Nano drug delivery system as well as preparation method and application thereof

Abstract

The invention discloses a nano drug delivery system and a preparation method and application thereof.The nano drug delivery system comprises nano particles, a platelet membrane, a tissue-type plasminogen activator and a thrombin specific aptamer, the nano particles are at least partially coated with the platelet membrane, and the thrombin specific aptamer is at least partially coated with the tissue-type plasminogen activator. The tissue-type plasminogen activator and the thrombin specific aptamer are connected to the platelet membrane. The nano drug delivery system disclosed by the invention has good targeting property, a rapid thrombolysis effect and a local anticoagulation effect, and is beneficial to avoiding secondary coagulation and reembolism risks.

Description

Technical Field

[0001] The present application relates to the field of biomedicine technology, and in particular to a nano drug delivery system and a preparation method and application thereof. Background Art

[0002] Thrombotic diseases are a type of disease in which blood clots (thrombi) form abnormally within blood vessels, leading to partial or complete blockage of blood vessels. These clots can occur in arteries, veins, or the heart and can cause serious health problems, including acute ischemic stroke and myocardial infarction.

[0003] Tissue plasminogen activator (tPA) is a widely accepted thrombolytic drug in clinical practice. However, its short half-life, lack of targeting, and susceptibility to systemic bleeding complications significantly limit its clinical efficacy and applicability. Therefore, the development of novel drug delivery systems that can precisely deliver tPA while improving both efficacy and safety has become a hot topic of research. In recent years, the rapid development of targeted drug delivery technologies has provided new insights into the precision treatment of stroke. Natural platelets, in particular, have attracted considerable attention from researchers due to their inherent biocompatibility and specific targeting properties.

[0004] However, current targeted drug delivery technologies typically only have a single therapeutic mechanism—thrombolysis. During the thrombolysis process, as the clot dissolves, some coagulation factors and platelets trapped within the clot may be released, which may temporarily increase the local blood's tendency to coagulate and increase the risk of re-embolism. Summary of the Invention

[0005] The present application provides a nano drug delivery system and its preparation method and application, which are used to solve the problem that the existing anti-thrombotic mechanism is single and the anti-thrombotic effect is poor.

[0006] One aspect of the present application discloses a nano drug delivery system comprising nanoparticles, platelet membranes, tissue-type plasminogen activator and thrombin-specific aptamers, wherein the platelet membranes at least partially cover the nanoparticles, and the tissue-type plasminogen activator and the thrombin-specific aptamer are connected to the platelet membranes. It should be noted that in the nano-drug delivery system of the present application, the nano-scale particle size is conducive to ensuring its stability and safety in the body's circulation; the platelet membrane can highly specifically identify and target the thrombus site, significantly enhancing the nano-drug delivery system's ability to accurately enrich thrombus tissue, effectively improving the utilization efficiency of thrombolytic drugs and reducing systemic side effects; tissue-type plasminogen activator (tPA) is directly loaded on the outer membrane surface without entering the internal structure of the carrier, so after the nanoparticles come into contact with the thrombus, they can be quickly released and exert thrombolytic activity, shortening the lag time of drug release, increasing the speed and efficacy of acute thrombus clearance, and exerting a rapid thrombolytic effect; in addition, the thrombin-specific aptamer (ThrombinAptamer) has extremely high thrombin-specific recognition ability and high affinity, and can accurately identify and efficiently bind thrombin at the thrombus site, while exerting a local anticoagulant effect, inhibiting thrombin activity after thrombolytic therapy, avoiding the risk of secondary coagulation and re-embolism, and further improving the safety and stability of thrombolytic therapy.

[0007] In one implementation of the present application, the nanoparticles are made of poly(lactic-co-glycolic acid) copolymer. It should be noted that poly(lactic-co-glycolic acid) copolymer (PLGA), as the core material of the nanocarrier, has good biocompatibility and biodegradability, and its degradation products are non-toxic and non-irritating, making it safe for clinical use.

[0008] In one implementation of the present application, the particle size of the nanoparticles is 80 nm to 150 nm.

[0009] In one implementation of the present application, the particle size of the nano drug delivery system is 100 nm to 200 nm.

[0010] In one implementation of the present application, the zeta potential of the nano-drug delivery system is -30mV to -20mV. It should be noted that the specific particle size range and surface potential parameters of the nano-drug delivery system give it excellent stability in the blood circulation, significantly prolonging its half-life in the body, enhancing its ability to target and accumulate in thrombotic tissue, and at the same time helping to avoid rapid clearance by the reticuloendothelial system.

[0011] In one implementation of the present application, the thrombin-specific aptamer and the platelet membrane are connected via a thiol-maleimide cross-linking method or a PEG-NHS cross-linking method.

[0012] In an implementation form of the application, the sequence of the thrombin-specific aptamer comprises 5'-GGTTGGTGTGGTTGG-3' (SEQ ID NO. 1). It should be noted that the sequence of the thrombin-specific aptamer designed in the application has excellent thrombin-specific binding ability, can effectively recognize thrombin and inhibit its function.

[0013] In an implementation form of the application, the tissue-type plasminogen activator and the platelet membrane are connected by EDC / NHS cross-linking.

[0014] Another aspect of the application discloses a preparation method of a nano drug delivery system, comprising: coating a nanoparticle with a platelet membrane; connecting a tissue-type plasminogen activator to the platelet membrane; and connecting a thrombin-specific aptamer to the platelet membrane.

[0015] Still another aspect of the application discloses an application of the nano drug delivery system in preparing a drug for treating a patient with thrombotic disease.

[0016] The application has the following beneficial effects:

[0017] It should be noted that in the nano drug delivery system of the application, the nanoscale particle size is conducive to ensuring the stability and safety of its in vivo circulation; the platelet membrane can specifically recognize and target the thrombus site, significantly enhancing the precise enrichment capacity of the nano drug delivery system for thrombotic tissue, effectively improving the utilization efficiency of thrombolytic drugs and reducing systemic side effects; the tissue-type plasminogen activator (tPA) is directly loaded on the outer membrane surface without entering the internal structure of the carrier, so it can be quickly released and exert thrombolytic activity after the nanoparticle contacts the thrombus, shortening the lag time of drug release, improving the speed and efficacy of acute thrombus removal, and exerting rapid thrombolytic effect; in addition, the thrombin-specific aptamer (Thrombin Aptamer) has extremely high thrombin-specific recognition ability and high affinity, can accurately recognize and efficiently bind thrombin at the thrombus site, and at the same time exerts local anticoagulation effect, inhibits the activity of thrombin after thrombolytic therapy, avoids the risk of secondary coagulation and re-embolization, and further improves the safety and stability of thrombolytic therapy. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 is a structural schematic diagram of the nano drug delivery system involved in the application.

[0019] Figure 2 is a graph of the hemolysis rate experiment results of the nano drug delivery system involved in the embodiments of the application.

[0020] Figure 3 is a graph of the experiment results of the influence of the nano drug delivery system involved in the embodiments of the application on each organ. DETAILED DESCRIPTION

[0021] The present invention is further described in detail below by specific embodiments in conjunction with the accompanying drawings. In the following embodiments, many detailed descriptions are intended to enable the present application to be better understood. However, those skilled in the art can readily appreciate that some of the features may be omitted in different circumstances, or may be replaced by other materials or methods. In some cases, some operations related to the present application are not shown or described in the specification. This is to avoid the core of the present application being overwhelmed by excessive descriptions, and for those skilled in the art, it is not necessary to describe these related operations in detail. They can fully understand the related operations based on the description in the specification and the general technical knowledge in the art.

[0022] In addition, the features, operations, or characteristics described in the specification may be combined in any appropriate manner to form various embodiments. Furthermore, the steps or actions in the method description may be reordered or adjusted in a manner readily apparent to those skilled in the art. Therefore, the various sequences in the specification and drawings are provided solely for the purpose of clearly describing a particular embodiment and are not intended to be mandatory, unless otherwise specified.

[0023] The serial numbers assigned to the components in this document, such as "first", "second", etc., are only used to distinguish the objects described and do not have any order or technical meaning.

[0024] Acute ischemic stroke is a leading cause of death and disability worldwide, accounting for over 80% of all strokes. Tissue plasminogen activator (tPA) is currently the only widely accepted thrombolytic drug in clinical practice. However, its short half-life, lack of targeting, and susceptibility to systemic bleeding complications significantly limit its clinical efficacy and applicability. The development of novel drug delivery systems that can precisely deliver tPA while improving both efficacy and safety has become a hot research topic. In recent years, the rapid development of nanoparticle drug delivery technology has provided new avenues for precision treatment of stroke. In particular, biomimetic nanomaterials, due to their inherent biocompatibility and specific targeting properties, have attracted significant attention from researchers. Platelet membranes, as natural biomimetic materials, are considered ideal shell materials for constructing thrombolytic drug carriers due to their inherent ability to target and aggregate thrombi. Aptamers, on the other hand, are high-affinity, highly specific single-stranded nucleic acid ligands that can specifically recognize coagulation-related targets, such as thrombin, exerting a local anticoagulant effect. Therefore, by combining the biomimetic targeting properties of platelet membrane, the thrombolytic pharmacological effects of tPA and the anticoagulant ability of thrombin Aptamer, a new ternary composite nanodrug delivery system is designed, which is expected to achieve rapid thrombolysis and local anticoagulation at the same time, minimize adverse reactions and improve clinical efficacy.

[0025] Against the above background, the present application proposes a new platelet membrane-Aptamer-tPA ternary composite nano-drug delivery system with precise targeting, efficient thrombolysis and local anticoagulation synergistic therapeutic functions, aiming to effectively overcome the clinical bottleneck problems such as low thrombolysis efficiency, short treatment window and high bleeding risk in the current tPA treatment process. The purpose of this application is to provide a platelet membrane-Aptamer-tPA ternary composite nano-drug delivery system with precise thrombus targeting, rapid thrombolysis ability and local anticoagulation function, so as to significantly improve the thrombolytic treatment effect of acute ischemic stroke and effectively reduce the risk of systemic bleeding. The nano-drug delivery system proposed in this application overcomes the clinical difficulties of traditional thrombolytic drugs such as insufficient targeting, slow release rate and high risk of re-embolism through innovative structural design and functional integration, and has important clinical application potential.

[0026] This application provides a nano-drug delivery system and its preparation method and application. It has the following advantages: 1. Rapid and precise thrombolysis: By directly loading tPA onto the platelet membrane surface, the drug can quickly contact the thrombus and quickly release its thrombolytic activity; 2. Efficient thrombus targeting: The natural biomimetic properties of the platelet membrane enable the nanosystem to actively identify and enrich at the site of the thrombus; 3. Local synergistic anticoagulation: The thrombin aptamer specifically recognizes thrombin and inhibits its function, effectively preventing secondary coagulation and embolism after thrombolysis; 4. High safety: The system has good biocompatibility and significantly reduces the risk of systemic bleeding.

[0027] Figure 1 This is a schematic diagram of the structure of the nano drug delivery system involved in this application. Figure 1 As shown, in one embodiment, the nano drug delivery system includes nanoparticles, platelet membranes, tissue-type plasminogen activator, and thrombin-specific aptamers. The platelet membranes at least partially cover the nanoparticles, and the tissue-type plasminogen activator and thrombin-specific aptamers are attached to the platelet membranes.

[0028] In one embodiment, the platelet membrane can coat the nanoparticles to form core-shell nanoparticles.

[0029] In a specific embodiment, the particle size of the nanoparticles may be 80 nm to 150 nm.

[0030] In one embodiment, the average particle size of the nanoparticles may be 120 nm.

[0031] In one embodiment, the surface of the nanoparticles is negatively charged. It should be noted that the electrostatic adsorption between the positive charges on the platelet membrane surface and the negative charges on the PLGA can be used to form a biomimetic core-shell structure.

[0032] In one embodiment, the nanoparticle material may include poly(lactic-co-glycolic acid) (PLGA). It should be noted that poly(lactic-co-glycolic acid) (PLGA), as the core material of the nanocarrier, has good biocompatibility and biodegradability, and its degradation products are non-toxic and non-irritating, making it safe for clinical use.

[0033] In one embodiment, the weight average molecular weight of PLGA may be 10 kDa to 70 kDa.

[0034] In one embodiment, PLGA nanoparticles can be prepared by an emulsion-solvent evaporation method, thereby being able to prepare PLGA nanoparticles with controllable particle size, uniformity, and stability.

[0035] In one embodiment, the platelet membrane is a human platelet membrane. It should be noted that the human platelet membrane retains its natural surface proteins, such as CD41, CD42b, GPIIb / IIIa, etc., which can recognize and target thrombus sites with high specificity.

[0036] In a specific embodiment, the drug release behavior of tissue plasminogen activator complies with the first-order kinetic model, and the cumulative drug release within 30 minutes is greater than 80%.

[0037] In one embodiment, tissue plasminogen activator can be immobilized on the amino or carboxyl groups on the platelet membrane surface via 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) / N-hydroxysuccinimide (NHS) chemical cross-linking.

[0038] In one embodiment, the thrombin-specific aptamer (Aptamer) is an antithrombin aptamer.

[0039] In a specific embodiment, the nucleic acid sequence of the thrombin-specific aptamer includes 5'-GGTTGGTGTGGTTGG-3' (SEQ ID NO. 1) or a functionally modified derivative thereof.

[0040] In one embodiment, the thrombin-specific aptamer (Aptamer) is modified on the platelet membrane surface by thiol-maleimide cross-linking or PEG-NHS cross-linking.

[0041] In a specific embodiment, the particle size of the nano drug delivery system is 100 nm to 200 nm, and the Zeta potential is -20 mV to -30 mV.

[0042] In one embodiment, the nano-drug delivery system exhibits significantly better thrombolytic efficiency and safety compared to free tissue plasminogen activator in the treatment of acute ischemic stroke.

[0043] In a specific embodiment, the preparation method of the nano drug delivery system may include: coating nanoparticles with platelet membranes; linking tissue plasminogen activator to the platelet membranes; and linking thrombin-specific aptamers to the platelet membranes.

[0044] In one embodiment, platelet membranes can be extracted by a freeze-thaw method, which can include the following steps: collecting fresh human whole blood, centrifuging to obtain platelets, repeatedly freezing and thawing the platelets, and physically breaking the platelets to release the membrane structure to obtain the platelet membranes.

[0045] In one embodiment, platelets can be coated onto the surface of nanoparticles through a physical extrusion method. Specifically, the method includes: mixing nanoparticles and platelets, extruding the mixture through a polycarbonate membrane with a pore size of 200 nm, and utilizing electrostatic attraction between the positive charge on the platelet membrane surface and the negative charge carried by the nanoparticles to coat the platelets onto the surface of the nanoparticles, forming a biomimetic core-shell structure.

[0046] In one embodiment, tissue-type plasminogen activator can be linked to the platelet membrane via DEC / NHS chemical crosslinking. Specifically, this includes adding EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and Sulfo-NHS (sodium N-hydroxysuccinimide sulfonate) to core-shell particles, and then adding tissue-type plasminogen activator to perform a crosslinking reaction.

[0047] In one embodiment, a thrombin-specific aptamer can be attached to the platelet membrane via thiol-maleimide crosslinking. Specifically, this can include adding maleimide-PEG-phospholipid (DSPE-PEG-Mal) to core-shell particles, anchoring the maleimide group to the platelet membrane surface via thin film hydration, and then adding a thrombin-specific aptamer containing a terminal thiol group to react to form a thioether bond.

[0048] In a specific embodiment, a nano drug delivery system is provided for use in preparing a drug for treating patients with thrombotic diseases.

[0049] In a specific embodiment, the thrombotic disease may include at least one of acute ischemic stroke, myocardial infarction, etc.

[0050] The present invention is further described in detail below by means of specific examples. The following examples are only provided to further illustrate the present invention and should not be construed as limiting the present invention. In the present examples, unless otherwise specified, the reagents and instruments used are all commercially available, and the experimental procedures are all carried out in accordance with the product specifications and conventional experimental specifications.

[0051] Example 1: Preparation of Nano-drug Delivery System

[0052] (1) Preparation of platelet membrane:

[0053] Fresh anticoagulated whole blood is collected from healthy individuals, and high-purity platelets are obtained through gradient centrifugation (150×g initial centrifugation to separate the platelet-rich plasma layer, followed by secondary centrifugation to purify the platelet precipitate); the platelet suspension is then subjected to repeated freeze-thaw treatment (freezing at -80°C for 30 minutes, thawing in a 37°C water bath for 10 minutes, and repeated three cycles) to release the membrane structure through physical disruption; then, ultrasonic vibration (350W treatment for 10 minutes) is combined to further disrupt cell fragments, and then repeated extraction is performed using a needle filter (400nm pore size) to remove residual intracellular substances and form platelet membrane vesicles; finally, the precipitate is collected by ultracentrifugation (such as 4000×g centrifugation for 3 minutes), and intact platelet membranes are obtained after washing with PBS containing protease inhibitors. This method achieves efficient platelet separation through gradient centrifugation, combines freeze-thaw-ultrasound synergistic action to disrupt cells, and ultimately obtains functional platelet membranes that retain membrane protein markers such as CD41 and CD61.

[0054] (2) Preparation of platelet membrane core-shell structure:

[0055] Using an emulsion-solvent evaporation method, poly(lactic-co-glycolic acid) (PLGA) was dissolved in dichloromethane (20-50 mg / mL) to form an oil phase. This was then added dropwise to an aqueous phase containing polyvinyl alcohol (PVA, 2-5% w / v). Ultrasonic emulsification (30-70W, 20kHz, 30 seconds to 2 minutes) was performed to form a stable O / W emulsion. After 3-5 hours of magnetic stirring to evaporate the dichloromethane, PLGA nanoparticles solidified and precipitated. Free PVA and residual solvent were removed by differential centrifugation (4000×g, 5 minutes), yielding uniform nanoparticles with a diameter of approximately 120 nm and a negative surface charge (zeta potential approximately -20 mV). The PLGA nanoparticles were then mixed with platelet membranes at a mass ratio of 40-60:1 and extruded 10-15 times through a 200 nm pore-size polycarbonate membrane. Electrostatic attraction between the positive surface charges of the platelet membranes and the negative charges of the PLGA nanoparticles formed a biomimetic core-shell structure.

[0056] (3) Loading and modification of tPA and Aptamer:

[0057] The core-shell nanoparticles were dispersed in 50 mM MES buffer (pH 6.0), and EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and 5 mM Sulfo-NHS (sodium N-hydroxysuccinimide sulfonate) were added to a final concentration of 2 mM. The reaction was stirred at room temperature for 15 minutes. rt-PA (recombinant tissue plasminogen activator) was then dissolved in 0.1 M sodium phosphate buffer (pH 7.5) and added to the activated nanoparticle suspension at a mass ratio of 1:1-2 (rt-PA: nanoparticles). The amide bond coupling was completed at room temperature for 2 hours. Unbound tPA was removed by ultrafiltration (100 kDa molecular weight cutoff), and the loading capacity was confirmed to be 50 μg / mg of nanoparticles by BCA protein quantification. Maleimide-PEG-phospholipid (DSPE-PEG-Mal) was added to the tPA-loaded nanoparticles, and the maleimide groups were anchored to the platelet membrane surface via thin film hydration (molar ratio 1:50). A thrombin-specific aptamer containing a terminal thiol group (such as HD1 or HD22) was synthesized and purified by HPLC to ensure thiol activity. The thiol-modified aptamer (nucleotide sequence: 5'-GGTTGGTGTGGTTGG-3') was mixed with the tPA-modified nanoparticles at a molar ratio of 10:1 in PBS buffer (pH 7.4) and allowed to react in the dark for 4 hours to form a stable thioether bond. After ultrafiltration and centrifugation to remove free aptamer, the coupling efficiency was verified by fluorescence labeling (FITC-labeled aptamer) to obtain platelet membrane-aptamer-tPA nanoparticles, also known as PLPA@PFP nanoparticles.

[0058] Example 2: In vitro verification of the targeting of the nano-drug delivery system

[0059] After immobilizing C57 mice, the eyes were quickly enucleated and peripheral blood was drawn into a centrifuge tube containing EDTA-K2 anticoagulant (approximately 500-600 μL). The blood and anticoagulant were gently mixed and centrifuged to obtain the upper platelet-rich plasma and the middle layer (the buffy coat, after separation of the upper layer containing platelets and white blood cells). A secondary purification was then performed to obtain a platelet pellet, which was resuspended in PBS containing 0.3% heat-inactivated serum. The platelet suspension was then mixed with collagen (10 μg / mL) and ADP (10 μM) to induce platelet activation and aggregation. Thrombin (2 U / mL) was then added and incubated (37°C, 5% CO2, 1 hour) to promote fibrin cross-linking and the formation of a dense thrombus-like structure. Platelet-rich static thrombi were mixed with 1 mL of saline, 1 mL of CY5.5-labeled rt-PA (10 μg / mL), and 1 mL of CY5.5-labeled platelet membrane-Aptamer-tPA nanodrug delivery system (nanodrug delivery system concentration of 50 μg / mL, containing t-PA at a concentration of 10 μg / mL) to obtain saline control group, rt-PA group, and platelet membrane-Aptamer-tPA group. The mixture was then incubated with the thrombi at 37°C for 1 hour. After incubation, the thrombi were removed and washed three times with PBS buffer. The targeting ability of the PLPA@PFP nanoparticles was verified using fluorescence analysis on a small animal imaging system (IVIS).

[0060] The results showed that the adhesion ability of the nanosystem of the present application to thrombus was 3.6 times that of the control group (rt-PA group), and there was a significant difference (p<0.01).

[0061] Example 3: In vitro verification of the thrombolytic effect of the nano-drug delivery system

[0062] Thrombi were prepared according to the method of Example 1. The standardized thrombus samples were then subjected to thrombolysis experiments in sterile test tubes with three treatment solutions: 1 mL of normal saline, 1 mL of 10 μg / mL rt-PA, and 1 mL of 50 μg / mL platelet membrane-Aptamer-tPA (containing 10 μg / mL rt-PA). The experimental system was incubated in a 37°C constant temperature shaking water bath. The following measurements were performed at 0, 1, 2, and 3 hours: thrombus volume reduction and fibrin network dissociation were recorded using an optical microscope; the supernatant was collected and the absorbance at 410 nm and 540 nm was measured using a microplate reader.

[0063] The results showed no significant change in absorbance in the saline group, indicating no spontaneous thrombus lysis. The rt-PA group showed an approximately 45% increase in absorbance at 410 nm within 3 hours, demonstrating partial fibrin degradation activity. At the same time point, the platelet membrane-Aptamer-tPA group showed an 82% increase in absorbance at 410 nm, with a significant increase in the 540 nm signal. This suggests that the synergistic effect of aptamer-mediated targeted thrombin binding and platelet membrane biomimetic delivery significantly enhanced the local concentration and thrombolytic efficiency of rt-PA. It is important to note that 540 nm is the absorption peak of hemoglobin during red blood cell release. If red blood cells are present in the thrombus and are destroyed by tPA, releasing hemoglobin, the absorbance at 540 nm increases. In this experiment, the increased 540 nm signal indicates more complete disruption of the thrombus structure (including red blood cells), indirectly indicating the depth and efficiency of thrombolysis.

[0064] Example 4: In vivo verification of the thrombolytic effect of the nano-drug delivery system

[0065] C57BL / 6 mice were anesthetized with 2% isoflurane inhalation anesthesia and fixed in the prone position. Hair was removed with a shaver and disinfected with iodine. A midline skin incision was made along the neck, and tissue dissection was performed to expose the left common, external, and internal carotid arteries. The common and internal carotid arteries were briefly ligated, and a cannula was placed through the external carotid artery into the internal carotid artery until it reached the middle cerebral artery. Blood from inbred mice was then slowly infused to create a thrombus in vitro. A middle cerebral artery occlusion model was established using thromboembolism. Following modeling, mice were injected via the tail vein with drugs. The mice were divided into the following experimental groups: saline group, rt-PA group, or platelet membrane-Aptamer-tPA group. Each group received the following treatments: 1 mL of saline, 1 mL of 10 μg / mL rt-PA, or 1 mL of 50 μg / mL platelet membrane-Aptamer-tPA (containing 10 μg / mL rt-PA), respectively. Neurological function scores and brain tissue TTC staining were performed 24 hours after administration. The results showed that the cerebral blood flow recovery rate in the nanodrug delivery system treatment group was 92%, significantly higher than the 68% in the free tPA group, and the infarct volume was significantly reduced.

[0066] Example 5: Biosafety of Nanodrug Delivery Systems

[0067] (1) Platelet membrane-Aptamer-tPA was co-incubated with red blood cells to observe whether it had a hemolytic effect and verify the biocompatibility of PLPA@PFP nanoparticles. The specific method was as follows: fresh mouse blood was collected using an EP tube containing heparin. After standing at room temperature for 1 hour, it was centrifuged at 3000 rpm for 20 minutes. The supernatant was removed, and 2-3 times the amount of physiological saline was added to resuspend the precipitate and centrifuged again. Repeat 3-4 times until the supernatant was transparent. The supernatant was discarded to obtain the red blood cell pellet. The red blood cells were mixed with physiological saline to form a 4% suspension for later use. 400uL of platelet membrane-Aptamer-tPA with concentrations of 0.1, 1, 10, 100, 500, and 1000μg / mL was added to the red blood cells and incubated at 37℃ for 3h. Physiological saline was used as a negative control and distilled water as a positive control. Three replicates were performed for each group. After incubation, the solution was centrifuged at 3000rpm for 20 minutes. The supernatant was taken and the absorbance value at 540nm was measured using an enzyme-linked microplate reader. Figure 2 This is a graph showing the hemolysis rate experimental results of the nano drug delivery system involved in the embodiments of the present application.

[0068] (2) Platelet membrane-Aptamer-tPA was co-incubated with human umbilical vein endothelial cells to verify whether it had an effect on the activity of endothelial cells. The specific method was as follows: Human umbilical vein endothelial cells (HUVECs) were seeded in a 96-well plate at 5,000 cells per well and cultured at 37°C and 5% carbon dioxide using DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin double antibody solution. When the cells reached the logarithmic growth phase, the original culture medium in the 96-well plate was removed, and nanodrug solutions were prepared using DMEM medium at concentrations of 0, 1, 10, 100, 500, and 1000 ug / mL and added to each well. The 0 μg / mL nanodrug solution was used as the control group and co-cultured with the cells for 24 hours. After 24 hours, the culture medium was discarded and each well was gently rinsed with 200uL of physiological saline, repeated twice, and then 100uL of 10% CCK-8 solution was added to each well. The cells were incubated in the dark for 1 hour, and then the OD absorbance value of each well at 415nm was detected using a microplate reader to calculate the cell activity.

[0069] (3) Platelet membrane-Aptamer-tPA was injected into the tail vein of mice to observe the biosafety of the drug in vivo. The specific method is as follows: healthy mice were randomly divided into 5 groups, namely the control group and the 1, 7, 14, and 21 day groups. The control group was given 200ul of normal saline, and the other groups were injected with 200ul of drug. The drug was injected into the mouse body through the tail vein. Euthanasia was performed 1, 3, 7, 14, and 21 days after injection. Blood was then collected for routine blood tests and blood biochemical tests. The main organs: heart, liver, spleen, lung, and kidney were collected, paraffin-embedded and sectioned, and H&E staining was performed to observe the pathological changes of the main organs. Figure 3Figure 1 is a graph showing the experimental results of the influence of the nano drug delivery system involved in the embodiments of the present application on various organs.

[0070] The above results show that the hemolysis rate of the platelet membrane-Aptamer-tPA nanoparticles prepared in the present application is very low, and has basically no effect on cell activity, does not cause pathological changes in organs, is safe and non-toxic, and has high biocompatibility.

[0071] The above is a further detailed description of the present application in combination with specific embodiments, and cannot be considered as limiting the specific implementation of the present application to these descriptions. For ordinary skilled persons in the technical field to which the present application belongs, some simple deductions or substitutions can be made without departing from the concept of the present application.

Claims

1. A nano drug delivery system, characterized in that: The invention comprises nanoparticles, platelet membranes, tissue-type plasminogen activator and thrombin-specific aptamers, wherein the platelet membranes at least partially cover the nanoparticles, and the tissue-type plasminogen activator and the thrombin-specific aptamers are connected to the platelet membranes.

2. The nano drug delivery system according to claim 1, characterized in that The material of the nanoparticles includes poly(lactic acid-co-glycolic acid).

3. The nano drug delivery system according to claim 1, characterized in that The particle size of the nanoparticles is 80nm to 150nm.

4. The nano drug delivery system according to claim 1, wherein: The particle size of the nano drug delivery system is 100nm to 200nm.

5. The nano drug delivery system according to claim 1, wherein: The zeta potential of the nano drug delivery system is -30mV to -20mV.

6. The nano drug delivery system according to claim 1, characterized in that: The thrombin-specific aptamer and the platelet membrane are connected through a thiol-maleimide cross-linking method or a PEG-NHS cross-linking method.

7. The nano drug delivery system according to claim 1 or 6, characterized in that: The sequence of the thrombin-specific aptamer includes 5'-GGTTGGTGTGGTTGG-3'.

8. The nano drug delivery system according to claim 1, wherein: The tissue plasminogen activator and the platelet membrane are connected by EDC / NHS cross-linking.

9. A method for preparing a nano drug delivery system, characterized in that: include: Coating the platelet membrane with nanoparticles; allowing tissue plasminogen activator to attach to the platelet membrane; A thrombin-specific aptamer is attached to the platelet membrane.

10. Use of the nano drug delivery system according to any one of claims 1 to 8 in the preparation of a drug for treating patients with thrombotic diseases.

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