Drug coating balloon of rapamycin-loaded metal polyphenol nano-particles as well as preparation method and application of drug coating balloon

Abstract

The invention discloses a drug coating balloon of metal polyphenol nanoparticles loaded with rapamycin as well as a preparation method and application of the drug coating balloon. According to the invention, a new generation of drug balloon coating is obtained through a method of combining the construction of a functionalized drug loading system and a drug coating balloon coating technology. According to the invention, the metal organic framework nanoparticles realize high-load and high-stability encapsulation of rapamycin; the tannic acid is used for etching to form the metal polyphenol hollow shell structure loaded rapamycin, and the particle size of the nano-particles is not obviously influenced before and after the tannic acid is used for etching the metal organic framework nano-particles. The nanoparticles with the metal polyphenol hollow shell structure have good biocompatibility and have an obvious inhibition effect on proliferation of smooth muscle cells, and meanwhile, under the same drug concentration, compared with a single rapamycin group, immunofluorescence staining shows that the drug concentration of the nanoparticles is obviously reduced, and the stability of the nanoparticles is improved. The rapamycin-loaded metal polyphenol nanoparticle group has better inhibition effect on smooth muscle cell proliferation, smooth muscle cell contraction is more obvious, and specific protein expression quantity is less.

Description

Technical Field

[0001] The present invention relates to the technical field of medical devices, and in particular to a drug-coated balloon of metal polyphenol nanoparticles loaded with rapamycin, and a preparation method and application thereof. Background Art

[0002] Cardiovascular disease is currently the number one cause of death in the world, with global morbidity and mortality rates increasing dramatically, surpassing cancer and other diseases. Atherosclerosis is a major cause of cardiovascular disease, caused by excessive proliferation of smooth muscle cells. Lipid deposition and foam cell formation on the vascular wall trigger a persistent chronic inflammatory response. The significant increase in oxidative stress and inflammatory response can lead to cell damage, hinder endothelial function, and worsen the process of intimal hyperplasia. Since the 1990s, balloon dilatation has been one of the most commonly used strategies for the treatment of cardiovascular disease. Drug-coated balloons bring antiproliferative drugs to the vascular wall and release them evenly, inhibiting smooth muscle cell proliferation and restenosis. They have the advantages of no intervention and no implantation of metal wires and polymer coatings that remain in the vascular wall for a long time. They are expected to accelerate arterial healing, retain and restore normal vascular morphology and function early, and inhibit stent thrombosis and restenosis. However, the drug-coated balloons currently used lose too much drug during delivery and expansion, and only about 20-30% of the drug reaches the target lesion vessel wall, which greatly limits the bioavailability of the drug. Therefore, there is an urgent need to explore the next generation of drug-coated balloons to meet related clinical applications.

[0003] Metal ions are involved in many metabolic processes in the human body, and their homeostasis is essential for life. In cardiovascular diseases, the balance of metal ions is often broken, which is associated with various disturbances in physiological processes that lead to abnormal cardiac function. Exogenous supplementation of metal ions has the potential to serve as a therapeutic strategy for the treatment of cardiovascular diseases. Compared with other therapeutic drugs, metal ions have wide availability, good stability and safety, and diverse drug delivery strategies. The delivery strategy of metal ions is very important to exert their therapeutic effects and reduce potential toxic side effects in cardiovascular applications, which are also receiving increasing attention. Controllable local delivery strategies of metal ions based on various biomaterials are constantly being designed.

[0004] Currently, various nanocarriers have been explored in drug delivery systems, such as inorganic nanomaterials, biopolymers, liposome particles, etc. Among these nanocarriers, metal organic frameworks (MOFs) are a class of organic-inorganic hybrid materials that have attracted considerable attention since their introduction. They have been applied in various fields such as sensing, bioimaging, and drug delivery. Zeolitic imidazolate frameworks (ZIFs), a subclass of MOFs, are non-toxic, biocompatible, have excellent drug loading capacity, and are pH-sensitive, offering great potential for drug delivery.

[0005] Natural polyphenols (tannic acid) and metal ions are simply mixed in the presence of a matrix. This rapid, low-cost method is used for conformal coatings on different substrates, including bulk materials, nanomaterials, and biointerfaces. Tannic acid is approved by the FDA as a safe food additive and is one of the most important ingredients in traditional medicines. Studies have shown that tannic acid has a wide range of pharmacological activities, such as anti-inflammatory, neuroprotective, anti-tumor, cardioprotective, and anti-pathogenic effects. The adjacent hydroxyl groups on tannic acid provide chelating sites for metal ions, and the large number of gallic acid groups on tannic acid promotes efficient coordination-driven cross-linking, resulting in a three-dimensional stable metal polyphenol network. Summary of the invention

[0006] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide a method for preparing rapamycin-loaded metal polyphenol nanoparticles.

[0007] Another object of the present invention is to provide rapamycin-loaded metal polyphenol nanoparticles obtained by the above preparation method.

[0008] Another object of the present invention is to provide application of the above-mentioned metal polyphenol nanoparticles loaded with rapamycin.

[0009] The purpose of the present invention is achieved through the following technical solutions:

[0010] A method for preparing rapamycin-loaded metal polyphenol nanoparticles comprises the following steps: adding rapamycin to a metal organic framework precursor solution to prepare the rapamycin-loaded metal organic framework nanoparticles; then adding the obtained metal organic framework nanoparticle dispersion to a tannic acid solution, rapidly stirring, and after the stirring is completed, collecting the nanoparticles by centrifugation, washing, and obtaining the rapamycin-loaded metal polyphenol nanoparticles.

[0011] Furthermore, the metal organic framework includes any one or more of ZIF-8, ZIF-67, HKUST-1, UiO-66, and MIL-88A.

[0012] Furthermore, the solvent of the metal organic framework precursor solution is methanol.

[0013] Furthermore, the amount of rapamycin added is calculated based on a ratio of 8-14 mg:25 mL of the solvent of the metal organic framework precursor solution; preferably, it is calculated based on 12 mg:25 mL.

[0014] Furthermore, the concentration of the metal organic framework nanoparticles is controlled to be 4±1 mg / mL.

[0015] Furthermore, the concentration of the tannic acid is controlled to be 10±1 mg / mL.

[0016] When the metal organic framework is ZIF-8, the preparation method comprises the following steps:

[0017] Step 1: Weigh zinc nitrate hexahydrate and rapamycin and dissolve them in methanol;

[0018] Step 2: Weigh 2-methylimidazole and dissolve it in water, quickly add it to the solution obtained in step 1, mix and stir, collect the nanoparticles by centrifugation after stirring, and wash them to obtain ZIF-8 nanoparticles loaded with rapamycin;

[0019] Step 3: dispersing the rapamycin-loaded ZIF-8 nanoparticles obtained in step 2 in ultrapure water to obtain a nanoparticle dispersion;

[0020] Step 4: Weigh tannic acid and fully dissolve it in ultrapure water to obtain a tannic acid solution;

[0021] Step 5: Add the tannic acid solution obtained in step 4 to the nanoparticle dispersion obtained in step 3, and stir rapidly. After stirring, collect the nanoparticles by centrifugation, wash, and obtain metal polyphenol nanoparticles loaded with rapamycin.

[0022] Furthermore, the weight ratio of zinc nitrate hexahydrate, rapamycin and 2-methylimidazole in step 1 and step 2 is 100-200:8-14:1000-2000.

[0023] Furthermore, the ratio of zinc nitrate hexahydrate, rapamycin and 2-methylimidazole in step 1 and step 2 is 100:12:1000 by mass.

[0024] Furthermore, the stirring time in step 2 is 30±5 min.

[0025] Furthermore, the concentration of the nanoparticles described in step 5 is controlled to be 4±1 mg / mL, and the concentration of tannic acid is controlled to be 10±1 mg / mL.

[0026] Furthermore, the concentration of the nanoparticles described in step 5 is controlled to be 4 mg / mL, and the concentration of tannic acid is controlled to be 10 mg / mL.

[0027] Furthermore, the stirring time in step 5 is 5±1 min.

[0028] Furthermore, the centrifugation conditions in step 2 and step 5 are 10000±2000g, 10±2min.

[0029] Furthermore, the cleaning described in step 2 and step 5 refers to cleaning with methanol 2-4 times.

[0030] A metal polyphenol nanoparticle loaded with rapamycin is obtained by the preparation method.

[0031] A drug-coated balloon comprises a balloon body and a drug coating at least partially covering the surface of the balloon body, wherein the drug coating is the above-mentioned metal polyphenol nanoparticles loaded with rapamycin.

[0032] The preparation method of the drug-coated balloon is to fill the metal polyphenol nanoparticle suspension loaded with rapamycin into a syringe, apply an electromagnetic field inside or outside the syringe for magnetic stirring, ultrasonically spray the uniformly stirred suspension onto the outer surface of a rotating inflatable balloon body, and dry and fold the sprayed balloon to form a drug-loaded coating.

[0033] Application of the drug-coated balloon in the preparation of drugs for treating atherosclerosis.

[0034] Application of the drug-coated balloon in the preparation of drugs for inhibiting smooth muscle cell proliferation.

[0035] Compared with the prior art, the present invention has the following advantages and effects:

[0036] The present invention provides a nano drug delivery system of rapamycin loaded with a metal polyphenol hollow structure, and obtains a new generation of drug balloon coating by combining a functionalized drug-carrying system with a coating technology of a drug-coated balloon.

[0037] The research and development of the new generation of drug balloon coatings of the present invention combines the pathological characteristics of atherosclerosis to construct a more functional drug delivery system, and multiple channels achieve efficient therapeutic effects. Metal organic framework nanoparticles achieve high load and high stability encapsulation of rapamycin; the metal organic framework nanoparticles are then etched with tannic acid to form a metal polyphenol hollow shell structure to load rapamycin, and the tannic acid has no significant effect on the particle size of the nanoparticles before and after etching the metal organic framework nanoparticles. The nanoparticles of the metal polyphenol hollow shell structure of the present invention have good biocompatibility and have a significant inhibitory effect on the proliferation of smooth muscle cells. At the same time, immunofluorescence staining shows that under the same drug concentration, compared with the rapamycin group alone, the metal polyphenol nanoparticle group loaded with rapamycin has a better and excellent inhibitory effect on the proliferation of smooth muscle cells, and the smooth muscle cells contract more significantly and the expression of specific proteins is less. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 is a particle size distribution and dispersity diagram of ZIF-8 nanoparticles loaded with rapamycin in Example 1;

[0039] Figure 2 is a graph showing the particle size distribution and dispersibility of the metal polyphenol nanoparticles loaded with rapamycin in Example 1;

[0040] Figure 3 is a transmission electron microscopy image of the ZIF-8 nanoparticles loaded with rapamycin in Example 1;

[0041] Figure 4 is a transmission electron microscopy morphology image of the metal polyphenol nanoparticles loaded with rapamycin in Example 1;

[0042] Figure 5 This is a graph showing the results of measuring the cytotoxicity of metal polyphenol nanoparticles at different concentrations on smooth muscle cells;

[0043] Figure 6 This is a graph showing the results of measuring the inhibitory effect of metal polyphenol nanoparticles loaded with different concentrations of rapamycin on the proliferation of smooth muscle cells;

[0044] Figure 7 is the immunofluorescence staining result of the control group, the metal polyphenol nanoparticle group, the rapamycin group, and the metal polyphenol nanoparticle group loaded with rapamycin;

[0045] Figure 8 is a graph showing the drug loading rate of ZIF-8 nanoparticles loaded with rapamycin in Example 1 and Comparative Example 1;

[0046] Fig. 9 is the drug loading rate diagram of ZIF-8 nanoparticles loaded with rapamycin at different dosages;

[0047] Fig.10 is rapamycin and the supernatant of the first methanol washing of Example 1 and Example 7 1 H NMR spectra;

[0048] Fig.11 is a diagram of drug loading efficiency of rapamycin-loaded metal polyphenol nanoparticles at different dosages;

[0049] Fig.12 These are the XRD results of ZIF-8, ZIF-8 loaded with rapamycin, and metal polyphenol nanoparticles loaded with rapamycin. DETAILED DESCRIPTION

[0050] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0051] Example 1: Preparation method of metal polyphenol hollow structure loaded with rapamycin

[0052] Weigh 100 mg of zinc nitrate hexahydrate and 12 mg of rapamycin and dissolve them in 5 mL of methanol; weigh 1000 mg of 2-methylimidazole and dissolve it in 5 mL of water, and add 2-methylimidazole to zinc nitrate hexahydrate; mix and stir quickly for 30 minutes, collect nanoparticles by centrifugation (10000 g, 10 minutes) after stirring, wash three times with methanol to remove excess materials, and obtain ZIF-8 nanoparticles loaded with rapamycin. The obtained ZIF-8 nanoparticles loaded with rapamycin are dispersed in ultrapure water; weigh tannic acid and fully dissolve it in ultrapure water, add the tannic acid solution to the solution of the nanoparticles, control the nanoparticle concentration to 4 mg / mL, and the tannic acid concentration to 10 mg / mL; stir quickly for 5 minutes, collect nanoparticles by centrifugation (10000 g, 10 minutes) after stirring, and wash three times with methanol to remove excess materials to obtain metal polyphenol nanoparticles loaded with rapamycin.

[0053] Figure 1 The particle size distribution and dispersibility of ZIF-8 nanoparticles loaded with rapamycin, Figure 2 The particle size distribution and dispersibility of metal polyphenol nanoparticles loaded with rapamycin, Figure 3 Transmission electron microscopy image of ZIF-8 nanoparticles loaded with rapamycin. Figure 4 The transmission electron microscopy image of metal polyphenol nanoparticles loaded with rapamycin. It can be observed from the image that tannic acid etches ZIF-8 nanoparticles to form a hollow shell structure, and there is no obvious effect on the particle size and uniformity of the nanoparticles before and after etching. Fig.12 The XRD results of ZIF-8, ZIF-8 loaded with rapamycin, and metal polyphenol nanoparticles loaded with rapamycin prove the successful synthesis of ZIF-8; loading rapamycin will not change the crystal structure of ZIF-8; the crystal structure disappears after tannic acid etching of ZIF-8 nanoparticles, and the etching is successful.

[0054] Example 2: Preparation method of metal polyphenol hollow structure loaded with rapamycin

[0055] 200 mg of zinc nitrate hexahydrate and 12 mg of rapamycin were weighed and dissolved in 5 mL of methanol; 2000 mg of 2-methylimidazole was weighed and dissolved in 5 mL of water, 2-methylimidazole was added to zinc nitrate hexahydrate, and the mixture was rapidly mixed and stirred for 30 min. After the stirring was completed, nanoparticles were collected by centrifugation (10000 g, 10 min), and washed three times with methanol to remove excess materials to obtain ZIF-8 nanoparticles loaded with rapamycin. The obtained ZIF-8 nanoparticles loaded with rapamycin were dispersed in ultrapure water; tannic acid was weighed and fully dissolved in ultrapure water, and the tannic acid solution was added to the solution of the nanoparticles, and the concentration of the nanoparticles was controlled to be 4 mg / mL and the concentration of the tannic acid was 10 mg / mL; the mixture was rapidly stirred for 5 min. After the stirring was completed, the nanoparticles were collected by centrifugation (10000 g, 10 min), and washed three times with methanol to remove excess materials to obtain metal polyphenol nanoparticles loaded with rapamycin.

[0056] Electron microscopy results showed that tannic acid etched ZIF-8 nanoparticles to form a hollow shell structure, and there was no significant effect on the particle size and uniformity of the nanoparticles before and after etching. XRD results proved the successful synthesis of ZIF-8; loading rapamycin did not change the crystal structure of ZIF-8; the crystal structure of ZIF-8 nanoparticles disappeared after tannic acid etching, indicating successful etching.

[0057] Example 3: A method for preparing a hollow metal polyphenol structure loaded with rapamycin, comprising the following steps:

[0058] 100 mg of zinc nitrate hexahydrate and 8 mg of rapamycin were weighed and dissolved in 5 mL of methanol; 1000 mg of 2-methylimidazole was weighed and dissolved in 5 mL of water, 2-methylimidazole was added to zinc nitrate hexahydrate, and the mixture was rapidly mixed and stirred for 30 min. After the stirring was completed, nanoparticles were collected by centrifugation (10000 g, 10 min), and washed three times with methanol to remove excess materials to obtain ZIF-8 nanoparticles loaded with rapamycin. The obtained ZIF-8 nanoparticles loaded with rapamycin were dispersed in ultrapure water; tannic acid was weighed and fully dissolved in ultrapure water, and the tannic acid solution was added to the solution of the nanoparticles, and the nanoparticle concentration was controlled to be 4 mg / mL and the tannic acid concentration was 10 mg / mL; the mixture was rapidly stirred for 5 min. After the stirring was completed, the nanoparticles were collected by centrifugation (10000 g, 10 min), and washed three times with methanol to remove excess materials to obtain metal polyphenol nanoparticles loaded with rapamycin.

[0059] Electron microscopy results showed that tannic acid etched ZIF-8 nanoparticles to form a hollow shell structure, and there was no significant effect on the particle size and uniformity of the nanoparticles before and after etching. XRD results proved the successful synthesis of ZIF-8; loading rapamycin did not change the crystal structure of ZIF-8; the crystal structure of ZIF-8 nanoparticles disappeared after tannic acid etching, indicating successful etching.

[0060] Example 4: A method for preparing a hollow metal polyphenol structure loaded with rapamycin, comprising the following steps:

[0061] 100 mg of zinc nitrate hexahydrate and 10 mg of rapamycin were weighed and dissolved in 5 mL of methanol; 1000 mg of 2-methylimidazole was weighed and dissolved in 5 mL of water, 2-methylimidazole was added to zinc nitrate hexahydrate, and the mixture was rapidly mixed and stirred for 30 min. After the stirring was completed, nanoparticles were collected by centrifugation (10000 g, 10 min), and washed three times with methanol to remove excess materials to obtain ZIF-8 nanoparticles loaded with rapamycin. The obtained ZIF-8 nanoparticles loaded with rapamycin were dispersed in ultrapure water; tannic acid was weighed and fully dissolved in ultrapure water, and the tannic acid solution was added to the solution of the nanoparticles, and the concentration of the nanoparticles was controlled to be 4 mg / mL and the concentration of the tannic acid was 10 mg / mL; the mixture was rapidly stirred for 5 min. After the stirring was completed, the nanoparticles were collected by centrifugation (10000 g, 10 min), and washed three times with methanol to remove excess materials to obtain metal polyphenol nanoparticles loaded with rapamycin.

[0062] Electron microscopy results showed that tannic acid etched ZIF-8 nanoparticles to form a hollow shell structure, and there was no significant effect on the particle size and uniformity of the nanoparticles before and after etching. XRD results proved the successful synthesis of ZIF-8; loading rapamycin did not change the crystal structure of ZIF-8; the crystal structure of ZIF-8 nanoparticles disappeared after tannic acid etching, indicating successful etching.

[0063] Example 5: A method for preparing a hollow metal polyphenol structure loaded with rapamycin, comprising the following steps:

[0064] 100 mg of zinc nitrate hexahydrate and 14 mg of rapamycin were weighed and dissolved in 5 mL of methanol; 1000 mg of 2-methylimidazole was weighed and dissolved in 5 mL of water, 2-methylimidazole was added to zinc nitrate hexahydrate, and the mixture was rapidly mixed and stirred for 30 min. After the stirring was completed, nanoparticles were collected by centrifugation (10000 g, 10 min), and washed three times with methanol to remove excess materials to obtain ZIF-8 nanoparticles loaded with rapamycin. The obtained ZIF-8 nanoparticles loaded with rapamycin were dispersed in ultrapure water; tannic acid was weighed and fully dissolved in ultrapure water, and the tannic acid solution was added to the solution of the nanoparticles, and the concentration of the nanoparticles was controlled to be 4 mg / mL and the concentration of the tannic acid was 10 mg / mL; the mixture was rapidly stirred for 5 min. After the stirring was completed, the nanoparticles were collected by centrifugation (10000 g, 10 min), and washed three times with methanol to remove excess materials to obtain metal polyphenol nanoparticles loaded with rapamycin.

[0065] Electron microscopy results showed that tannic acid etched ZIF-8 nanoparticles to form a hollow shell structure, and there was no significant effect on the particle size and uniformity of the nanoparticles before and after etching. XRD results proved the successful synthesis of ZIF-8; loading rapamycin did not change the crystal structure of ZIF-8; the crystal structure of ZIF-8 nanoparticles disappeared after tannic acid etching, indicating successful etching.

[0066] Comparative Example 1

[0067] 200 mg of zinc nitrate hexahydrate and 10 mg of rapamycin were weighed and dissolved in 5 mL of methanol; 440 mg of 2-methylimidazole was weighed and dissolved in 5 mL of methanol, and the mixture was rapidly mixed and stirred for 30 min. After the stirring was completed, the nanoparticles were collected by centrifugation (10000 g, 10 min), and washed three times with methanol to remove excess materials to obtain ZIF-8 nanoparticles loaded with rapamycin. The obtained ZIF-8 nanoparticles loaded with rapamycin were dispersed in ultrapure water; tannic acid was weighed and fully dissolved in ultrapure water, and the tannic acid solution was added to the solution of the nanoparticles, and the concentration of the nanoparticles was controlled to be 4 mg / mL and the concentration of the tannic acid was 10 mg / mL; the mixture was rapidly stirred for 5 min. After the stirring was completed, the nanoparticles were collected by centrifugation (10000 g, 10 min), and washed three times with methanol to remove excess materials to obtain metal polyphenol nanoparticles loaded with rapamycin.

[0068] Embodiment 6:

[0069] 249 mg of cobalt nitrate hexahydrate and 10 mg of rapamycin were weighed and dissolved in 25 mL of methanol; 328 mg of 2-methylimidazole was weighed and dissolved in 25 mL of methanol, and the latter was introduced into the former under magnetic stirring to mix, and the mixed solution was allowed to stand at room temperature for 24 hours. Then, the nanoparticles were collected by centrifugation (10000 g, 10 minutes) and washed three times by centrifugation with methanol.

[0070] The obtained ZIF-67 nanoparticles loaded with rapamycin were dispersed in ultrapure water; tannic acid was weighed and fully dissolved in ultrapure water, and the tannic acid solution was added to the nanoparticle solution to control the nanoparticle concentration to be 4 mg / mL and the tannic acid concentration to be 10 mg / mL; the mixture was rapidly stirred for 5 minutes. After the stirring was completed, the nanoparticles were collected by centrifugation (10000 g, 10 minutes), and washed three times with methanol to remove excess materials to obtain metal polyphenol nanoparticles loaded with rapamycin.

[0071] Embodiment 7:

[0072] 1.22 g of copper nitrate trihydrate, 10 mg of rapamycin, and 0.58 g of trimesic acid were weighed and dissolved in 5 g of dimethyl sulfoxide to obtain a precursor solution. Then, 1 mL of the precursor solution was added to 50 mL of methanol within 1 minute, and after stirring for 20 minutes, the formed blue nanoparticles were collected by centrifugation (10000 g, 10 minutes), and washed three times by centrifugation with methanol.

[0073] The obtained HKUST-1 nanoparticles loaded with rapamycin were dispersed in ultrapure water; tannic acid was weighed and fully dissolved in ultrapure water, and the tannic acid solution was added to the nanoparticle solution to control the nanoparticle concentration to be 4 mg / mL and the tannic acid concentration to be 10 mg / mL; the mixture was rapidly stirred for 5 minutes, and after the stirring was completed, the nanoparticles were collected by centrifugation (10000g, 10 minutes), and washed three times with methanol to remove excess materials, thereby obtaining metal polyphenol nanoparticles loaded with rapamycin.

[0074] Embodiment 8:

[0075] 25.78 mg ZrCl4 (0.11 mmol), 10 mg rapamycin, and 13.29 mg 1,4-benzenedicarboxylic acid (0.08 mmol) were weighed and dissolved in 10 mL DMF solution; then 1.441 g acetic acid (0.024 mol) was added to the above solution. The mixed solution was placed in an oven for 24 hours, cooled to room temperature, and then centrifuged and washed three times with methanol.

[0076] The obtained UiO-66 nanoparticles loaded with rapamycin were dispersed in ultrapure water; tannic acid was weighed and fully dissolved in ultrapure water, and the tannic acid solution was added to the nanoparticle solution to control the nanoparticle concentration to be 4 mg / mL and the tannic acid concentration to be 10 mg / mL; the mixture was rapidly stirred for 5 minutes. After the stirring was completed, the nanoparticles were collected by centrifugation (10000 g, 10 minutes), and washed three times with methanol to remove excess materials to obtain metal polyphenol nanoparticles loaded with rapamycin.

[0077] Embodiment 9:

[0078] 42 mg of fumaric acid, 10 mg of rapamycin, and 160 mg of Fe(NO3)3 9H2O were weighed and dissolved in 8 mL of DMF solution; the resulting mixture was placed in an oil bath (110°C) for 30 min. After the reaction mixture was cooled to room temperature, it was centrifuged and washed three times with methanol.

[0079] The obtained MIL-88A nanoparticles loaded with rapamycin were dispersed in ultrapure water; tannic acid was weighed and fully dissolved in ultrapure water, and the tannic acid solution was added to the nanoparticle solution to control the nanoparticle concentration to be 4 mg / mL and the tannic acid concentration to be 10 mg / mL; the mixture was rapidly stirred for 5 minutes. After the stirring was completed, the nanoparticles were collected by centrifugation (10000 g, 10 minutes), and washed three times with methanol to remove excess materials to obtain metal polyphenol nanoparticles loaded with rapamycin.

[0080] Example 10: Method for constructing a functionalized drug-carrying system combined with coating technology for drug-coated balloons

[0081] A suspension of metal polyphenol nanoparticles loaded with rapamycin prepared in any one of Examples 1-5 is selected and filled into a syringe, an electromagnetic field is applied inside or outside the syringe for magnetic stirring, the uniformly stirred suspension is ultrasonically atomized and sprayed onto the outer surface of a rotating inflatable balloon body, the sprayed balloon is dried and folded, placed in a protective sleeve and sterilized with ethylene oxide to form a drug-carrying coating.

[0082] Example 11: Preparation method of hollow metal polyphenol structure

[0083] 100 mg of zinc nitrate hexahydrate was weighed and dissolved in 5 mL of methanol; 1000 mg of 2-methylimidazole was weighed and dissolved in 5 mL of water, 2-methylimidazole was added to zinc nitrate hexahydrate, and the mixture was rapidly mixed and stirred for 30 min. After the stirring was completed, nanoparticles were collected by centrifugation (10000 g, 10 min), and washed three times with methanol to remove excess materials to obtain ZIF-8 nanoparticles loaded with rapamycin. The obtained ZIF-8 nanoparticles loaded with rapamycin were dispersed in ultrapure water; tannic acid was weighed and fully dissolved in ultrapure water, and the tannic acid solution was added to the solution of the nanoparticles, and the concentration of the nanoparticles was controlled to be 4 mg / mL and the concentration of the tannic acid was 10 mg / mL; the mixture was rapidly stirred for 5 min. After the stirring was completed, the nanoparticles were collected by centrifugation (10000 g, 10 min), and washed three times with methanol to remove excess materials to obtain metal polyphenol nanoparticles.

[0084] Effect Example 1: Evaluation of the effect of rapamycin-loaded metal polyphenol nanoparticles on smooth muscle cells

[0085] Rat aortic smooth muscle cells were used as research cells, and CCK-8 was used to detect the cytotoxicity of metal polyphenol nanoparticles on smooth muscle cells; CCK-8 was used to detect the inhibitory function of metal polyphenol nanoparticles loaded with rapamycin on the proliferation of smooth muscle cells. The operation method is as follows:

[0086] 1. Add 100 μL of cells to each well of a 96-well plate (the blank group does not add cells, but adds the same volume of culture medium); culture the cells in a 5% CO2 cell culture incubator at 37°C for 24 h;

[0087] 2. Add 10 μL of different concentrations of experimental group stimulation to each well;

[0088] 3. Incubate the 96-well plate in a 5% CO2 cell culture incubator at 37°C for 24 hours;

[0089] 4. Add 10 μL of CCK-8 solution to each well and incubate in the incubator for 2 hours;

[0090] 5. Measure the absorbance at 450nm using an enzyme-labeled instrument;

[0091] 6. Result analysis:

[0092] The cell survival rate calculation formula is: cell survival rate = [(As-Ab) / (Ac-Ab)] × 100%; in this formula, As is the absorbance value of the test group, Ab is the absorbance value of the blank group, and Ac is the absorbance value of the control group.

[0093] At the same time, rat aortic smooth muscle cells were used as research cells, and immunofluorescence staining was used to determine the inhibitory effects of metal polyphenol nanoparticles (at a concentration of 3 μg / mL in terms of rapamycin), rapamycin (at a concentration of 3 μg / mL), and metal polyphenol nanoparticles loaded with rapamycin (at a concentration of 3 μg / mL in terms of rapamycin) on the proliferation of smooth muscle cells. The immunofluorescence staining operation method is as follows:

[0094] 1. Fixation: Fix the smooth muscle cells with 4% formaldehyde for 15 minutes, then rinse three times in PBS;

[0095] 2. Permeabilization: Incubate with 0.1-0.25% Triton X-100 for 5 minutes, then wash the cells 3 times with PBS;

[0096] 3. Blocking: Incubate with 4% BSA for 30 minutes;

[0097] 4. Incubate with alpha-SMA antibody at room temperature for 1 hour, then wash three times with PBS;

[0098] 5. Incubate with secondary antibody (Goat Anti-Rabbit IgG) at room temperature for 1 hour (protected from light), then wash three times with PBS;

[0099] 6. DAPI counterstaining, then washing 3 times with PBS;

[0100] Figure 5 This is a graph showing the results of measuring the cytotoxicity of metal polyphenol nanoparticles at different concentrations on smooth muscle cells; Figure 6 This is a graph showing the results of measuring the inhibitory effect of metal polyphenol nanoparticles loaded with different concentrations of rapamycin on the proliferation of smooth muscle cells; Figure 7 The following are the results of immunofluorescence staining of the control group, metal polyphenol nanoparticle group, rapamycin group, and metal polyphenol nanoparticle group loaded with rapamycin. It can be seen from the figure that the metal polyphenol nanoparticle loaded with rapamycin has good biocompatibility and has a significant inhibitory effect on the proliferation of smooth muscle cells. At the same time, immunofluorescence staining shows that under the same drug concentration, compared with the rapamycin group alone, the metal polyphenol nanoparticle group loaded with rapamycin has a better inhibitory effect on the proliferation of smooth muscle cells, more obvious contraction of smooth muscle cells, and less expression of specific proteins.

[0101] Effect Example 2: Determination of drug loading rate of ZIF-8 nanoparticles loaded with rapamycin

[0102] The rapamycin-loaded ZIF-8 nanoparticles prepared in Examples 1, 3, 4, 5 and Comparative Example 1 were ultrasonically dispersed in a methanol solution, 1 mL was added to a centrifuge tube, 1 mmol of hydrochloric acid solution was added dropwise until the emulsion became clear and transparent, the absorbance at 277 nm was measured using an ultraviolet spectrophotometer, and the rapamycin concentration loaded on the ZIF-8 nanoparticles was calculated using the rapamycin standard curve.

[0103] Drug loading rate = mass of rapamycin loaded on ZIF-8 nanoparticles / mass of rapamycin loaded on ZIF-8 nanoparticles.

[0104] Figure 8 1 is a graph showing the drug loading rate of ZIF-8 nanoparticles loaded with rapamycin in Example 1 and Comparative Example 1. Compared with Comparative Example 1, Example 1 achieves a high loading rate for rapamycin.

[0105] Fig. 9 is the drug loading rate of rapamycin-loaded ZIF-8 nanoparticles with different dosages.

[0106] Effect Example 3: Determination of drug loading rate of metal polyphenol nanoparticles loaded with rapamycin

[0107] The supernatant after the first centrifugation of the metal polyphenol nanoparticles loaded with rapamycin washed with methanol in Example 1 was collected; and the supernatant after the first centrifugation of the metal polyphenol nanoparticles washed with methanol in Example 7 was collected. 1 H NMR analysis, the results are as follows Fig.10 It can be seen that there is no rapamycin in the supernatant after centrifugation of the metal polyphenol nanoparticles loaded with rapamycin after the first washing with methanol in Example 1, and TA will not cause leakage of rapamycin during the reaction of ZIF-8 nanoparticles loaded with rapamycin, so the content of rapamycin loaded by the metal polyphenol nanoparticles is consistent with the content of rapamycin loaded by the ZIF-8 nanoparticles.

[0108] Drug loading rate = mass of rapamycin loaded on ZIF-8 nanoparticles / mass of metal polyphenol nanoparticles loaded with rapamycin

[0109] The method for determining the drug loading rate of other rapamycin-loaded metal polyphenol nanoparticles is the same as above. Fig.11 : is the drug loading rate of rapamycin-loaded metal polyphenol nanoparticles (Examples 3, 4, 1, and 5) at different dosages.

[0110] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing rapamycin-loaded metal polyphenol nanoparticles, characterized in that: Rapamycin is added to a metal organic framework precursor solution to prepare metal organic framework nanoparticles loaded with rapamycin; then the obtained dispersion of the metal organic framework nanoparticles is added to a tannic acid solution, and the mixture is rapidly stirred. After the stirring is completed, the nanoparticles are collected by centrifugation and washed to obtain metal polyphenol nanoparticles loaded with rapamycin.

2. The method for preparing rapamycin-loaded metal polyphenol nanoparticles according to claim 1, characterized in that: The metal organic framework includes any one or more of ZIF-8, ZIF-67, HKUST-1, UiO-66, and MIL-88A.

3. The method for preparing rapamycin-loaded metal polyphenol nanoparticles according to claim 1 or 2, characterized in that: The solvent of the metal organic framework precursor solution is methanol; The amount of rapamycin added is calculated based on the ratio of 8 to 14 mg to 25 mL of the solvent of the metal organic framework precursor solution; The concentration of the metal organic framework nanoparticles is controlled to be 4±1 mg / mL; The concentration of the tannic acid is controlled to be 10±1 mg / mL.

4. The method for preparing rapamycin-loaded metal polyphenol nanoparticles according to claim 1, characterized in that: The metal organic framework is ZIF-8, and the preparation method comprises the following steps: Step 1: Weigh zinc nitrate hexahydrate and rapamycin and dissolve them in methanol; Step 2: Weigh 2-methylimidazole and dissolve it in water, quickly add it to the solution obtained in step 1, mix and stir, collect the nanoparticles by centrifugation after stirring, and wash them to obtain ZIF-8 nanoparticles loaded with rapamycin; Step 3: dispersing the rapamycin-loaded ZIF-8 nanoparticles obtained in step 2 in ultrapure water to obtain a nanoparticle dispersion; Step 4: Weigh tannic acid and fully dissolve it in ultrapure water to obtain a tannic acid solution; Step 5: Add the tannic acid solution obtained in step 4 to the nanoparticle dispersion obtained in step 3, and stir rapidly. After stirring, collect the nanoparticles by centrifugation, wash, and obtain metal polyphenol nanoparticles loaded with rapamycin.

5. The method for preparing rapamycin-loaded metal polyphenol nanoparticles according to claim 4, characterized in that: The weight ratio of zinc nitrate hexahydrate, rapamycin and 2-methylimidazole in step 1 and step 2 is 100-200:8-14:1000-2000.

6. The method for preparing rapamycin-loaded metal polyphenol nanoparticles according to claim 5, characterized in that: The weight ratio of zinc nitrate hexahydrate, rapamycin and 2-methylimidazole in step 1 and step 2 is 100:12:1000; The concentration of the nanoparticles described in step 5 is controlled to be 4 mg / mL, and the concentration of tannic acid is controlled to be 10 mg / mL; The stirring time in step 2 is 30±5min; The stirring time in step 5 is 5±1min; The centrifugation conditions in step 2 and step 5 are 10000±2000g, 10±2min; The cleaning described in step 2 and step 5 refers to cleaning with methanol 2-4 times.

7. A metal polyphenol nanoparticle loaded with rapamycin, characterized in that: The method is obtained by the preparation method described in any one of claims 1 to 6.

8. A drug-coated balloon, characterized in that: The invention comprises a balloon body and a drug coating at least partially covering the surface of the balloon body, wherein the drug coating is the metal polyphenol nanoparticles loaded with rapamycin as described in claim 7.

9. The method for preparing the drug-coated balloon according to claim 8, characterized in that: The rapamycin-loaded metal polyphenol nanoparticle suspension is filled into a syringe, an electromagnetic field is applied inside or outside the syringe for magnetic stirring, the uniformly stirred suspension is ultrasonically sprayed on the outer surface of a rotating inflatable balloon body, and the sprayed balloon is dried and folded to form a drug-loaded coating.

10. Use of the drug-coated balloon according to claim 8 in preparing a drug for treating atherosclerosis.

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