CN112823791A

CN112823791A - Bionic nano drug delivery system for protein thrombolytic drug and application of bionic nano drug delivery system

Info

Publication number: CN112823791A
Application number: CN201911134484.8A
Authority: CN
Inventors: 陆伟跃; 王松立; 王睿峰; 谢操
Original assignee: Fudan University
Current assignee: Fudan University
Priority date: 2019-11-19
Filing date: 2019-11-19
Publication date: 2021-05-21

Abstract

The invention belongs to the field of pharmacy, relates to a bionic nano drug delivery system for a protein thrombolytic drug, and particularly relates to a nano drug delivery system for a protein thrombolytic drug coated by a biological membrane, and preparation and application thereof. The protein-loaded thrombolytic drug nanoparticles are used as the skeleton kernel to be supported and filled in the biomembrane sac to prepare the protein-loaded thrombolytic drug bionic nano drug delivery system which has good biocompatibility and long systemic circulation time and can deliver drugs to focus positions in a targeted manner.

Description

Bionic nano drug delivery system for protein thrombolytic drug and application of bionic nano drug delivery system

Technical Field

The invention belongs to the field of pharmacy, and relates to a bionic nano drug delivery system of a protein thrombolytic drug, preparation of the drug delivery system, and application of the drug delivery system in treatment of arterial thrombotic diseases.

Background

Thrombotic diseases seriously endanger human health and life, and comprise a plurality of common diseases and frequently encountered diseases such as ischemic cerebral apoplexy, myocardial infarction, pulmonary thromboembolism and the like, and become a high-mortality disease after tumors. The immediate detection and administration of thrombolytic drugs are the best way to improve the survival rate of patients and reduce the incidence rate, and from the 80 s in the 20 th century, thrombolytic drugs are applied to the treatment of thromboembolic diseases, and clinically, the thrombolytic drugs are developed from the first generation of streptokinase and urokinase to the current reteplase and tenecteplase, and the specificity, half-life period, thrombolytic efficiency and the like are obviously improved. However, the development of clinical thrombolytic drugs for decades still cannot avoid the occurrence of side effects such as bleeding. The current targeted treatment strategies such as thrombolysis catheters and the like are expensive, are limited by early drugs, and are not easy to control the drug concentration. Thus, there are still many problems to be solved in relation to the efficacy and potential adverse effects of thrombolytic drugs.

Research on thrombolysis has shown that safety problems caused by off-target are reduced and the circulation time in blood is increased by encapsulating a thrombolytic drug in a targeting nanocarrier, or that thrombolytic effect is improved by coupling the thrombolytic drug with an antibody or ligand that specifically binds to a thrombus component (platelet or fibrin), but targeted delivery of thrombolytic drugs relies on exogenous short peptide modification, such as liposome surface modification RGD to achieve platelet-specific targeting; research shows that the introduction of exogenous short peptide can activate immune system, increase phagocytosis of macrophage to drug delivery system to reduce biological half-life period, and further influence thrombolysis effect; antibodies or ligands modifying thrombolytic drug molecules may also affect their activity and may cause rapid induction of inactivation or clearance of plasma.

Based on the current situation of the prior art, the invention aims to provide a bionic nano drug delivery system of a protein thrombolytic drug. The protein thrombolytic drug bionic nano drug delivery system has good biocompatibility, can deliver drugs to thrombus sites in a targeted manner, improves the drug concentration and thrombolytic effect of the thrombus sites, reduces side effects generated by the drugs, and has wide application prospects.

Disclosure of Invention

The invention aims to overcome the defects of the existing protein thrombolytic drugs and preparations based on the current state of the prior art, and provides a bionic nano drug delivery system for the protein thrombolytic drugs, which has good biocompatibility, can deliver the drugs to thrombus parts in a targeted manner, improves the thrombolytic effect, and reduces side effects.

Specifically, the biomimetic nano drug delivery system for the protein thrombolytic drug provided by the invention comprises a surface biological membrane and a skeleton kernel carrying the protein thrombolytic drug, and the particle size is 50-1000nm, preferably 100-200 nm.

The biological membrane related by the invention is a natural cell membrane or an artificial biological membrane or a mixed membrane consisting of the natural cell membrane and the artificial biological membrane. The natural cell membrane is erythrocyte membrane, platelet membrane, macrophage membrane or leukocyte membrane; the artificial biomembrane is liposome membrane; the mixed membrane composed of natural cell membrane and artificial biomembrane is a hybrid membrane.

The thrombolytic drug of the invention is tissue plasminogen activator, plasminogen kinase such as urokinase, and/or fibrin hydrolase such as lumbrokinase.

The skeleton inner core of the protein-carried thrombolytic drug is a nanoparticle of the protein-carried thrombolytic drug. The nanoparticles are nanoparticles prepared from organic polymer materials or monomers or inorganic materials by a certain physical and/or chemical method.

The biological membrane on the surface of the bionic nano drug delivery system of the protein thrombolytic drug comprises endogenous P-selectin protein and/or self-recognition regulatory immune proteins such as CD47, CD55, CD59 and the like.

The invention provides preparation of a biomimetic nano drug delivery system of a protein thrombolytic drug and application of the biomimetic nano drug delivery system in treating arterial thrombotic diseases.

The invention carries out the experiment:

1. preparation and characterization of bionic nano drug delivery system of protein thrombolytic drug

The skeleton kernel of the protein-loaded thrombolytic drug nanoparticle is prepared by a multiple emulsion method, and then a biomembrane is coated on the surface of the skeleton kernel by ultrasound to characterize the physicochemical properties of the skeleton kernel and investigate the serum stability and the drug release characteristics of the skeleton kernel.

2. Pharmacokinetic investigation of biomimetic nano-drug delivery system of protein thrombolytic drugs

The protein thrombolytic drug bionic nano drug delivery system is marked by fluorescein, blood is taken from the orbit after tail vein injection of a mouse, the in vivo pharmacokinetics behavior of the protein thrombolytic drug bionic nano drug delivery system is inspected, and the tissue distribution of the protein thrombolytic drug bionic nano drug delivery system is inspected.

3. In vitro targeting of bionic nano drug delivery system of protein thrombolytic drug

The adhesion capability of the biomimetic nano drug delivery system of the protein thrombolytic drug is evaluated by the in vitro combination of the fluorescence labeling method and fibrin and Human Umbilical Vein Endothelial Cells (HUVEC), and the affinity capability of the biomimetic nano drug delivery system of the protein thrombolytic drug to arterial thrombus is evaluated by the combination of the fluorescence labeling method and platelet rich plasma clot.

4. In vivo targeting of bionic nano drug delivery system of protein thrombolytic drug

The target ability and specificity of the protein thrombolytic drug bionic nano drug delivery system to thrombus in vivo are evaluated by adopting a fluorescein labeled protein thrombolytic drug bionic nano drug delivery system and a carotid artery thrombus model mouse.

5. Drug effect evaluation of protein thrombolytic drug bionic nano drug delivery system

The therapeutic effect of the protein thrombolytic drug bionic nano drug delivery system on the carotid artery thrombus after tail vein administration is examined through a carotid artery thrombus model mouse.

6. Safety evaluation of bionic nano drug delivery system of protein thrombolytic drug

By measuring the bleeding time and the fibrinogen content in plasma, the side effect of the thrombolytic protein drug bionic nano drug delivery system on the blood coagulation system is investigated.

The experimental result shows that the bionic nano drug delivery system for the protein thrombolytic drug has an obvious core-shell structure under a transmission electron microscope and has good stability in 20% serum; in vivo and in vitro targeting experiments prove that the bionic nano drug delivery system for the protein thrombolytic drugs can deliver the protein thrombolytic drugs to thrombus parts in a targeted manner through adhesive proteins on the surfaces of biological membranes, can effectively improve the drug concentration and thrombolytic effect of the thrombus parts, reduce the drug dosage, reduce side effects such as systemic hemorrhage related to the dosage and the like, and has good application prospect.

Drawings

FIG. 1: the characterization result of the poly (acetic acid-glycolic acid) nano-particle coated by the Lumbrukinase (LBK) biomembrane, wherein,

FIG. A is an electron microscope picture of poly (glycolic acid-co-glycolic acid) (PLGA-NPs/LBK) loaded with lumbrokinase, FIG. B is an electron microscope picture of poly (glycolic acid-co-glycolic acid) (PEG-NPs/LBK) modified by polyethylene glycol loaded with lumbrokinase, FIG. C is an electron microscope picture of PLGA nano particle (RBC-NPs/LBK) coated by erythrocyte membrane loaded with lumbrokinase, FIG. D is an electron microscope picture of PLGA nano particle (PNPs/LBK) coated by platelet membrane loaded with lumbrokinase, FIG. E is a particle size result of different nano particles, and FIG. F is a Zeta potential result of different nano particles;

as shown in the figure, the particle size of PLGA-NPs/LBK is about 190nm, the particle size is increased to 210nm after coating a platelet membrane, and the same result can be obtained after coating a erythrocyte membrane; the zeta potential of PNPs/LBK is close to that of platelets and is obviously different from that of PLGA-NPs/LBK; the transmission electron microscope image of PNPs/LBK can observe an obvious core-shell structure of the membrane-coated nanoparticles, and proves that the platelet membrane is successfully coated on the PLGA nanoparticles.

FIG. 2: the serum stability and the in vitro drug release characteristics of the lumbrokinase-loaded biomembrane coated poly (acetic acid-glycolic acid) nanoparticle are provided, wherein,

FIG. A is serum stability of PEG-NPs/LBK, RBC-NPs/LBK, PNPs/LBK at 37 deg.C, and FIG. B is in vitro release curve of lumbrokinase in the above nanoparticles;

the stability results show that the three groups of nanoparticles have better stability in 20% serum, the particle size is slightly increased after the nanoparticles are placed at 37 ℃ for 48 hours, the PEG-NPs/LBK group is increased from 190nm to about 210nm, the RBC-NPs/LBK group is increased from 220nm to about 250nm, and the PNPs/LBK group is increased from 215nm to about 260 nm;

the in vitro release shows that the lumbrokinase in the PLGA nanoparticles has no burst release phenomenon, the release reaches nearly 50% within 4h, and the cumulative release curve of the nanoparticles after 24 hours tends to be flat and reaches about 70%. The coated biofilm did not affect the release of lumbrokinase and released most of lumbrokinase within 5 hours.

FIG. 3: the result of the pharmacokinetics and tissue distribution of the nanometer particle of the biological film coating poly acetic acid-glycolic acid in vivo, wherein,

the graph A and the graph B are the results of fluorescence distribution in different organs after 4 hours and 8 hours of tail vein injection of PEG-NPs/DiD, RBC-NPs/DiD, PNPs/DiD, respectively, and the graph C is the pharmacokinetic curve of PEG-NPs/DiD, RBC-NPs/DiD, PNPs/DiD in vivo; the result shows that after tail vein injection, PEG-NPs/DiD, RBC-NPs/DiD and PNPs/DiD are mainly distributed in organs such as liver, spleen and the like, and are rarely accumulated in kidney, the brain and lung tissues are rarely distributed, and the accumulation amount in the organs is increased along with the time extension; relative to the difference in the rate of in vivo clearance of PEG-NPs/DiD, there was still about 50% residue in blood after 7 hours of injection in the RBC-NPs/DiD and PNPs/DiD groups.

FIG. 4: evaluation of cytotoxicity and hemolytic toxicity of the biomembrane-coated poly (acetic acid-glycolic acid) nanoparticle, wherein,

the graph A is the hemolytic toxicity test result of PEG-NPs, RBC-NPs and PNPs with different PLGA concentrations on erythrocytes, and the graph B is the toxicity test result of PEG-NPs, RBC-NPs and PNPs with different PLGA concentrations on HUVEC; as shown in the figure, PEG-NPs, RBC-NPs and PNPs do not generate obvious hemolysis when the concentration of PLGA reaches 0.5 mg/mL. Similarly, PEG-NPs, RBC-NPs, PNPs did not significantly affect HUVEC survival at PLGA concentrations of 0.0312 mg/mL.

FIG. 5: the biological membrane coated poly (acetic acid) -glycolic acid nanoparticles are combined with in vitro arterial thrombus components (vascular endothelial cells, fibrin) and platelet rich plasma clots,

panel A and B are the results of the binding capacity of PEG-NPs/DiD, RBC-NPs/DiD, PNPs/DiD to fibrin and platelet rich clot, respectively, and panel C and panel D are the results of the binding capacity of PEG-NPs/DiD, RBC-NPs/DiD, PNPs/DiD to HUVEC in the inactivated and activated states, respectively; the result shows that compared with PEG-NPs/DiD and RBC-NPs/DiD, the binding capacity of PNPs/DiD to fibrin and platelet rich plasma clot is obviously enhanced; PNPs/DiD have strong binding activity to HUVEC activated by tumor necrosis factor alpha (TNF-alpha), but no binding activity to non-activated HUVEC.

FIG. 6: the biological membrane coated poly acetic acid-glycolic acid nanoparticles have targeting property on a carotid artery thrombosis model mouse induced by ferric chloride, wherein,

the left column is a photo detected by photoacoustic imaging after 30 minutes of tail vein injection administration; the right column is a photograph of an isolated carotid artery thrombus tissue section 30 minutes after tail vein injection administration; the result shows that the PEG-NPs/DiR and RBC-NPs/DiR groups show weak fluorescence signals at the thrombus part of the mouse, and the fluorescence signals shown by the PNPs/DiR groups are obviously enhanced, so that the PNPs/DiR has stronger affinity to the thrombus, and the fluorescence imaging of the surface of the blood vessel cavity of the longitudinal section of the carotid artery of the mouse further confirms the result.

FIG. 7: the lumbrokinase-loaded biomembrane coated polyacetic acid-glycolic acid nanoparticle has thrombolytic effect on carotid artery thrombosis model mice, wherein,

panel A is an image of H & E section of mouse carotid artery cross section after PEG-NPs/LBK, RBC-NPs/LBK, PNPs/LBK, LBK (8 x 10^4U/kg), LBK (2.4 x 10^5U/kg) treatment, panel B is the ratio of thrombus area to blood vessel lumen area shown by H & E section of each group of carotid arteries after treatment, and panel C is the absorbance value of 280nm of each group of isolated carotid lysate; the results show that LBK (8X 10^4U/kg) slightly dissolves thrombus; the thrombus-targeted PNPs/LBK can obviously reduce or even disappear the area of carotid thrombus, and the effect is equivalent to that generated by high-dose LBK (2.4 multiplied by 10^ 5U/kg); only a certain degree of thrombolysis can be observed by PEG-NPs/LBK and RBC-NPs/LBK; the same results were obtained for the absorbance results of carotid lysates, with the lowest OD in the thrombus-targeted PNPs/LBK group, similar to the level produced by high dose LBK (2.4X 10^5U/kg), indicating that PNPs/LBK had the best thrombolytic effect.

FIG. 8: the in vivo safety evaluation of the poly (acetic acid-glycolic acid) nano particle coated by the lumbrokinase biomembrane is carried out, wherein,

FIG. A shows the bleeding time of mice 30 minutes after caudal vein injection of PEG-NPs/LBK, RBC-NPs/LBK, PNPs/LBK, LBK (8 x 10^4U/kg), LBK (2.4 x 10^5U/kg), and FIG. B shows the fibrinogen content of the blood of the mice 2 hours after caudal vein injection of PEG-NPs/LBK, RBC-NPs/LBK, PNPs/LBK, LBK (8 x 10^4U/kg), LBK (2.4 x 10^ 5U/kg); the results show that free lumbrokinase administration resulted in a significant prolongation of tail bleeding time compared to PBS-injected mice, and that bleeding time was positively correlated with lumbrokinase dosage, LBK (8X 10^4U/kg) was prolonged to about 250 seconds, LBK (2.4X 10^5U/kg) was prolonged to about 430 seconds, while PNPs/LBK had minimal effect on bleeding time, slightly increased over PBS group; in addition, after administration, the fibrinogen content in blood of LBK (2.4 x 10^5U/kg) is reduced from the normal value to 1.005mg/mL, the fibrinogen content in blood of LBK (8 x 10^4U/kg) group is reduced to 1.821mg/mL, and the PNPs/LBK is only reduced to 2.651mg/mL, which shows that the influence on the fibrinogen content in blood is small.

Detailed Description

The embodiments of the present invention will be described in detail with reference to specific examples, but the present invention is not limited to the following ranges.

Example 1

Preparation and characterization of lumbrokinase-loaded biomembrane coated polyacetic acid-glycolic acid nanoparticles

1) Preparation and characterization of lumbrokinase-loaded erythrocyte membrane-coated polyacetic acid-glycolic acid nanoparticles

Taking male ICR mouse whole blood, centrifuging at the temperature of 1000 g/min and 4 ℃ for 5 minutes, removing upper serum and leukocyte layers, washing lower red blood cells by using 1 XPBS, then re-suspending in 0.25 XPBS at the temperature of 4 ℃ for 30 minutes, centrifuging (15000 g/min and 4 ℃) for 7 minutes to remove hemoglobin, re-suspending the obtained light red cell membrane and storing in double distilled water, and detecting the concentration of the membrane protein by using a BCA kit; preparing PLGA nano-particles (PLGA-NPs/LBK) carrying lumbrokinase by a multiple emulsion method: dissolving a proper amount of PLGA in dichloromethane, then mixing with a lumbrokinase solution dissolved in water, ultrasonically treating the mixture to form primary emulsion, adding the primary emulsion into a proper amount of external water phase, ultrasonically treating the mixture to form multiple emulsion, stirring, centrifuging and washing the mixture with water to obtain the PLGA nano-particle carrying lumbrokinase; then mixing the obtained mixture with a certain amount of erythrocyte membranes, and coating the erythrocyte membranes on the surface of the PLGA nanoparticles by ultrasound to obtain lumbrukinase erythrocyte membrane coated poly (acetic acid) -glycolic acid nanoparticles (RBC-NPs/LBK). The morphology was observed by electron microscopy and the particle size and zeta potential were measured by a laser scattering particle sizer (as shown in FIG. 1).

2) Preparation and characterization of lumbrokinase-loaded platelet membrane-coated poly (acetic acid-glycolic acid) nanoparticles

Taking male ICR mouse whole blood, centrifuging at 300 g/min and 4 ℃ for 5 minutes, taking supernatant, centrifuging at 2000g/min for 5 minutes, discarding the supernatant, washing lower-layer platelets with double distilled water, repeatedly freezing and thawing for 3 times, centrifuging at 20000 g/min for 7 minutes, resuspending the obtained white platelet membrane, preserving the membrane protein in the double distilled water, and detecting the membrane protein concentration by using a BCA kit. Other preparation methods of RBC-NPs/LBK are adopted to obtain platelet membrane coated lumbrokinase polyacetic acid-glycolic acid nanoparticles (PNPs/LBK), and the characterization results are shown in figure 1.

3) Preparation of fluorescein (DiD or DiR) -labeled biofilm-coated polyacetic acid-glycolic acid nanoparticles

Preparing fluorescein labeled PLGA nanoparticles by a multiple emulsion method: dissolving a proper amount of PLGA and fluorescein (DiD or DiR) in dichloromethane, then mixing with pure water, performing ultrasonic treatment to form primary emulsion, adding the primary emulsion into a proper amount of external water phase, performing ultrasonic treatment to form multiple emulsion, stirring, centrifuging and washing with water to obtain the fluorescein labeled PLGA nano-particles. Then mixing the mixture with a certain amount of erythrocyte membranes or platelet membranes, and coating the surfaces of the PLGA nanoparticles with cell membranes by ultrasound to obtain fluorescein-labeled erythrocyte membrane-coated poly (acetic acid-glycolic acid) nanoparticles (RBC-NPs/DiD or RBC-NPs/DiR) or fluorescein-labeled platelet membranes-coated poly (acetic acid-glycolic acid) nanoparticles (PNPs/DiD or PNPs/DiR).

4) Preparation of polyethylene glycol (PEG) -modified polyacetic acid-glycolic acid nanoparticles

Preparing PEG modified PLGA nanoparticles by a multiple emulsion method: dissolving a proper amount of PLGA and mPEG2000-DSPE in dichloromethane, mixing with pure water, performing ultrasonic treatment to form primary emulsion, adding the primary emulsion into a proper amount of external water phase, performing ultrasonic treatment to form multiple emulsion, stirring, centrifuging, and washing with water to obtain the PEG modified PLGA nanoparticles (PEG-NPs).

Example 2

In vitro release of lumbrokinase-loaded biofilm-coated poly (acetic acid) -glycolic acid nanoparticle drug delivery system

PLGA-NPs/LBK, PEG-NPs/LBK, RBC-NPs/LBK, PNPs/LBK, each 8mg, were dispersed in 4mL EP tubes, 3mL volumes. Samples were taken at 0.25, 0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12, 18, 24, 48 hours in a constant temperature shaker at 37 ℃ with constant shaking at 120rpm and a sample volume of 200. mu.L supplemented with the same volume of PBS. The sample was centrifuged at 15000rpm at 10 ℃ for 30 minutes, the supernatant was separated, 100. mu.L of the supernatant was sampled, the lumbrokinase concentration was measured according to the lumbrokinase substrate activity assay, and the cumulative release percentage was calculated, and the results are shown in FIG. 2B.

Example 3

Pharmacokinetics and tissue distribution in vivo of poly (acetic acid-glycolic acid) nanoparticles coated by lumbrokinase biomembrane

Normal ICR mice were used to examine pharmacokinetic behavior and tissue distribution in vivo. 200 mu LPEG-NPs/DiD, RBC-NPs/DiD and PNPs/DiD were injected into the tail vein, respectively, 50. mu.L of blood was collected from the orbit at 5, 30 minutes, 1, 3, 7, 24, and 48 hours, diluted 3-fold with PBS, the fluorescence intensity of the blood sample was measured at 644nm/663nm, and the change curve of the fluorescence intensity with time was plotted. Furthermore, pentobarbital was intraperitoneally administered 4 to 8 hours after tail vein injection, blood was collected, organs (heart, liver, spleen, lung, kidney and brain) of the mouse were isolated after heart perfusion, and the tissue was homogenized and the fluorescence intensity of the tissue homogenate was measured at 644nm/663nm, and the results are shown in FIG. 3.

Example 4

Hemolytic toxicity and cytotoxicity of biomembrane coated poly (acetic acid) -glycolic acid nanoparticles

Hemolytic toxicity: a series of PLGA concentration gradient biofilm-coated nanoparticles were incubated with freshly prepared mouse erythrocyte suspensions (5% erythrocyte concentration, V/V) for 2 hours at 37 ℃. PBS and pure water were set as negative and positive controls, respectively. The microplate reader measures absorbance at 541nm to check the blood compatibility of the sample. The results are shown in FIG. 4A;

cytotoxicity: cytotoxicity was assessed with HUVEC cells. Cells were seeded in 96-well plates and cultured overnight. And then, incubating the culture solution containing the biomembrane coated nanoparticles with a series of PLGA concentration gradients with cells, replacing the culture solution containing the nanoparticles with a DMEM (DMEM) culture medium containing 10% FBS (fetal bovine serum) after 4 hours, adding MTT (methyl thiazolyl tetrazolium) after 24 hours, and measuring the absorbance at 490 nm. Untreated cells were set as a negative control group. The results are shown in FIG. 4B.

Example 5

In-vitro targeting of biological membrane coated poly (acetic acid) -glycolic acid nanoparticles

1) Fibrin binding Capacity Studies

A fibrin clot was prepared from a 96-well plate, and 80. mu.L of PBS, PEG-NPs/DiD, RBC-NPs/DiD, PNPs/DiD (DiD concentration 1mg/mL) mixed in an equal volume to 50% serum was added, followed by incubation at 4 ℃ for 2 hours. Washing with cold PBS for 3 times, drying by filter paper, adding 150 μ L DMSO into each well, and measuring the fluorescence intensity (644nm/663nm) of each well solution after 30 min, the result is shown in FIG. 5A;

2) examination of the binding Capacity of platelet-rich plasma clot

Platelet rich plasma was added to 48 well plates at 150 μ L per well. Then 30. mu.L of thrombin solution (3BP/mL), 10. mu.L of calcium chloride solution (0.5mol/L), and 20. mu.L of ADP solution (5. mu. mol/L) were added and incubated at 37 ℃ for 30 minutes to form a platelet-rich plasma clot. PEG-NPs/DiD, RBC-NPs/DiD, PNPs/DiD (DiD concentration 1mg/mL) were then added and incubated at 37 ℃ for 30 minutes. Then, the wells were washed 3 times with cold PBS, after blotting with filter paper, 150. mu.L of DMSO was added to each well, and after 30 minutes, the fluorescence intensity (644nm/663nm) of each well solution was measured, and the results are shown in FIG. 5B;

3) examination of vascular endothelial cell binding Capacity

HUVC cells were subcultured in medium and HUVEC cells were activated by stimulating them with 50ng/mL of TNF- α for 24 hours. Activated or non-activated HUVEC cells were fixed with 4% paraformaldehyde at 4 ℃ for 30 minutes and blocked with 20% mouse serum for 30 minutes, then incubated with PBS, PEG-NPs/DiD, RBC-NPs/DiD, PNPs/DiD (DiD concentration 1mg/mL) at 4 ℃ for 30 minutes, the cells were washed three times with cold PBS, collected, examined by flow cytometry, and the data analyzed using Flowjo software. The results are shown in FIG. 5C, D.

Example 6

Targeting of biological membrane coated poly (acetic acid) -glycolic acid nanoparticles to thrombosis model mouse

ICR mice were anesthetized and fixed in supine position. After the cervical skin depilating treatment, the mouse neck muscles were dissected away with surgical scissors and the left carotid artery was exposed to the visual field. Carotid artery was then damaged with paper soaked in ferric chloride solution to form carotid thrombus. 5 minutes after thrombosis, 200. mu.L of different formulation groups containing 1mg/mL DiR (PEG-NPs/DiR, RBC-NPs/DiR, PNPs/DiR) were injected into the tail vein. 30 minutes after dosing, ICR mouse carotid artery thrombi were ultrasonically imaged using an ultrasonic imaging system (Visualsonics, Toronto, Canada), while photoacoustic imaging was performed at 748nm using a Vevo LAZR photoacoustic imaging system (Visualsonics, Toronto, Canada) with an LZ400 transducer (30MHz), with the results shown in fig. 6A;

the targeting effect was evaluated by confocal micrographs of paraffin sections of longitudinal sections of carotid arteries. 200 mu L of 1mol/L rhodamine 6G is injected into the tail vein to mark the platelet, and after 5 minutes, the thrombus model is prepared by the same experimental method as the previous experimental method and the tail vein is injected for administration. The carotid artery was dissected 30 minutes after injection of the nanoparticle solution. The carotid artery injury site was ligated with an operation thread, the ligation distance was controlled to 5mm, and then the carotid artery injury site was cut. The carotid artery after cutting was immersed in 4% paraformaldehyde, incubated overnight at 4 ℃ to prepare a paraffin section of a longitudinal section of the carotid artery, and observed by a confocal microscope. The results are shown in FIG. 6B.

Example 7

Thrombolytic effect of lumbrokinase-loaded biomembrane coated polyacetic acid-glycolic acid nanoparticles on carotid artery thrombosis model mice

A thrombus model was constructed as in example 6, and after 5 minutes, 200. mu.L of PEG-NPs/LBK, RBC-NPs/LBK, PNPs/LBK, LBK (8X 10^4U/kg) and LBK (2.4X 10^5U/kg) were injected into the tail vein. After 24 hours, the mice were anesthetized with pentobarbital, and the carotid artery containing the thrombus was harvested and controlled to 5mm in length. H & E staining the excised carotid artery, and observing under an optical microscope; or the excised carotid artery was placed in a solution containing 1mg/mL collagenase and 2mg/mL proteinase K, incubated at 37 ℃ for 2 days with shaking at 140rpm to lyse blood cells in the thrombus, and the absorbance was measured at 280nm, as shown in FIG. 7.

Example 8

In vivo safety of poly (acetic acid-glycolic acid) nanoparticles coated by lumbrokinase biomembrane

After anesthetizing mice with pentobarbital, 200. mu.L of PEG-NPs/LBK, RBC-NPs/LBK, PNPs/LBK, LBK (8 x 10^4U/kg) and LBK (2.4 x 10^5U/kg) were injected into the caudal vein. After 30 minutes, a cut was made at a distance of 2cm from the tail of the mouse, and the cut tail was immersed in warm saline (37 ℃) and the time required for hemostasis was recorded, as shown in fig. 8A;

at the same time, fibrinogen in the blood of the mice was measured. After 2 hours of tail vein administration, 500. mu.L of blood was collected from the eyelid, centrifuged at 2000g/min for 10 minutes, and 200. mu.L of the supernatant was measured for fibrinogen content in plasma by a full-automatic blood biochemical analyzer, and the results are shown in FIG. 8B.

Claims

1. A bionic nano drug delivery system of a protein thrombolytic drug is characterized by consisting of a biological membrane on the surface and a skeleton kernel carrying the protein thrombolytic drug; the biological membrane is a natural cell membrane or an artificial biological membrane or a mixed membrane consisting of the natural cell membrane and the artificial biological membrane.

2. The biomimetic nano drug delivery system of claim 1, wherein the natural cell membrane is a erythrocyte membrane, a platelet membrane, a macrophage membrane or a leukocyte membrane.

3. The natural cell membrane of claim 1, wherein the artificial biofilm is a liposome membrane.

4. The artificial biofilm according to claim 1, wherein the mixed membrane of natural cell membranes and artificial biofilms is a hybrid membrane.

5. The mixed membrane of natural cell membranes and artificial biofilms according to claim 1, said biofilms comprising endogenous P-selectin protein.

6. The biomimetic nano drug delivery system of claim 1, wherein the biological membrane comprises self-recognition regulatory immune proteins such as CD47, CD55, CD59, and the like.

7. The biomimetic nano drug delivery system of a protein thrombolytic drug of claim 1, wherein the backbone core of the protein-loaded thrombolytic drug is a protein-loaded thrombolytic drug nanoparticle.

8. The biomimetic nano drug delivery system of the protein thrombolytic drug according to claim 7, wherein the nanoparticles of the matrix core carrying the protein thrombolytic drug are nanoparticles prepared from organic polymer materials or monomers or inorganic materials by a certain physical and/or chemical method.

9. The biomimetic nano drug delivery system for thrombolytic protein drugs according to claim 1, wherein the nano drug delivery system has a particle size of 50-1000nm, preferably 100-200 nm.

10. The biomimetic nano drug delivery system of the thrombolytic drug of claim 1, wherein the thrombolytic drug is a tissue plasminogen kinase such as plasminogen activator and urokinase, and/or a fibrinolytic enzyme such as lumbrokinase.

11. The use of the biomimetic nano drug delivery system of a thrombolytic protein drug according to claim 1 for the preparation of a drug for the treatment of arterial thrombotic diseases.