Furthermore, alteplase in the present invention can also be replaced by other protein drugs. Preferably, the protein drugs include at least one of cytokine drugs, enzyme drugs, antibody drugs, hormone drugs, and vaccine protein drugs.

[0018] 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, pancreatic enzyme, and tereplase; and the hormone drugs include at least one of insulin, growth hormone, and gonadotropin.

[0019] The third aspect of the present invention provides the use of the recombinant human serum albumin-alteplase nanomedicine or the composition in the preparation of a drug for preventing and / or treating thrombotic diseases.

[0020] Furthermore, 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, and antiphospholipid antibody syndrome.

[0021] In a fourth aspect, the present invention provides a method for preparing the recombinant human serum albumin-alteplase nanodrug, the method comprising the following steps:

[0022] S1: Human serum albumin adhesion peptide and alteplase are linked together by cross-linking or recombinant genetic engineering technology;

[0023] S2: The HSAbp-rt-PA prepared in step S1 is mixed with the recombinant human serum albumin solution to prepare the recombinant human serum albumin-alteplase nanodrug.

[0024] 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.

[0025] Furthermore, the crosslinking agent of the chemical crosslinking method includes at least one of glutaraldehyde, genipin, and carbodiimide (EDC / NHS); the physical crosslinking method includes at least one of ionic crosslinking and thermal crosslinking; and the enzyme of the enzymatic crosslinking method includes at least one of transglutaminase and horseradish peroxidase (HRP).

[0026] Furthermore, the specific operation of linking the human serum albumin adhesion peptide and alteplase together by recombinant genetic engineering technology in step S1 is:

[0027] S1-1: connecting a human serum albumin adhesion peptide to the N-terminus or C-terminus of alteplase to obtain the amino acid sequence of the fusion protein;

[0028] S1-2: Optimizing the nucleotide sequence of the fusion protein according to the codon preference of the recombinant cell;

[0029] S1-3: The optimized fusion protein nucleotides are introduced into recombinant cells to express the corresponding fusion protein.

[0030] Furthermore, the method also includes the step of purifying the recombinant human serum albumin-alteplase nanomedicine.

[0031] Furthermore, the method also includes the step of freeze-drying and refrigerating the recombinant human serum albumin-alteplase nanomedicine.

[0032] The beneficial effects of the present invention include but are not limited to:

[0033] The recombinant human serum albumin-alteplase nanodrug disclosed in the present invention is connected via human serum albumin adhesive peptides, avoiding the step of chemical cross-linking, and has a simple preparation method. The recombinant human serum albumin-alteplase nanodrug of the present invention has a half-life of up to 29 minutes, has a better therapeutic effect, and has broad application prospects in the treatment of thrombotic diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0035] Figure 1 This is a gel electrophoresis diagram of the nano drug (rHSA-HSAbp-rt-PA) in an embodiment of the present invention.

[0036] Figure 2 Schematic diagram of the results of the rHSA-HSAbp-rt-PA and HSAbp-rt-PA cytotoxicity experiments in the examples of the present invention.

[0037] Figure 3 It is a schematic diagram of the results of the “blood activity-time curve” of rHSA-HSAbp-rt-PA and HSAbp-rt-PA in the examples of the present invention. DETAILED DESCRIPTION

[0038] The present invention is described in detail below with reference to the examples, but the present invention is not limited to these examples. Unless otherwise specified, the raw materials and catalysts in the examples of the present invention are purchased through commercial channels.

[0039] 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.

[0040] Alteplase: Alteplase was purchased from Boehringer Ingelheim.

[0041] Example 1: Screening of human serum albumin binding peptides

[0042] Using bioinformatics methods, a series of peptides that bind to HSA were designed by analyzing the spatial structural characteristics of monoclonal antibodies or nanobodies against HSA and the surface charge characteristics of human serum albumin. These peptides were directly synthesized using chemical synthesis, and their affinity for HSA was measured using surface plasmon resonance (SPR). The specific steps were as follows: EDC / NHS mixture (1:1 ratio) was injected at a flow rate of 10 µL / min to activate the carboxyl groups on the chip surface, forming reactive ester groups. HSA solution was then injected at a flow rate of 10 µL / min onto the activated chip surface to covalently bind to the activated carboxyl groups. Unreacted ester groups were blocked by injection of 1 M ethanolamine (pH 8.5) at a flow rate of 10 µL / min to prevent nonspecific binding. The chip surface was rinsed with PBS buffer at a flow rate of 10 µL / min until the baseline stabilized. A 0.1 µM peptide solution was then injected at a flow rate of 30 µL / min onto the activated chip, and the binding signal was monitored. Human serum albumin adhesion peptide sequences with equilibrium dissociation constants less than 0.01µM were screened.

[0043] After the above steps, we screened out the human serum albumin adhesion peptide VGPLGPHYYYCAADLWRL (SEQ ID NO. 1) and named it HSAbp.

[0044] Example 2 Preparation of fusion protein HSAbp-rt-PA

[0045] HSAbp was linked to the N-terminus of recombinant tissue plasminogen activator (sequence shown in SEQ ID NO. 2) to form a fusion protein HSAbp-rt-PA (sequence shown in SEQ ID NO. 3).

[0046] The nucleotide sequence encoding the fusion protein HSAbp-rt-PA was codon-optimized according to the preference of Escherichia coli to obtain the corresponding nucleotide sequence (the sequence is shown in SEQ ID NO.4).

[0047] A biological company was commissioned to clone the fusion protein HSAbp-rt-PA into the expression vector pET-22b (containing a His tag), and the expression vector pET-22b was used to transform BL21 (DE3) Escherichia coli. The culture was spread on LB agar plates containing ampicillin and incubated at 37°C for 12 hours.

[0048] Pick a single colony and inoculate it into 5 mL of LB liquid medium (containing Amp), shake and culture at 37℃ for 12 hours. Transfer it to 500 mL of LB medium at a ratio of 1:100 and culture at 37℃ until OD 600 =0.6. IPTG was added to a final concentration of 0.5 mM. After cold induction at 25°C for 16 hours, the cells were harvested and centrifuged at 8,000 × g for 10 minutes at 4°C. The supernatant was discarded and the pellet washed with PBS (pH 7.4). The cells were resuspended in lysis buffer (containing 1 mM PMSF and 10 mM imidazole) and sonicated on ice (300 W power, 3 seconds on / 5 seconds off, for 20 minutes). The cells were centrifuged at 12,000 × g for 30 minutes at 4°C, and the supernatant was collected.

[0049] A Ni-NTA column was equilibrated with binding buffer (20 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole, pH 8.0). The crude extract was applied to the column at a flow rate of 1 mL / min to bind the His-tagged protein. Nonspecifically bound proteins were eluted with buffers containing 20 mM and then 50 mM imidazole. The nanobody was eluted with a buffer containing 250 mM imidazole, and the eluted peak was collected. The eluate was transferred to a dialysis bag (molecular weight cutoff 25 kDa) and dialyzed against PBS for 24 hours (4°C, with three changes of buffer) to remove the imidazole and obtain the purified fusion protein HSAbp-rt-PA. Gel electrophoresis revealed that the molecular weight of the fusion protein HSAbp-rt-PA was approximately 61 kDa.

[0050] Example 3 Construction of Nanodrug (rHSA-HSAbp-rt-PA)

[0051] Weigh 100 mg of rHSA and dissolve it in 10 mL of PBS (pH 7.4) to prepare a 10 mg / mL rHSA solution. Sterilize the solution by filtration through a 0.22 μm filter and store at 4°C until use.

[0052] Weigh 100 mg of the fusion protein HSAbp-rt-PA and dissolve it in 10 mL of PBS (pH 7.4) to prepare a 10 mg / mL HSAbp-rt-PA solution. Sterilize the solution by filtration through a 0.22 μm filter and store at 4°C until needed.

[0053] HSAbp-rt-PA and rHSA were mixed at a molar ratio of 0.5 to 3:1 and reacted for 3-5 minutes. The reaction solution was placed in a dialysis bag (MWCO 100 kDa) to obtain the (rHSA-HSAbp-rt-PA) stock solution, which was dialyzed in 1 L of PBS for 24 hours (changing the solution three times). The purified nanoparticle suspension was detected by gel electrophoresis, then frozen (-80°C, 2 hours), and freeze-dried for 24 hours to obtain a solid powder.

[0054] The results are as follows Figure 1 shown.

[0055] Depend on Figure 1 As shown, HSAbp-rt-PA completely binds to rHSA at a 1:1 molar ratio of HSAbp-rt-PA:rHSA. At 2:1 and 3:1 molar ratios, a light-colored band of approximately 190 kDa appears. Therefore, it is speculated that HSA contains two binding sites for the fusion protein HSAbp-rt-PA, but one has weaker binding ability. The rHSA-HSAbp-rt-PA with a 1:1 molar ratio of HSAbp-rt-PA:rHSA was selected for subsequent experiments.

[0056] Example 4 Cytotoxicity assay of rHSA-HSAbp-rt-PA and HSAbp-rt-PA (CCK-8 method)

[0057] MTT / CCK-8 method: HUVEC cells were seeded (10 4Cells were placed in the blank group (no cells), the cell control group (HUVEC cells + complete culture medium), the alteplase group (1, 2.5, 5, 10, 20, 50 μg / mL), the HSAbp-rt-PA fusion protein group (equivalent to rt-PA concentrations of 1, 2.5, 5, 10, 20, 50 μg / mL), the rHSA-HSAbp-rt-PA nanoparticle group (equivalent to rt-PA concentrations of 1, 2.5, 5, 10, 20, 50 μg / mL), and the rHSA group (drug-free rHSANPs to exclude carrier toxicity). After 24 h of incubation, 10 μL of CCK-8 working solution (10% CCK-8 + 90% serum-free culture medium) was added to each well, and the absorbance at 450 nm was measured 1 h later. The experiment was repeated ≥3 times to exclude edge effects. The experimental results were statistically analyzed using SPSS, as shown in Table 1 and Table 2. Figure 2 shown.

[0058] Calculate cell viability:

[0059]

[0060] Table 1

[0061]

[0062] 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.

[0063] 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 was much less than that of the rt-PA group.

[0064] Example 5 rHSA-HSAbp-rt-PA and HSAbp-rt-PA activity experiments

[0065] The activity of rHSA-HSAbp-rt-PA and HSAbp-rt-PA solutions was detected using a human t-PA ELISA kit (purchased from Shanghai Chebang Biotechnology 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.

[0066] Table 2

[0067]

[0068] The results showed that the activities of rHSA-HSAbp-rt-PA and HSAbp-rt-PA were not much different from those of rt-PA.

[0069] Example 6 In vitro thrombolysis detection of rHSA-HSAbp-rt-PA and HSAbp-rt-PA

[0070] Fresh blood from a healthy individual was collected and placed in an anticoagulation tube. Thrombin (1-5 U / mL) and CaCl2 (10-20 mM) were added to the anticoagulated whole blood. After mixing, the mixture was injected into a silicone tube or 96-well plate and allowed to stand at 37°C for 1-2 hours to form a thrombus. The thrombus was gently removed, rinsed three times with PBS, and the surface moisture was blotted dry with filter paper and weighed (recorded as W0). The thrombus was then placed in a PBS solution containing a thrombolytic drug; an equal volume of PBS was used for the control group. The tube was incubated at 37°C with constant shaking (50-100 rpm) for 2 hours. The reaction was terminated, and the remaining thrombus was removed, rinsed with PBS, blotted dry, and weighed (recorded as W1). The thrombus lysis rate was calculated. The experimental groups were rHSA-HSAbp-rt-PA and HSAbp-rt-PA, and the positive control was alteplase (rt-PA). The concentrations of rHSA-HSAbp-rt-PA, HSAbp-rt-PA, and rt-PA, the active ingredient in alteplase, were the same (10 μg / mL). The negative control was an equal volume of PBS solution. Formula: Thrombolysis rate (%) = , the results are shown in Table 3.

[0071] Table 3

[0072]

[0073] **Indicates P value ≤ 0.01 compared with the PBS group.

[0074] As shown in Table 3, after statistical analysis, there was no significant difference in the in vitro thrombolytic effect among rHSA-HSAbp-rt-PA, HSAbp-rt-PA and alteplase.

[0075] Example 7 Detection of the Half-Life of rHSA-HSAbp-rt-PA and HSAbp-rt-PA

[0076] Twenty adult SD rats, half male and half female, were divided into two groups of 10. Each group received an intravenous bolus of rHSA-HSAbp-rt-PA or HSAbp-rt-PA (rt-PA clinical equivalent dose: 0.9 mg / kg). Blood was collected from the rat tail vein at 0 (baseline), 2, 5, 10, 15, 20, 25, 30, 40, 50, 60, 80, 100, and 120 minutes, and serum was collected. The retained rt-PA biological activity in the serum was detected using a human t-PA ELISA kit (purchased from Shanghai Chebang Biotechnology Co., Ltd.). A "blood drug activity-time curve" was plotted, and software was used for data fitting and pharmacokinetic parameter analysis. The results are shown in Table 4 and Table 5. Figure 3 shown.

[0077] Table 4

[0078]

[0079] From Table 4 and Figure 3 It can be seen that the half-life of rHSA-HSAbp-rt-PA and HSAbp-rt-PA is significantly increased relative to that of rt-PA (half-life 4-5min), and the half-life of rHSA-HSAbp-rt-PA is increased relative to that of HSAbp-rt-PA. It is speculated that the single HSAbp-rt-PA is partially cleared before binding to HSA in plasma after entering the blood.

[0080] Example 8 rHSA-HSAbp-rt-PA and HSAbp-rt-PA Animal Model Experiments

[0081] Middle cerebral artery occlusion (MCAO) model: Before the experiment, adult mice were fasted for 12 hours but not water. Mice were anesthetized with 5% chloral hydrate (0.1 mL / 10 g body weight) via intraperitoneal injection. After anesthesia took effect, the mice were secured in a supine position 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 isolated. The ECA was ligated proximally, and a suture was threaded under the CCA and ICA for later use. A small incision was made in the ECA approximately 3-4 mm from the bifurcation. A pre-treated suture (approximately 0.26-0.28 mm in diameter) was inserted through the ECA incision into the ICA to a depth of approximately 18-20 mm, occluding blood flow in the middle cerebral artery and inducing cerebral ischemia. Ischemia was maintained for 60 minutes, after which the suture was slowly withdrawn to allow reperfusion.

[0082] Drug administration: The experimental groups received rHSA-HSAbp-rt-PA and HSAbp-rt-PA, while the positive control received alteplase (rt-PA). The concentrations of rHSA-HSAbp-rt-PA, HSAbp-rt-PA, and rt-PA, the active ingredient in alteplase, were the same. The negative control group received an equal volume of PBS. During cerebral ischemia-reperfusion, each drug was injected via the tail vein at a dose of 0.9 mg / kg body weight. Twenty-four hours after reperfusion, the rats were anesthetized with 5% chloral hydrate, and the brains were removed by decapitation. The brains were sectioned in the trough and stained in 2% TTC solution for 8 minutes in a 37°C incubator protected from light. The rats were photographed, and the infarct volume was calculated.

[0083] Percentage of infarct volume = (volume of the contralateral hemisphere - volume of the non-infarcted area of ​​the ipsilateral hemisphere) / volume of the contralateral infarcted hemisphere × 100%.

[0084] The results are shown in Table 5.

[0085] Table 5

[0086]

[0087] 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.

[0088] As shown in Table 5, the effects of rHSA-HSAbp-rt-PA and HSAbp-rt-PA in treating cerebral infarction were significantly better than those of the rt-PA group.

[0089] The foregoing is merely an embodiment of the present invention. The scope of protection of the present invention is not limited by these specific embodiments but is determined by the claims of the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, improvements, etc. made within the technical concepts and principles of the present invention are intended to be included within the scope of protection of the present invention.

Claims

1. A recombinant human serum albumin-alteplase nanodrug, characterized in that: The nanomedicine 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), the alteplase and human serum albumin adhesion peptide are connected by means of a fusion protein, and the molar ratio of the recombinant human serum albumin to the fusion protein is 0.5:1~3:

1.

2. The recombinant human serum albumin-alteplase nanodrug according to claim 1, characterized in that: The molar ratio of the recombinant human serum albumin to the fusion protein is 1:

1.

3. The recombinant human serum albumin-alteplase nanodrug according to claim 1, characterized in that: The human serum albumin adhesion peptide is connected to the recombinant human serum albumin through a non-chemical bond.

4. A composition, characterized in that The composition at least comprises the recombinant human serum albumin-alteplase nanomedicine according to any one of claims 1 to 3 and additives acceptable in the medical field.

5. The composition according to claim 4, characterized in that The dosage form of the composition includes at least one of a nanosuspension, a nanoemulsion, and a freeze-dried powder.

6. Use of the recombinant human serum albumin-alteplase nanodrug according to any one of claims 1 to 3 or the composition according to claim 4 or 5 in the preparation of a medicament for preventing and / or treating thrombotic diseases.

7. The use according to claim 6, characterized in that 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, and antiphospholipid antibody syndrome.

8. A method for preparing the recombinant human serum albumin-alteplase nanodrug according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: S1: Human serum albumin adhesion peptide and alteplase are linked together by cross-linking or recombinant genetic engineering technology; S2: The HSAbp-rt-PA prepared in step S1 is mixed with the recombinant human serum albumin solution to prepare the recombinant human serum albumin-alteplase nanodrug.

9. The method according to claim 8, characterized in that The method further comprises the step of purifying the recombinant human serum albumin-alteplase nanomedicine.

Images