CN111840527B - Preparation method and application of shear-responsive nano drug delivery system - Google Patents

Abstract

The invention relates to a preparation method and application of a shear-responsive nano drug delivery system, which can effectively solve the problems of targeting thrombus capacity, reducing the toxic and side effects of drugs, improving the curative effect, carrying out responsive slow drug release under the physiological characteristic high shear force of thrombus parts and realizing the site-specific release of the drugs at the thrombus parts. beta-CD and adamantane are respectively grafted on PLGA and fucoidin molecular skeletons, the beta-CD vesicular-containing fucoidin supramolecular self-assembly nanoparticles are prepared by utilizing the host-guest inclusion action of the beta-CD and adamantane, urokinase is encapsulated in the network structure of the polysaccharide nanoparticles in the process of mediating self-assembly, and an antiplatelet drug and thrombolytic drug co-transport nano targeted drug delivery system with shear force response drug release characteristics is formed.

Description

A kind of preparation method of shear-responsive nano-drug delivery system and its application

technical field

The invention relates to medicine, in particular to a preparation method and application of a shear-responsive nano-drug delivery system using a shear-responsive nano-drug delivery system of cyclodextrin vesicles to cross-link fucoidan.

Background technique

Thromboembolic disease has become another major killer after tumors. The thrombus that causes this disease is actually a blood clot, mainly composed of insoluble fibrin, deposited platelets, accumulated white blood cells and trapped red blood cells. Existing antithrombotic drugs, such as streptokinase, urokinase, etc., have certain bleeding risks and other toxic side effects due to their active drugs all over the body. How to overcome these defects, selectively target drugs to the site of blood flow obstruction, and release active drugs in this region is the key technical problem that needs to be solved urgently. The study found that the blood vessels at the thrombus site show different physical characteristics from the normal vascular system. The shear stress of normal blood flow is lower than 70 dyne/cm ² , but at the thrombus site, due to the occlusion of the blood vessel, the fluid shear stress can locally increase by one or two. orders of magnitude, even reaching 1000 dyne/cm ² . Inspired by this natural physical mechanism, it is possible to develop a nano-drug delivery system that can target clots, stenosis, or abnormally constricted vascular regions to treat thrombus using local high shear stress as a general mechanism.

Fucoidan is a sulfated polysaccharide rich in fucose, mainly composed of fucose, sulfate groups and a certain proportion of D-xylose, D-mannose, L-rhamnose, glucose, D-glucuronan and acetyl groups. Years of research have found that fucoidan not only has anticoagulant and antithrombotic effects, but also can bind to the highly expressed P-selectin receptor on activated platelets at the thrombus site, inhibiting thrombus formation and also having thrombus targeting effects. β-Cyclodextrin is a compound in which 7 glucose units are combined into a ring by 1,4-glycosidic bonds. Since the glycosidic bonds connecting the glucose units cannot rotate freely, cyclodextrin is not a cylindrical molecule, but a slightly conical shape. The ring of β-cyclodextrin, in which the primary hydroxyl group is located on the outer surface of the narrow face, and the secondary hydroxyl group is located on the outer surface of the broad face, resulting in the structure of β-cyclodextrin showing the particularity of "outer hydrophilic, inner hydrophobic". The internal hydrophobic cavity can encapsulate hydrophobic guest molecules. Therefore, the hydrophobic guest molecules are modified on fucoidan, and cyclodextrin derivatives are used to form cyclodextrin nanovesicles. The guest molecules can realize self-assembly based on host-guest interaction, and the prepared nano-drug delivery system has thrombus targeting ability and shear stress response properties. After the system reaches the thrombus site, under the local high shear force of the thrombus, the drug encapsulated in the nanoparticle delivery system is released to achieve a good thrombolytic effect. This drug delivery system prepared by utilizing physiological characteristics is a safer and more effective thrombolysis, but there has been no public report so far.

SUMMARY OF THE INVENTION

In view of the above situation, in order to overcome the defects of the prior art, the purpose of the present invention is to provide a preparation method and application of a shear-responsive nano-drug delivery system, which can effectively solve the ability of targeting thrombosis, reduce the toxic and side effects of drugs and improve the curative effect. , responsive and slow drug release under the high shear force, which is the physiological characteristic of the thrombus site, realizes the problem of fixed-point release of the drug at the thrombus site.

The technical solution solved by the present invention is a preparation method of a shear-responsive nano-drug delivery system. Fucoidan supramolecular self-assembled nanoparticles containing β-CD vesicles were prepared by host-guest inclusion, and urokinase was encapsulated in the network structure of polysaccharide nanoparticles during the process of mediating self-assembly, forming a shear The antiplatelet drug and thrombolytic drug co-transport nano-targeted drug delivery system with force-responsive drug release characteristics includes the following steps:

(1) Synthesis of fucoidan modified adamantane: add 10-100 mg of fucoidan to 1-10 mL of formamide, dissolve by ultrasonic, and add 4-40 mg of 4-dimethylaminopyridine, 10- 100 μL of triethylamine and 5-50 mg of adamantanecarbonyl chloride were reacted for 24-48 h, then immersed in isopropanol, precipitated at 4 °C for 2-4 h, centrifuged at 8000-12000 rpm for 10-15 min, discarded the supernatant, collected the precipitate, Then wash with isopropanol for at least two times, dissolve the precipitate with water, dialyze with MWCO of 1000 (dialysis bag or dialysis membrane, the same below) for 1-2 days, freeze-dry to obtain fucoidan modified adamantane;

(2) Synthesis of PLGA-βCD (βCD is β-cyclodextrin): Dissolve 1.5-3 g of polylactic-glycolic acid (PLGA, 15KD) in 15-30 mL of N,N-dimethylformamide ( DMF), add 38.34-76.68 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 23.02-46.04 mg of N-hydroxysuccinimide during stirring at room temperature , activate the carboxyl group for 30-60min; add pre-dissolved 235.4-470.8mg mono(6-ethylenediamino-6-deoxy)-β-cyclodextrin (EDA-β-CD) solution, react for 72h, slowly add Ultrapure water to make the solution turbid (that is, no phase separation occurs), centrifuge at 8000rpm for 30min, collect the precipitate, dialyze the precipitate to 3500 MWCO for 1-2 days, freeze-dry to obtain PLGA-βCD dried product;

(3) Preparation of cyclodextrin nanovesicles: Dissolve 10-50 mg of PLGA-βCD dry matter in 1-5 mL of acetone solution, and slowly add it dropwise to 5-10 mL of 1% mass concentration at a rate of 0.5 mL/min. Polyether (F68, 1%) solution, dialyzed with MWCO of 8000-14000, freeze-dried to form cyclodextrin nanovesicles, and the particle size of cyclodextrin nanovesicles is 50-200 nm;

(4) Construction of nano-drug delivery system: Weigh 20 mg of fucoidan-modified adamantane and dissolve it in 1 mL of ultrapure water, and add cyclodextrin with a weight ratio of fucoidan-modified adamantane to cyclodextrin nanovesicles of 1:1. Fine nanovesicles, under magnetic stirring, add 0.5-2mg urokinase, stir and cross-link for 48h, centrifuge and wash to collect nanoparticles, freeze-dry to obtain a nano-drug delivery system, the particle size of the nano-drug delivery system is 100-500nm.

The application of the shear-responsive nano-drug delivery system prepared by the method in the preparation of drugs for the treatment of atherosclerosis, thrombosis, pulmonary embolism, vascular embolic diseases and arthritis, as well as in the preparation of antithrombotic drug injections and freeze-dried powder injections Applications.

The preparation method of the invention is simple, the raw materials are abundant, and the production and preparation are easy. The prepared nano-drug delivery system has the ability to target thrombus, can reduce the toxic and side effects of the drug, improve the curative effect, and can perform responsiveness under the high shear force of the physiological characteristics of the thrombus site. The slow release of the drug realizes the fixed-point release of the drug at the thrombus site, and opens up a new way for the treatment of atherosclerosis, thrombosis, pulmonary embolism, vascular embolic diseases and arthritis drugs. It is a major innovation in sexually transmitted diseases and arthritis drugs, with significant economic and social benefits.

Detailed ways

The specific embodiments of the present invention will be described in detail below with reference to the examples.

The invention is given in the following examples in its specific implementation.

Example 1

In the specific implementation of the present invention, a preparation method of a shear-responsive nano-drug delivery system comprises the following steps:

(1) Synthesis of fucoidan-modified adamantane: 10 mg of fucoidan was added to 1 mL of formamide, dissolved by ultrasonic, and 4 mg of 4-dimethylaminopyridine, 10 μL of triethylamine and 5 mg of adamantane were added under stirring at room temperature. Formyl chloride was reacted for 24h, immersed in isopropanol, precipitated at 4°C for 2h, centrifuged at 8000rpm for 15min, discarded the supernatant, collected the precipitate, washed the precipitate with isopropanol at least twice, dissolved the precipitate with water, and used MWCO as 1000 (dialysis bag or dialysis membrane, the same below), dialyzed for 1-2 days, freeze-dried to obtain fucoidan-modified adamantane;

(2) Synthesis of PLGA-βCD (βCD is β-cyclodextrin): Dissolve 1.5 g of polylactic-glycolic acid (PLGA, 15KD) in 15 mL of N,N-dimethylformamide (DMF) , 38.34-76.68 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 23.02 mg of N-hydroxysuccinimide were added during stirring at room temperature to activate the carboxyl group for 30 min; Add pre-dissolved 235.4mg mono(6-ethylenediamino-6-deoxy)-β-cyclodextrin (EDA-β-CD) solution, react for 72h, slowly add ultrapure water to make the solution cloudy (ie No phase separation occurs), centrifuge at 8000rpm for 30min, collect the precipitate, dialyze the precipitate at 3500 MWCO for 1-2 days, freeze-dry, and obtain PLGA-βCD dry matter;

(3) Preparation of cyclodextrin nanovesicles: Dissolve 10 mg of PLGA-βCD dried in 1 mL of acetone solution, and slowly add it dropwise to 5 mL of polyether (F68,1 %) solution, dialyzed with MWCO of 8000-14000, freeze-dried to form cyclodextrin nanovesicles;

(4) Construction of nano-drug delivery system: Weigh 20 mg of fucoidan-modified adamantane and dissolve it in 1 mL of ultrapure water, add 20 mg of cyclodextrin nanovesicles, and add 0.5 mg of urokinase for cross-linking under magnetic stirring for 48 hours. , centrifugal washing to collect nanoparticles, freeze-drying, to obtain a nano-drug delivery system.

Example 2

In the specific implementation of the present invention, a preparation method of a shear-responsive nano-drug delivery system comprises the following steps:

(1) Synthesis of fucoidan-modified adamantane: 50 mg of fucoidan was added to 5 mL of formamide, dissolved by ultrasonication, and 20 mg of 4-dimethylaminopyridine, 50 μL of triethylamine and 25 mg of adamantane were added under stirring at room temperature. Formyl chloride was reacted for 36 h, immersed in isopropanol, precipitated at 4°C for 3 h, centrifuged at 10,000 rpm for 12 min, discarded the supernatant, collected the precipitate, washed the precipitate with isopropanol at least twice, dissolved the precipitate with water, and used MWCO as 1000 (dialysis bag or dialysis membrane, the same below), dialyzed for 1-2 days, freeze-dried to obtain fucoidan-modified adamantane;

(2) Synthesis of PLGA-βCD (βCD is β-cyclodextrin): 2.25g of polylactic-glycolic acid (PLGA, 15KD) was dissolved in 25mL of N,N-dimethylformamide (DMF) , 57.51 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 34.53 mg of N-hydroxysuccinimide were added during stirring at room temperature to activate the carboxyl group for 45 min; Dissolved 353mg of mono(6-ethylenediamino-6-deoxy)-β-cyclodextrin (EDA-β-CD) solution, reacted for 72h, slowly added ultrapure water to make the solution turbid (that is, no phase occurred). Separation), then centrifuged at 8000rpm for 30min, collected the precipitate, dialyzed the precipitate to 3500 MWCO for 1-2 days, freeze-dried to obtain PLGA-βCD dried product;

(3) Preparation of cyclodextrin nanovesicles: Dissolve 25 mg of the dried PLGA-βCD in 2.5 mL of acetone solution, and slowly add it dropwise to 7.5 mL of polyether (F68 with a mass concentration of 1% at a rate of 0.5 mL/min) , 1%) solution, dialyzed against MWCO of 10,000, freeze-dried to form cyclodextrin nanovesicles;

(4) Construction of nano-drug delivery system: Weigh 20 mg of fucoidan-modified adamantane and dissolve it in 1 mL of ultrapure water, add 20 mg of cyclodextrin nanovesicles, and add 1 mg of urokinase for cross-linking under magnetic stirring for 48 h. The nanoparticles are collected by centrifugation and washing, and freeze-dried to obtain a nano-drug delivery system.

Example 3

In the specific implementation of the present invention, a preparation method of a shear-responsive nano-drug delivery system comprises the following steps:

(1) Synthesis of fucoidan-modified adamantane: 100 mg of fucoidan was added to 10 mL of formamide, dissolved by ultrasonic, and 40 mg of 4-dimethylaminopyridine, 100 μL of triethylamine and 50 mg of adamantane were added under stirring at room temperature. Formyl chloride was reacted for 48h, immersed in isopropanol, precipitated at 4°C for 4h, centrifuged at 12000rpm for 10min, discarded the supernatant, collected the precipitate, washed the precipitate with isopropanol at least twice, dissolved the precipitate with water, and used MWCO as 1000 dialyzed for 1-2 days, freeze-dried to obtain fucoidan modified adamantane;

(2) Synthesis of PLGA-βCD (βCD is β-cyclodextrin): Dissolve 3g of polylactic-glycolic acid (PLGA, 15KD) in 30mL of N,N-dimethylformamide (DMF), During stirring at room temperature, 76.68 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 46.04 mg of N-hydroxysuccinimide were added to activate the carboxyl group for 60 min; 470.8mg of mono(6-ethylenediamino-6-deoxy)-β-cyclodextrin (EDA-β-CD) solution was reacted for 72h, and ultrapure water was slowly added to make the solution turbid (that is, no phase occurred). Separation), then centrifuged at 8000rpm for 30min, collected the precipitate, dialyzed the precipitate to 3500 MWCO for 1-2 days, freeze-dried to obtain PLGA-βCD dried product;

(3) Preparation of cyclodextrin nanovesicles: Dissolve 50 mg of PLGA-βCD dried in 5 mL of acetone solution, and slowly add it dropwise to 10 mL of polyether (F68,1 %) solution, dialyzed against MWCO of 14,000, freeze-dried, and formed into cyclodextrin nanovesicles;

(4) Construction of nano-drug delivery system: Weigh 20 mg of fucoidan-modified adamantane, dissolve it in 1 mL of ultrapure water, add 20 mg of cyclodextrin nanovesicles, add 2 mg of urokinase under magnetic stirring, and stir for cross-linking for 48 h. The nanoparticles are collected by centrifugation and washing, and freeze-dried to obtain a nano-drug delivery system.

The shear-responsive nano-drug delivery systems prepared by the methods of the above examples 1-3 are effectively used for the preparation of drugs for the treatment of atherosclerosis, thrombosis, pulmonary embolism, vascular thromboembolic diseases and arthritis, as well as for the preparation of antithrombotic drug injections, lyophilized powders The application of the shear-responsive nano-drug delivery system in the preparation of drugs for the treatment of atherosclerosis, thrombosis, pulmonary embolism, vascular embolism and arthritis, as well as in the preparation of antithrombotic drug injections and freeze-dried powder injections .

In the present invention, fucoidan that can be combined with P-selectin highly expressed on activated platelets is used as a material for targeting thrombus, adamantane is modified on the fucoidan, and cyclodextrin derivatives form nanovesicles to retain surface cavities. Using adamantane as a guest molecule, the cavity on the surface of cyclodextrin nanoparticles can contain guest molecule adamantane, then cross-link polysaccharide and coat urokinase to obtain a nano-drug delivery system; the prepared cyclodextrin nanovesicles have a particle size of The particle size of the nano-drug delivery system is between 50-200nm, and the particle size of the nano-drug delivery system is between 100-500nm; the system has the following characteristics: 1) The fucoidan in this system has the ability to target the thrombus site, reducing the use of drugs and reducing toxic side effects 2) The prepared nano-drug delivery system is responsive to shear force, and when the shear force is higher than 100 dyne/cm ² , the drug in the system can be released responsively; 3) The prepared nano-drug delivery system has a biological phase. Capacitive and versatile.

The invention relates to the construction of the shear-responsive nano-drug delivery system. First, cyclodextrin was esterified with PLGA with a terminal carboxyl group, and then nanovesicles with a cavity structure on the surface were synthesized by nanocoprecipitation method. After that, the hydrophobic cavity of the nanovesicles can be cross-linked with the adamantane molecules modified on the fucoidan, and coat the thrombolytic drug urokinase to obtain a nano-drug delivery system. Fucoidan is first targeted at the thrombus site. Under the high shear force of the thrombus, the cross-linking of the host and the guest is broken to release urokinase for thrombolysis.

The fucoidan used in the present invention is a kind of polysaccharide containing fucose and sulfuric acid groups, and has various biological functions, such as anti-coagulation, anti-tumor, anti-thrombosis, anti-virus and the like. P-selectin exists on the platelet α-granule membrane and can bind to P-selectin glycoprotein ligand 1 (PSG1) and mediate platelet adhesion. Fucoidan can bind to P-selectin, target thrombus and inhibit the aggregation of activated platelets. After the prepared nano-drug delivery system is targeted to the thrombus site, under the action of high shear force at the vascular blockage site, the encapsulated drug is released to achieve the effect of thrombolysis. Consistent results have been obtained, and the relevant experimental data are as follows (taking Example 2 as an example):

Experiment 1: Rheometer Experiment

The drug-loaded nanoparticle solution (5 mg/mL) was sheared for 1 min in a rheometer (A2-G2 TA Instruments) using a 20 mm cone-plate structure. The supernatant was then collected, the drug release was determined, and its concentration was calculated and normalized to the highest shear level (1000 dyne/cm ² ) value.

The above experiments were set up in four different groups of 1, 10, 100, and 1000 dyne/cm ² . By measuring the release of the drug in the supernatant, it was calculated that the drug release ratio was lower than 20% when the shear force was 10 dyne/cm ² . The drug release ratio reached 70% at 100 dyne/cm ² . The results showed that the drug release amount of the prepared nano-drug delivery system increased significantly when the shear force was 100 dyne/cm ² , and with the increase of the shear force, the drug release amount also increased. increase, suggesting the shear responsiveness of the drug delivery system.

Experiment 2: In vitro thrombolysis

Blood clots were prepared by mixing 100 μL of whole blood with 50 U of thrombin solution followed by continuous shaking at 50 rpm for 30 min at 37°C, and then transferring the clot from the tube to a 24-well plate. The prepared blood clot was treated with PBS, free uPA, blank carrier, drug-loaded nanoparticles, and pre-sheared drug-loaded nanoparticles, and the clot was continuously oscillated at 50 rpm for 300 minutes at 37°C. At 0, 30, 90, 180, and 300 minutes after treatment, 200 μl of the supernatant was put into 96 multi-well petri dishes, and the optical density (OD415) of hemoglobin released during the lysis process was measured using a microplate reader. The dissolution of the blood clot was recorded.

The results showed that the optical density (OD415) of the hemoglobin released during the dissolution process of the pre-sheared nanoparticles group was significantly higher than that of the other groups, and the dissolution of blood clots was more obvious than that of the other groups, suggesting that the nanoparticles were sheared during shearing. Thrombolytic drug release under force.

Experiment 3: Hemolysis experiment

The fresh rat blood collected in an EDTA anticoagulant tube was centrifuged at 2500 g for 10 min, the supernatant was removed, and the pellet was resuspended in PBS and centrifuged again, and repeated twice to obtain red blood cell pellet. Different concentrations of drug-loaded nanoparticles were added to red blood cells and incubated for 4 h by rotation. PBS was used as a negative control and ultrapure water was used as a positive control. Each group was repeated three times. After incubation, the solution was centrifuged at 10,000 g for 1 min, and the absorbance of the supernatant was measured at a wavelength of 541 nm. The higher the light absorption, the more obvious the hemolytic effect of the carrier material, and the lower the light absorption, the lower the hemolytic effect of the carrier material.

Experiments show that the hemolytic effect of different concentrations of the carrier on red blood cells is similar to that of the negative control group PBS, and the light absorbance is lower and there is no significant difference, that is, no hemolytic effect is produced.

Experiment 4: In vitro targeting

First, a platelet-rich thrombus model was prepared in vitro. The whole blood anticoagulated with 3.2% sodium citrate was centrifuged at 1800g for 10 min. Three layers were observed in the centrifuge tube with the naked eye. The surface layer was the platelet-rich plasma layer (PRP layer). The middle layer is the white blood cell layer, and the bottom layer is the red blood cell layer. Aspirate 180 μL of the supernatant and a small amount of platelet-rich boundary layer, mix it, and distribute it into a 96-well plate, and add calcium chloride (CaCl ₂ 0.4mM) and thrombin (0.5U/mL) in turn. Incubate in a 37°C incubator for 40min. 1 min after the thrombus model was formed, different concentrations of fluorescently labeled nanocarriers were added to co-culture with the thrombus, and then the thrombus surface was washed with 0.9% NaCl, the fluorescence intensity was observed, the images were recorded, and the results were analyzed. Set up a PBS control group.

The results showed that the nanoparticle-treated thrombus had a higher fluorescence signal than the control group, and the fluorescence signal intensity was about 3 times higher than that of the untargeted group, which proved the thrombus targeting ability of the prepared targeting nano-drug delivery system.

Experiment 5: Arterial Injury Model

SD male rats were used for carotid artery injury to establish an in vivo thrombosis model. The SD male rats were divided into four groups, all of which were anesthetized with 10% chloral hydrate (0.5mL/100g), the carotid artery was isolated from one side of the rat, and the filter paper ring infiltrated with ₁₀ % FeCl solution was wrapped in the separation. the carotid artery, and use a plastic pad under the artery to protect the surrounding tissue from damage. The filter paper ring was placed for 10 minutes and removed to observe the thrombosis. After thrombus formation, uPA, blank nanoparticles, drug-loaded nanoparticles and normal saline control solution were injected through the tail vein respectively (in which the administration concentration of uPA was 0.2 mg/mL), and the arterial thrombus was removed 90 minutes later for H&E section staining. Watch for blood clots.

The results showed that there were many large clots in the saline group, which almost completely occupied the lumen; the slices of the free uPA group showed that there were still some clots occupying about half of the lumen, on the contrary, only a few scattered in the preparation group. Blood clot, thrombolytic effect is obvious.

Experiment 6: In vivo tissue distribution

The nanocarriers were labeled with the near-infrared dye IR783, and the in vivo distribution of the delivery system was monitored in real time by the FX PRO in vivo imager. 6-7 week-old male rats with the previously described thrombus model were selected and randomly divided into three groups: 1) IR783 group; 2) IR783-blank nanoparticles group; 3) IR783-drug-loaded nanoparticles group. The administration concentration of IR783 was 0.2 mg/mL. The above drugs were injected into the rats by tail vein injection. At 10 min, 30 min, 60 min, and 90 min, the model rats were placed in the FX PRO fluorescent in vivo imager. Fluorescence and X-Ray imaging were performed in the experiment, and the parameters were set to the excitation wavelength of 720 nm and the emission wavelength of 790 nm. The processed data were recorded and the results were analyzed. After 90 minutes, the rats in each group were sacrificed to take out the heart, liver, spleen, lung, and kidney. After soaking and washing in PBS, the filter paper absorbed excess water, and placed it in a fluorescent in vivo imager to scan and take pictures, and record and analyze the preparation distribution of each tissue and organ. .

Fluorescence imaging showed that the fluorescence intensity of nanoparticles at the thrombus site gradually increased with time, and the fluorescence intensity at 90 min was about twice that of free IR83, suggesting that the nanoparticles can efficiently accumulate at the thrombus site.

Experiment 7: In vivo safety evaluation

6-7 week old male rats with arterial thrombosis model were selected and randomly divided into three groups: 1) normal saline group, 2) free uPA group, 3) drug-loaded nanoparticles group. The coagulation indexes of the arterial thrombosis rats treated with granules, aPTT (activated partial thromboplastin time), FIB (fibrinogen), PT (prothrombin time) and TT (thrombin time) were analyzed and explained, and the The heart, liver, spleen, lung and kidney tissue were removed after sacrifice, and then embedded in H&E section and observed under the microscope. Among them, normal rats were used as negative controls.

Compared with the normal saline group, there was no significant difference in coagulation parameters, activated partial thromboplastin time, fibrinogen time, prothrombin time and thrombin time in the drug-loaded nanoparticle group after treatment. The obvious pathological changes indicated the in vivo safety of the nano-drug delivery system.

Experiment 8: Tail bleeding experiment

The hemostatic potential of rats was determined by the template tail bleeding time method. Five minutes after administration through the tail vein, a longitudinal incision of 2 mm in depth and 4 mm in length was made in the superficial tail vein along the left axis of the tail, starting at 10 mm proximal to the tail. Monitor the bleeding at the incision within 20 minutes, and pay attention to wipe the blood with filter paper in time.

In this experiment, saline, blank nanoparticles, drug-loaded nanoparticles, pre-sheared nanoparticles and urokinase solution were set up, and the tail bleeding time of each group was monitored and recorded.

The results showed that compared with the bleeding time of the normal saline group (about 3 min), the nano-delivery system group did not significantly prolong the tail bleeding time (about 4 min), and the tail bleeding time of the rats treated with urokinase was longer than the normal saline group by about 10min, indicating that the nano-delivery system significantly reduces the bleeding side effect of the drug.

Experiment 9: Cytotoxicity

Human umbilical vein endothelial cells (HUVEC) were seeded in 96-well plates at 5 x 10 ³ cells/well, and after culturing for 24 hours in an incubator at 37°C, 5% CO ₂ , 200 µL containing 100 µg/well were added to each well. mL, 50 µg/mL, 20 µg/mL, and 10 µg/mL blank nanoparticles were cultured for 24h and 48h, with 6 replicate wells for each concentration. Toxic effects on cells were investigated by SRB method.

The results showed that the carrier nanoparticles had no obvious toxic and side effects on HUVEC cells after 24h and 48h, the cell survival rate was not lower than 85%, and the effect on cells was small.

At the same time as the experiment of Example 2, the same experiment was also carried out on other examples, and the same and similar results were obtained, which are not listed here one by one.

Experiments show that the present invention has the following outstanding features and beneficial effects with respect to the prior art:

In the present invention, β-CD and adamantane are grafted on PLGA and fucoidan molecular skeleton respectively, and the host-guest inclusion effect of β-CD and adamantane is used to prepare fucoidan supramolecular self-assembly containing β-CD vesicles. Nanoparticles, in the process of mediating self-assembly, encapsulate urokinase in the network structure of polysaccharide nanoparticles to prepare antiplatelet drugs and thrombolytic drugs co-transport nano-targeted drug delivery with shear force-responsive drug release characteristics The system, which has not been publicly reported so far, is the applicant's initiative, has outstanding substantive features, and has achieved very good beneficial technical effects:

1. It can be targeted at the thrombus site, enhance the efficient accumulation of drugs at the thrombus site, and greatly improve the utilization rate and efficacy of the drug;

2. Shear-responsive drug release can reduce the systemic toxic and side effects of the drug, concentrate the active drug on the blood flow blockage, and give full play to the efficacy of the drug;

3. It has good biocompatibility and safety, and has a wide range of applications. It can be effectively used in the preparation of drugs for the treatment of atherosclerosis, thrombosis, pulmonary embolism, vascular thromboembolic diseases and arthritis, as well as in the preparation of antithrombotic drug injections, freeze-dried drugs Powder injection has opened up new ways to treat atherosclerosis, thrombosis, pulmonary embolism, vascular embolism disease and arthritis drugs, antithrombotic drug injections, and freeze-dried powder injection, which has significant economic and social benefits.

Claims (5)

1. A preparation method of a shear-responsive nano drug delivery system is characterized in that beta-CD and adamantane are respectively grafted on PLGA and fucoidin molecular skeletons, fucoidin supramolecular self-assembly nanoparticles containing beta-CD vesicles are prepared by utilizing the host-guest inclusion effect of the beta-CD and adamantane, urokinase is encapsulated in a network structure of the polysaccharide nanoparticles in the process of mediating self-assembly, and the antiplatelet drug and thrombolytic drug co-transport nano targeted drug delivery system with the shear-responsive drug release characteristic is formed and comprises the following steps:

(1) synthesis of fucoidan-modified adamantane: adding 10-100mg of fucoidin into 1-10mL of formamide, carrying out ultrasonic dissolution, adding 4-40mg of 4-dimethylaminopyridine, 10-100 muL of triethylamine and 5-50mg of adamantane formyl chloride under the condition of stirring at room temperature for reacting for 24-48h, adding isopropanol for immersion, precipitating for 2-4h at 4 ℃, centrifuging at 12000rpm for 10-15min at 8000 ℃, collecting the supernatant, discarding the precipitate, washing the precipitate at least twice with isopropanol, dissolving the precipitate with water, dialyzing for 1-2 days by using a dialysis bag or a dialysis membrane with MWCO of 1000, and carrying out freeze drying to obtain fucoidin-modified adamantane;

(2) synthesis of PLGA-. beta.CD: dissolving 1.5-3g of polylactic acid-glycolic acid in 15-30mL of N, N-Dimethylformamide (DMF), adding 38.34-76.68mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 23.02-46.04mg of N-hydroxysuccinimide during stirring at room temperature, and activating carboxyl for 30-60 min; adding pre-dissolved 235.4-470.8mg of mono (6-ethylenediamine-6-deoxy) -beta-cyclodextrin solution, reacting for 72h, slowly adding ultrapure water to enable the solution to be turbid, centrifuging at 8000rpm for 30min, collecting precipitate, dialyzing a dialysis bag or a dialysis membrane with the MWCO of the precipitate being 3500 for 1-2 days, and freeze-drying to obtain PLGA-beta CD dry matter;

(3) preparing cyclodextrin nano vesicles: dissolving 10-50mg of PLGA-beta CD dry matter in 1-5mL of acetone solution, slowly dripping the PLGA-beta CD dry matter into 5-10mL of polyether solution with the mass concentration of 1% at the speed of 0.5mL/min, dialyzing by using a dialysis bag or a dialysis membrane with MWCO of 8000-14000, and freeze-drying to form cyclodextrin nano vesicles with the particle size of 50-200 nm;

(4) construction of a nano drug delivery system: weighing 20mg of fucoidin-modified adamantane, dissolving the fucoidin-modified adamantane in 1mL of ultrapure water, adding the cyclodextrin nano vesicle at a weight ratio of the fucoidin-modified adamantane to the cyclodextrin nano vesicle of 1: 1, adding 0.5-2mg of urokinase under magnetic stirring, stirring and crosslinking for 48h, centrifuging, washing, collecting nanoparticles, and freeze-drying to obtain a nano drug delivery system with the particle size of 100-500 nm.

2. The method of preparing a shear-responsive nano-drug delivery system of claim 1, comprising the steps of:

(1) synthesis of fucoidan-modified adamantane: adding 10mg of fucoidin into 1mL of formamide, carrying out ultrasonic dissolution, adding 4mg of 4-dimethylaminopyridine, 10 mu L of triethylamine and 5mg of adamantane formyl chloride under the condition of stirring at room temperature for reacting for 24 hours, adding isopropanol for immersion, precipitating for 2 hours at 4 ℃, centrifuging for 15 minutes at 8000rpm, discarding the supernatant, collecting the precipitate, washing the precipitate with isopropanol at least two times, dissolving the precipitate with water, dialyzing for 1-2 days by using a dialysis bag or a dialysis membrane with MWCO of 1000, and carrying out freeze drying to obtain fucoidin modified adamantane;

(2) synthesis of PLGA-. beta.CD: dissolving 1.5g of polylactic acid-glycolic acid in 15mL of N, N-Dimethylformamide (DMF), adding 38.34-76.68mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 23.02mg of N-hydroxysuccinimide during stirring at room temperature, and activating carboxyl for 30 min; adding predissolved 235.4mg of mono (6-ethylenediamine-6-deoxidation) -beta-cyclodextrin solution, reacting for 72h, slowly adding ultrapure water to make the solution turbid, centrifuging for 30min at 8000rpm, collecting precipitate, dialyzing a dialysis bag or a dialysis membrane with the MWCO of the precipitate being 3500 for 1-2 days, and freeze-drying to obtain PLGA-beta CD dry matter;

(3) preparing cyclodextrin nano vesicles: dissolving 10mg of PLGA-beta CD dry matter in 1mL of acetone solution, slowly dripping the PLGA-beta CD dry matter into 5mL of polyether solution with the mass concentration of 1% at the speed of 0.5mL/min, dialyzing by using a dialysis bag or a dialysis membrane with MWCO of 8000-14000, and freeze-drying to form cyclodextrin nano vesicles;

(4) construction of a nano drug delivery system: weighing 20mg of fucoidin modified adamantane, dissolving the fucoidin modified adamantane in 1mL of ultrapure water, adding 20mg of cyclodextrin nano vesicle, adding 0.5mg of urokinase under magnetic stirring, stirring and crosslinking for 48 hours, centrifugally washing, collecting nanoparticles, and freeze-drying to obtain the nano drug delivery system.

3. The method of preparing a shear-responsive nano-drug delivery system of claim 1, comprising the steps of:

(1) synthesis of fucoidan modified adamantane: adding 50mg of fucoidin into 5mL of formamide, carrying out ultrasonic dissolution, adding 20mg of 4-dimethylaminopyridine, 50 mu L of triethylamine and 25mg of adamantane formyl chloride under the condition of stirring at room temperature to react for 36h, adding isopropanol to immerse, precipitating for 3h at 4 ℃, centrifuging for 12min at 10000rpm, discarding supernatant, collecting precipitate, washing the precipitate with isopropanol at least two times, dissolving the precipitate with water, dialyzing for 1-2 days by using a dialysis bag or a dialysis membrane with MWCO of 1000, and carrying out freeze drying to obtain fucoidin modified adamantane;

(2) synthesis of PLGA- β CD: dissolving 2.25g of polylactic acid-glycolic acid in 25mL of N, N-Dimethylformamide (DMF), adding 57.51mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 34.53mg of N-hydroxysuccinimide during stirring at room temperature, and activating carboxyl for 45 min; adding a pre-dissolved 353mg of mono (6-ethylenediamine-6-deoxy) -beta-cyclodextrin solution, reacting for 72h, slowly adding ultrapure water to make the solution turbid, centrifuging at 8000rpm for 30min, collecting precipitate, dialyzing a dialysis bag or a dialysis membrane with the MWCO precipitate of 3500 for 1-2 days, and freeze-drying to obtain PLGA-beta CD dried substance;

(3) preparing cyclodextrin nano vesicles: dissolving 25mg of PLGA-beta CD dry matter in 2.5mL of acetone solution, slowly dripping the PLGA-beta CD dry matter into 7.5mL of polyether solution with the mass concentration of 1% at the speed of 0.5mL/min, dialyzing by using a dialysis bag or a dialysis membrane with MWCO of 10000, and freeze-drying to form cyclodextrin nano vesicles;

(4) construction of a nano drug delivery system: weighing 20mg of fucoidin modified adamantane, dissolving the fucoidin modified adamantane in 1mL of ultrapure water, adding 20mg of cyclodextrin nano vesicle, adding 1mg of urokinase under magnetic stirring, stirring and crosslinking for 48 hours, centrifugally washing to collect nanoparticles, and freeze-drying to obtain the nano drug delivery system.

4. The method of preparing a shear-responsive nano-drug delivery system of claim 1, comprising the steps of:

(1) synthesis of fucoidan modified adamantane: adding 100mg of fucoidin into 10mL of formamide, carrying out ultrasonic dissolution, adding 40mg of 4-dimethylaminopyridine, 100 mu L of triethylamine and 50mg of adamantane carbonyl chloride under the condition of stirring at room temperature to react for 48 hours, adding isopropanol to immerse, precipitating for 4 hours at 4 ℃, centrifuging at 12000rpm for 10 minutes, removing supernate, collecting precipitate, washing the precipitate with isopropanol at least two times, dissolving the precipitate with water, dialyzing for 1-2 days by using a dialysis bag or a dialysis membrane with MWCO of 1000, and carrying out freeze drying to obtain fucoidin modified adamantane;

(2) synthesis of PLGA-. beta.CD: dissolving 3g of polylactic acid-glycolic acid in 30mL of N, N-Dimethylformamide (DMF), adding 76.68mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 46.04mg of N-hydroxysuccinimide during stirring at room temperature, and activating carboxyl for 60 min; adding predissolved 470.8mg of mono (6-ethylenediamine-6-deoxidation) -beta-cyclodextrin solution, reacting for 72h, slowly adding ultrapure water to make the solution turbid, centrifuging for 30min at 8000rpm, collecting precipitate, dialyzing a dialysis bag or a dialysis membrane with the MWCO of the precipitate being 3500 for 1-2 days, and freeze-drying to obtain PLGA-beta CD dry matter;

(3) preparing cyclodextrin nano vesicles: dissolving 50mg of PLGA-beta CD dry matter in 5mL of acetone solution, slowly dripping the solution into 10mL of polyether solution with the mass concentration of 1% at the speed of 0.5mL/min, dialyzing by using a dialysis bag or a dialysis membrane with MWCO of 14000, and freeze-drying to form cyclodextrin nano vesicles;

(4) construction of a nano drug delivery system: weighing 20mg of fucoidin modified adamantane, dissolving the fucoidin modified adamantane in 1mL of ultrapure water, adding 20mg of cyclodextrin nano vesicle, adding 2mg of urokinase under magnetic stirring, stirring and crosslinking for 48 hours, centrifuging, washing and collecting nanoparticles, and freeze-drying to obtain the nano drug delivery system.

5. Use of a shear-responsive nano-drug delivery system prepared by the method of any one of claims 1 to 4 for the preparation of a medicament for the treatment of atherosclerosis, thrombosis, pulmonary embolism, vascular embolic disease.