Preparation method of nano-drug for regulating atherosclerotic lesion as well as product and application of nano-drug

Abstract

The invention discloses a preparation method of a nano-drug for regulating atherosclerosis lesion and a product and application of the nano-drug, and belongs to the field of pharmaceutical chemicals, the method comprises the following steps: preparing a macrophage membrane, preparing plant polysaccharide nano-particles through a nano-precipitation method, and preparing the plant polysaccharide nano-particles through a nano-precipitation method; the plant polysaccharide nanoparticle comprises a polylactic acid-glycolic acid copolymer carrier, wherein plant polysaccharide is loaded on the polylactic acid-glycolic acid copolymer carrier; the plant polysaccharide nanoparticles are coated with a macrophage membrane. The preparation method is simple and remarkable in effect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine and chemical industry, and particularly relates to a preparation method of a nano drug for regulating atherosclerotic lesions, and a product and application thereof. BACKGROUND

[0002] Atherosclerosis (AS) is a lipid deposition in the intima of large and medium arteries, intimal thickening, and then gradually forming a plaque, making the artery elasticity decreased, the lumen narrowed, and the plaque rupture leading to thrombosis, causing arterial blood supply obstruction. Atherosclerosis is the main cause of coronary heart disease, stroke, peripheral vascular disease and other atherosclerotic vascular diseases. Vascular inflammation and plaque stability are the decisive factors that accelerate atherosclerotic lesions, and the vascular stability is determined by the proteinase secreted by macrophages, VSMC phenotype and elastin-rich extracellular matrix, and the fibrous cap composed of contractile SMC cells can protect and stabilize the atherosclerotic plaque rupture.

[0003] SMCs are a class of highly differentiated cells that are mainly responsible for the contraction and relaxation of blood vessels. Under normal physiological conditions, SMCs maintain a contractile phenotype and play a contractile function to regulate blood vessels and maintain vascular tension. Under pathological conditions such as vascular injury and inflammation, SMCs can transform into osteoblasts, adipocytes and macrophage foam cells. Studies have found that SMCs can transform into macrophage-like phenotype in atherosclerosis, and these cells express both macrophage and SMC markers. In human coronary artery sections, cell-specific markers were used to identify cell types, and the results showed that in foam cell-rich lesions, 50% of the foam cells were derived from SMCs; the markers CD68 and SMα-actin, which are markers of macrophages and SMCs, respectively, indicate that in advanced AS patients, 40% of CD68-positive cells are derived from SMCs, which indicates that the macrophages identified as monocyte-derived macrophages in AS plaques are actually derived from SMCs. Intervention of SMCs with ox-LDL can induce the formation of classic foam cells, and this foam formation is related to the transformation of SMCs into synthetic SMCs. Studies have also shown that in high-fat-fed ApoE− / − mice, foam cell formation is formed by activating ABCA1 expression in SMCs, which is independent of macrophages. Therefore, the use of macrophages (such as RAW264.7 and TPH-1) to induce differentiation cannot represent the formation of foam in atherosclerosis, and SMCs should be used to induce foam, which is more consistent with the pathogenesis of atherosclerosis foam. Therefore, studying how to regulate vascular smooth muscle cells to maintain a contractile phenotype and inhibit macrophage-like phenotype differentiation is an important research approach to alleviate atherosclerosis. SUMMARY

[0004] The present application aims to provide a preparation method of a nano drug for regulating atherosclerotic lesions to solve the problem of complex preparation of nano drugs in the prior art.

[0005] The present application is achieved by the following technical scheme, a preparation method of a nano drug for regulating atherosclerotic lesions, comprising the following steps: preparing macrophage membranes, preparing plant polysaccharide nanoparticles by a nano precipitation method, the plant polysaccharide nanoparticles comprising: a polylactic acid-glycolic acid copolymer carrier, the polylactic acid-glycolic acid copolymer carrier loaded with plant polysaccharides, and the plant polysaccharide nanoparticles coated with macrophage membranes.

[0006] Further, the preparation of macrophage membranes comprises the following steps: dispersing cells separated from a mouse macrophage cell line RAW 264.7 in a membrane protein extraction buffer and cooling in an ice bath for 15 min; then, treating the cell suspension with an ultrasonic cell crusher for 3 seconds to completely lyse the cells; and then centrifuging the obtained mixture, collecting the supernatant and centrifuging again to precipitate the cell membranes.

[0007] Further, the preparation of plant polysaccharide nanoparticles by a nano precipitation method comprises the following steps: dissolving the polylactic acid-glycolic acid copolymer carrier in DMSO, then adding plant polysaccharide active ingredients to fully dissolve them, to prepare a first mixed solution; adding the first mixed solution dropwise to ultrapure water and slowly stirring at room temperature for 1 h to evaporate the organic solvent, to prepare a second mixed solution; placing the second mixed solution in a dialysis bag in distilled water to remove free organic solvents, and freeze-drying to obtain plant polysaccharide nanoparticles.

[0008] Further, the coating of plant polysaccharide nanoparticles with macrophage membranes comprises: mixing the macrophage vesicle solution with the plant polysaccharide nanoparticles, and ultrasonically treating at 42 kHz, 100 W for 3 min, and extruding 30 times through a 400 nm polycarbonate porous membrane with a liposome micro-extruder.

[0009] Further, the plant polysaccharides comprise one or more combinations of wolfberry polysaccharides, black fungus polysaccharides, fucoidan, dandelion polysaccharides and Huoshan Dendrobium polysaccharides.

[0010] Another aspect of the present application provides a nano drug product for regulating atherosclerotic lesions, prepared according to the preparation method described above.

[0011] Another aspect of the present application also provides an application of a nano drug product for regulating atherosclerotic lesions, which comprises: (1) application in the treatment of atherosclerosis; and (2) application in the regulation of atherosclerotic lesions.

[0012] Compared with the prior art, the present application has the following advantages and beneficial effects:

[0013] The present application has the advantages of simple operation method, high preparation efficiency, and good curative effect of the prepared product. BRIEF DESCRIPTION OF DRAWINGS

[0014] The accompanying drawings, which are included to provide a further understanding of the embodiments of the application and are incorporated in and constitute a part of this application, illustrate embodiments of the application and together with the description serve to explain the principles of the application. In the drawings:

[0015] Figure 1 TEM image of blank carrier nanoparticles provided for Example 2 of the present application.

[0016] Figure 2 TEM image of plant polysaccharide nanoparticles provided for Example 2 of the present application.

[0017] Figure 3 TEM image of macrophage membrane-coated plant polysaccharide nanoparticles provided for Example 2 of the present application. DETAILED DESCRIPTION

[0018] In order to make the objects, technical schemes and advantages of the embodiments of the present application clearer, the technical schemes in the embodiments of the present application will be described clearly and completely below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are some embodiments of the present application, rather than all the embodiments of the present application.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All publications mentioned in this specification are herein incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any reference in this specification is not an admission that it is prior art. The use of the alternative "a" or "an" to describe a plurality of elements is intended to include both the singular and the plural unless otherwise indicated. The use of the term "about" in relation to a number is intended to include ±20% of the number unless otherwise indicated. In some embodiments, the term "about" in relation to a number is intended to include ±10% of the number. In some embodiments, the term "about" in relation to a number is intended to include ±5% of the number.

[0020] Example 1

[0021] The embodiment discloses a preparation method of a nano-drug product for regulating an atherosclerotic lesion, and the method comprises the following steps:

[0022] Step 1: cells isolated from a mouse macrophage cell line RAW 264.7 are dispersed in a membrane protein extraction buffer and cooled in an ice bath for 15 min; then, the cell suspension is treated with an ultrasonic cell crusher for 3 seconds to completely lyse the cells; then, the obtained mixture is centrifuged, the supernatant is collected and centrifuged again to precipitate the cell membrane, so that the macrophage membrane is prepared.

[0023] Step 2: plant polysaccharide nanoparticles are prepared by a nano-precipitation method, and the plant polysaccharide nanoparticles comprise: a polylactic acid-glycolic acid copolymer carrier, and the polylactic acid-glycolic acid copolymer carrier carries plant polysaccharides. The polylactic acid-glycolic acid copolymer carrier is dissolved in DMSO, and then the plant polysaccharide active ingredient is added and fully dissolved to prepare a first mixed solution; the first mixed solution is added dropwise into ultrapure water and slowly stirred at room temperature for 1 h, so that the organic solvent is evaporated to prepare a second mixed solution; the second mixed solution is placed in a dialysis bag and dialyzed in distilled water to remove free organic solvents, and then freeze-dried to obtain the plant polysaccharide nanoparticles.

[0024] Specifically, the plant polysaccharides comprise one or more of a combination of wolfberry polysaccharides, black fungus polysaccharides, fucoidan, dandelion polysaccharides and Dendrobium huoshanense polysaccharides.

[0025] Step 3: the plant polysaccharide nanoparticles are coated with the macrophage membrane. The macrophage vesicle solution is mixed with the plant polysaccharide nanoparticles, and ultrasonic treatment is performed at 42 kHz and 100 W for 3 min, and the plant polysaccharide nanoparticles are extruded 30 times through a 400 nm polycarbonate porous membrane by using a liposome micro-extruder.

[0026] Embodiment 2

[0027] The embodiment discloses a specific operation process of a preparation method of a nano-drug for regulating an atherosclerotic lesion, and the method comprises the following contents:

[0028] (1) Preparation of macrophage membranes: The macrophage membranes (MM) were isolated from mouse macrophage cell line RAW 264.7 cells using a membrane protein extraction kit. Briefly, the collected cells were dispersed in a membrane protein extraction buffer and cooled in an ice bath for 15 minutes. Thereafter, the cell suspension was treated with an ultrasonic cell disrupter (42 kHz, 100 W) for 3 seconds to completely lyse the cells. The resulting mixture was then centrifuged (4 °C, 700 g, 8 minutes), and the supernatant was collected and centrifuged again (4 °C, 14 000 g, 30 minutes) to precipitate the cell membranes. The total protein content of the macrophages was determined by the bicinchoninic acid protein assay. To obtain macrophage vesicles, the extracted macrophages were first subjected to ultrasonic treatment for 15 minutes, and then extruded 10 times through a 400 nm polycarbonate porous membrane using an Avestin micro-extruder (Avestin, LF-1, Canada). The harvested macrophage vesicles were stored in PBS at a temperature of 4 °C.

[0029] (2) Preparation of plant polysaccharide nanoparticles: The plant polysaccharide nanoparticles were prepared by the nanoprecipitation method. 10 mg of PLGA was dissolved in 1 mL of DMSO, and then 1 mg of the plant polysaccharide active ingredient was added to dissolve it completely, obtaining a solvent phase in which the polymer and the drug were miscible; 1 mL of the above solvent phase was added dropwise to 6 mL of ultrapure water under slow stirring. The mixture was stirred at room temperature for 1 h to evaporate the organic solvent. Then, the prepared plant polysaccharide nanoparticle solution was dialyzed in a dialysis bag (MWCO = 3500 Da) in distilled water for 12 h to remove free organic solvents, and freeze-dried to obtain plant polysaccharide nanoparticles for standby. If no plant polysaccharide active ingredient is added during preparation, blank carrier nanoparticles (PLGA NPs) are obtained.

[0030] Measurement of drug loading and encapsulation efficiency of plant polysaccharide nanoparticles: The freeze-dried plant polysaccharide nanoparticle powder was redissolved to obtain a plant polysaccharide nanoparticle solution of a certain concentration. According to the previously established standard curve of the plant polysaccharide active ingredient, the amount of active ingredient in the plant polysaccharide nanoparticle solution was measured using LC-MS, and the drug loading and encapsulation efficiency were calculated as follows:

[0031]

[0032] The amount of plant polysaccharide active ingredient contained in the plant polysaccharide nanoparticles, The total amount of plant polysaccharide nanoparticles.

[0033]

[0034] The amount of plant polysaccharide active ingredient in the plant polysaccharide nanoparticles, Total amount of plant polysaccharide active ingredient.

[0035] (4) Preparation of macrophage membrane-coated plant polysaccharide nanoparticles: 1 x 10 8 The MM vesicle solution obtained from 1 x 10

[0036] The TEM image of the blank carrier nanoparticles (PLGANPs) prepared in this example is shown in Figure 1 From Figure 1 it can be seen that the PLGANPs microspheres prepared in this example are about 100 nm in size, round, uniform in morphology, and have good dispersibility. Figure 2 The TEM image of the plant polysaccharide nanoparticles prepared in this example is Figure 3 The TEM image of the macrophage membrane-coated plant polysaccharide nanoparticles prepared in this example is

[0037] The above specific embodiments further illustrate the purposes, technical solutions and beneficial effects of the present application. It should be understood that the above description is only a specific embodiment of the present application and is not intended to limit the protection scope of the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing a nano-drug for regulating an atherosclerotic lesion, characterized by, The preparation method of the nanomedicine comprises: preparing macrophage membranes, plant polysaccharide nanoparticles are prepared by a nanometer precipitation method, and the plant polysaccharide nanoparticles comprise: a polylactic acid-glycolic acid copolymer carrier, and the polylactic acid-glycolic acid copolymer carrier is loaded with plant polysaccharides; the plant polysaccharide nanoparticles are coated with macrophage membranes.

2. The method of claim 1, wherein the nano-drug for regulating atherosclerotic lesion is prepared by the steps of: (a) mixing a biodegradable polymer, a drug, and a surfactant; (b) dissolving the mixture in a solvent; (c) removing the solvent; and (d) drying the mixture. The preparation of the macrophage membranes comprises the following steps: cells separated from a mouse macrophage cell line RAW 264.7 are dispersed in a membrane protein extraction buffer and cooled in an ice bath for 15 min; then, the cell suspension is treated with an ultrasonic cell crusher for 3 seconds to completely lyse the cells; the obtained mixture is then centrifuged, the supernatant is collected and centrifuged again to precipitate the cell membranes.

3. The method for preparing nanomedicine for regulating atherosclerotic lesions according to claim 1, characterized in that, The preparation of the plant polysaccharide nanoparticles by the nanometer precipitation method comprises the following steps: a polylactic acid-glycolic acid copolymer carrier is dissolved in DMSO, and then plant polysaccharide active ingredients are added to fully dissolve them, to prepare a first mixed solution; the first mixed solution is added dropwise to ultrapure water and slowly stirred at room temperature for 1 h to evaporate the organic solvent, to prepare a second mixed solution; the second mixed solution is placed in a dialysis bag and dialyzed in distilled water to remove free organic solvents, and then freeze-dried to obtain plant polysaccharide nanoparticles.

4. The method of claim 1, wherein the nano-drug for regulating atherosclerotic lesion is prepared by the steps of: (a) mixing a biodegradable polymer, a drug, and a surfactant; (b) dissolving the mixture in a solvent; (c) removing the solvent; and (d) drying the mixture. The coating of the plant polysaccharide nanoparticles with macrophage membranes comprises: the macrophage vesicle solution is mixed with the plant polysaccharide nanoparticles, and ultrasonic treatment is performed at 42 kHz and 100 W for 3 min, and the plant polysaccharide nanoparticles are extruded 30 times through a 400 nm polycarbonate porous membrane using a liposome micro-extruder.

5. The method of claim 1, wherein the nano-drug for regulating atherosclerotic lesion is prepared by the steps of: (a) mixing a biodegradable polymer, a drug, and a surfactant; (b) dissolving the mixture in a solvent; (c) removing the solvent; and (d) drying the mixture. The plant polysaccharides comprise: a combination of one or more of wolfberry polysaccharides, black fungus polysaccharides, fucoidan, dandelion polysaccharides, and Dendrobium huoshanense polysaccharides.

6. A nano-pharmaceutical product for modulating atherosclerotic lesions, characterized in that, The nanomedicine product is prepared according to the preparation method of any one of claims 1-5.

7. Use of a nanomedicine product for modulating atherosclerotic lesions, characterized in that, The nanomedicine product in claim 6 comprises the following applications: (1) application in treating atherosclerosis; (2) application in regulating atherosclerotic lesions.

Images