阅读说明：本技术 一种剪切响应性纳米递药系统的制备方法及其应用 (Preparation method and application of shear-responsive nano drug delivery system ) 是由 张慧娟 裴亚敏 朱玲 侯琳 张红岭 张振中 于 2020-06-15 设计创作，主要内容包括：本发明涉及剪切响应性纳米递药系统的制备方法及其应用,可有效解决靶向血栓能力,减少药物的毒副作用提高疗效,在血栓部位的生理特征高剪切力下进行响应性缓慢释药,实现药物在血栓部位定点释放的问题。将β-CD和金刚烷分别接枝于PLGA和岩藻多糖分子骨架上,利用β-CD和金刚烷的主客体包合作用制备含有β-CD囊泡的岩藻多糖超分子自组装纳米粒,在介导自组装的过程中将尿激酶包封于多糖纳米粒的网状结构中,构成具有剪切力响应释药特征的抗血小板药物和溶栓药物共转运纳米靶向递药系统,本发明制备方法简单,原料丰富,易生产制备,减少药物的毒副作用,提高疗效。(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 vesicle-containing fucoidin supramolecular self-assembly nanoparticles are prepared by utilizing the host-guest inclusion effect 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 the antiplatelet drug and thrombolytic drug co-transport nano targeted drug delivery system with the shear force response drug release characteristic is formed.)

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-;

(2) synthesis of PLGA- β 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 the precipitate MWCO at 3500 for 1-2 days, and freeze-drying to obtain PLGA-beta CD dried substance;

(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 MWCO of 8000-14000, and freeze-drying to form cyclodextrin nano vesicles, wherein the particle size of the cyclodextrin nano vesicles is 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 in 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, centrifugally washing to collect 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 to react for 24 hours, adding isopropanol to immerse, 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 with MWCO of 1000, and carrying out freeze drying to obtain fucoidin modified adamantane;

(2) Synthesis of PLGA- β 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 pre-dissolved 235.4mg 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 the precipitate MWCO at 3500 for 1-2 days, and freeze-drying to obtain PLGA-beta CD dried substance;

(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 MWCO of 8000-;

(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, centrifuging, 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 the supernatant, collecting the precipitate, washing the precipitate with isopropanol at least two times, dissolving the precipitate with water, dialyzing for 1-2 days 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 the precipitate MWCO for 3500 for 1-2 days, and freeze-drying to obtain a 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 MWCO as 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, centrifuging, washing and collecting 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 formyl 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 with MWCO of 1000, and carrying out freeze drying to obtain fucoidin modified adamantane;

(2) synthesis of PLGA- β 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 pre-dissolved 470.8mg 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 the precipitate MWCO at 3500 for 1-2 days, and freeze-drying to obtain PLGA-beta CD dried substance;

(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 MWCO as 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 the shear-responsive nano-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 diseases and arthritis.

6. The use of the shear-responsive nano drug delivery system prepared by the method of any one of claims 1 to 4 in the preparation of an antithrombotic drug injection and a lyophilized powder injection.

Technical Field

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

Background

Thromboembolic diseases have become a further killer after tumors, and the thrombus that causes the disease is in fact a blood clot, consisting mainly of insoluble fibrin, deposited platelets, accumulated white blood cells and entrapped red blood cells. The existing antithrombotic drugs, such as streptokinase, urokinase and the like, have certain bleeding risk and other toxic and side effects because the active drugs are distributed throughout the whole body. How to overcome the defects, selectively targeting the drug to the blood flow blockage site and intensively releasing the active drug in the area is a technical key which needs to be solved urgently. The research shows that the blood vessel at the thrombus site shows different physical characteristics from the normal blood vessel system, and the normal blood flow shearing force is lower than 70dyne/cm²However, at the thrombus site, the shear stress of the fluid due to the blood vessel blockage can be locally increased by one to two orders of magnitude, even up to 1000dyne/cm². Inspired by this natural physical mechanism, it became possible to develop a nano-drug delivery system that can target clots, stenoses or abnormally constricted vascular regions to treat thrombi with local high shear stress as a general mechanism.

Fucoidin is a sulfuric acid polysaccharide rich in fucose, and mainly comprises fucoidin, sulfuric acid groups, and a certain proportion of D-xylose, D-mannose, L-rhamnose, glucose, D-glucose aldehyde group and acetyl group. The research for many years finds that the fucoidin not only has the effects of anticoagulation, antithrombotic and the like, but also can be combined with a P selectin receptor highly expressed on activated platelets at a thrombus part, so that the fucoidin also has the thrombus targeting effect while inhibiting the formation of thrombus. Beta-cyclodextrin is a compound formed by combining 7 glucose units into a ring by 1, 4-glycosidic bonds, and because the glycosidic bonds connecting the glucose units cannot rotate freely, the cyclodextrin is not a cylindrical molecule but a slightly conical ring, wherein primary hydroxyl is positioned on the outer surface of a narrow face, and secondary hydroxyl is positioned on the outer surface of a wide face, so that the structure of the beta-cyclodextrin has the specificity of 'external hydrophilicity and internal hydrophobicity'. Hydrophobic object molecules can be encapsulated in the hydrophobic cavity inside the nanometer drug delivery system, so that the hydrophobic object molecules are modified on the fucoidan, cyclodextrin derivatives are used for forming cyclodextrin nanometer vesicles, the hydrophobic cavity on the surface is reserved, self-assembly based on host-object interaction can be realized with the object molecules on the polysaccharide, and the prepared nanometer drug delivery system has thrombus targeting capability and shearing force response characteristics. After the system reaches the thrombus part in a targeted manner, the medicament carried by the nanoparticle delivery system is released under the local high shear force of the thrombus, so that a good thrombolytic effect is achieved. The drug delivery system prepared by utilizing the physiological characteristics is safer and more effective thrombolysis, but has no public reports to date.

Disclosure of Invention

In view of the above situation, the present invention aims to overcome the defects of the prior art by providing a preparation method and an 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 high shear force of physiological characteristics of thrombus sites, and realizing the site-specific release of drugs at the thrombus sites.

The technical scheme includes that beta-CD and adamantane are respectively grafted to PLGA and fucoidin molecular frameworks, the fucoidin supramolecular self-assembly nanoparticles containing beta-CD vesicles are prepared by the inclusion of host and guest of the beta-CD and adamantane, urokinase is encapsulated in a network structure of the polysaccharide nanoparticles in the process of mediated self-assembly, and the nano targeting drug delivery system for co-transport of antiplatelet drugs and thrombolytic drugs with the shear force response 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-;

(2) Synthesis of PLGA- β CD (β CD, i.e., β -cyclodextrin): dissolving 1.5-3g of polylactic-co-glycolic acid (PLGA, 15 KD) 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 a pre-dissolved 235.4-470.8mg of mono (6-ethylenediamine-6-deoxy) -beta-cyclodextrin (EDA-beta-CD) solution, reacting for 72h, slowly adding ultrapure water to enable the solution to be turbid (namely, no phase separation occurs), centrifuging for 30min at 8000rpm, collecting precipitates, dialyzing the precipitates MWCO for 1-2 days for 3500, and freeze-drying to obtain a PLGA-beta CD dried substance;

(3) preparing cyclodextrin nano vesicles: dissolving 10-50mg of PLGA-beta CD dry matter in 1-5mL of acetone solution, slowly dropwise adding the PLGA-beta CD dry matter into 5-10mL of polyether (F68, 1%) solution with the mass concentration of 1% at the speed of 0.5mL/min, dialyzing 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 in 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, centrifugally washing to collect nanoparticles, and freeze-drying to obtain a nano drug delivery system with the particle size of 100-500 nm.

The shear response nano drug delivery system prepared by the method is applied to the preparation of drugs for treating atherosclerosis, thrombus, pulmonary embolism, vascular embolism diseases and arthritis, and is applied to the preparation of antithrombotic drug injections and freeze-dried powder injections.

The nano drug delivery system prepared by the method has the advantages of targeting thrombus capacity, reducing the toxic and side effects of the drug, improving the curative effect, carrying out responsive slow drug release under the physiological characteristic high shear force of a thrombus part, realizing the fixed-point release of the drug at the thrombus part, developing a new way for treating atherosclerosis, thrombus, pulmonary embolism vascular embolic disease and arthritis, being a great innovation for treating atherosclerosis, thrombus, pulmonary embolism vascular embolic disease and arthritis, and having remarkable economic and social benefits.

Detailed Description

The following examples are provided to explain the present invention in detail.

The invention is illustrated in the following examples.