Polydopamine nanoparticle-loaded extracellular vesicles and method for producing the same

Abstract

Polydopamine-loaded extracellular vesicles (EVVs) comprise M2 RAW264.7 macrophage-derived EVVs and polydopamine nanoparticles encapsulated within the EVVs as an active therapeutic component. The EVVs are prepared by treating RAW264.7 macrophages with IL-4 and IL-10 cytokines to polarize them into M2 macrophages, co-culturing the EVVs with polydopamine nanoparticles, and then extrusion-extracting the EVVs. The EVVs actively accumulate in the inflamed areas of atherosclerotic plaques, exerting antioxidant functions. These EVVs can be used to prepare anti-inflammatory drugs or formulations for atherosclerosis.

Description

[Technical Field]

[0001] The present invention claims priority to an application entitled "Polydopamine nanoparticle-loaded extracellular vesicles and methods for producing the same," application number CN2022110886583, filed by the applicant on September 7, 2022. The entire contents of the above application are incorporated herein by reference in their entirety.

[0002] The present invention relates to the technical field of atherosclerosis treatment, and in particular to an extracellular vesicle-polydopamine nanotargeting therapeutic platform, i.e., polydopamine nanoparticle-loaded extracellular vesicles and a method for preparing the same. [Background technology]

[0003] The description of the background art in the present invention belongs to the related art relevant to the present invention and is used only to facilitate the description and understanding of the inventive content of the present invention, and should not be construed as an indication that the applicant expressly believes or presumes that the present invention is prior art as of the filing date of the first application.

[0004] Atherosclerosis is the main cause of cardiovascular disease, which is one of the leading causes of morbidity and mortality worldwide. Inflammation is not only an important indicator of atherosclerosis but also promotes the overall progression of the disease. Therefore, anti-inflammation is considered a promising approach for the treatment of atherosclerosis.

[0005] However, anti-inflammatory drugs lack the ability to specifically target the inflammatory site and usually have a short half-life, which not only affects the actual therapeutic effect but also causes many side effects and may even lead to death.Nanomaterials with anti-inflammatory properties also have their own immunogenicity, toxicity, and biodistribution issues, limiting their application in the treatment of inflammation. Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide polydopamine-loaded extracellular vesicles that are capable of controlling the size of nanovesicles and have a high recovery rate, and a method for producing the same. [Means for solving the problem]

[0007] In order to solve the above problems in the prior art, the present invention provides the following technical solutions:

[0008] In a first aspect, the present invention provides a method for producing polydopamine-loaded extracellular vesicles, comprising: polarizing RAW264.7 macrophages into M2 macrophages by treating them with IL-4 and IL-10 cytokines; The method includes a step of co-culturing polydopamine nanoparticles with M2 macrophages, targeting the inflamed area, and obtaining polydopamine nanoparticle-loaded extracellular vesicles derived from M2 macrophages.

[0009] Furthermore, polydopamine nanoparticle-loaded extracellular vesicles derived from the M2 macrophages and targeting the inflamed area can be obtained by sequential extrusion of M2 macrophages co-cultured with polydopamine nanoparticles using a size-sequential extrusion method.

[0010] Furthermore, polydopamine nanoparticles are loaded into the extracellular vesicles using a size-sequential extrusion method.

[0011] Furthermore, the M2 macrophages were obtained by treating RAW264.7 macrophages with IL-4 and IL-10 cytokines, and were cultured in a cell culture incubator for at least 1 × 10 7 cells / ml of cells are co-cultured with 100 ug / mL of polydopamine nanoparticles.

[0012] Furthermore, the process of loading the polydopamine nanoparticles into the nano-extracellular vesicles specifically includes the steps of adding polydopamine nanoparticles to an M2 macrophage culture system, co-culturing the cells, collecting the cells using a cell scraper, and extruding the cell suspension through 1 μm, 400 nm, and 200 nm polycarbonate membranes in sequence using a microextruder for 5 to 6 consecutive extrusions, washing with PBS, and centrifuging to remove unloaded polydopamine nanoparticles, thereby finally obtaining polydopamine nanoparticle-loaded extracellular vesicles.

[0013] In a second aspect, the present invention provides polydopamine nanoparticle-loaded extracellular vesicles produced by the above-described production method.

[0014] The melatonin-loaded extracellular vesicles are produced by the above-mentioned production method. [Effects of the Invention]

[0015] The present invention has the following advantages over the prior art: In the method of the present invention, M2 macrophages are selected as the raw material for vesicles when selecting nanomaterials. M2 macrophages have the ability to target inflammatory environments and contain abundant anti-inflammatory factors. The vesicles from which they are derived not only contain abundant physiologically active substances from the parent cells, but also have the ability to actively target inflammatory environments, and have excellent biocompatibility and immune evasion capabilities, resulting in improved therapeutic effects.

[0016] In terms of manufacturing method, continuous extrusion, a physical method for obtaining vesicles, is used, which achieves controllable size and high yield of vesicles, simplifies the process of obtaining vesicles, and reduces costs.

[0017] In terms of functionality, the nanovesicles not only function as a carrier to deliver antioxidant polydopamine nanoparticles to inflammatory lesion sites and extend their circulation time in the body, but M2 macrophage-derived nanovesicles also have anti-inflammatory properties, and when combined with the delivery of anti-inflammatory particles, two therapeutic effects can be achieved with a single delivery.

[0018] The production method of the present invention is simple and effective, M2 macrophages can be easily obtained, and the physical method of continuous extrusion is easy to operate and allows for large-scale production.

[0019] Further advantages of the above-described non-conventional embodiments are described below with reference to the detailed description.

[0020] In order to more clearly describe the embodiments of the present application or the existing technical solutions, the following briefly describes the drawings that need to be used to describe the embodiments or the prior art. Obviously, the drawings described below are only a part of the embodiments described in the present application, and those skilled in the art can obtain other drawings based on these drawings without any creative work. [Brief explanation of the drawings]

[0021] [Figure 1] This figure shows the protein expression patterns of M2 macrophages obtained by treating RAW264.7 macrophages with IL-4 and IL-10 cytokines for 24 hours using the polydopamine nanoparticle-loaded extracellular vesicles and their manufacturing method of the present invention. Western blot analysis showed that the expression of Arg1, a protein marker for M2 macrophages, was upregulated, while the expression of iNOS, a protein marker for M1 macrophages, was downregulated, indicating that M2 macrophages were successfully obtained. (Arg1: arginase 1; iNOS: inducible nitric oxide synthase; Actin: actin; M0: unstimulated activated macrophages; M2: activated macrophage replacement.) [Figure 2] 1 shows a transmission electron microscope image of polydopamine nanoparticles in the polydopamine nanoparticle-loaded extracellular vesicles and the method for producing the same of the present invention. The transmission electron microscope image shows that the polydopamine nanoparticles are spherical, uniform in size, and evenly distributed, demonstrating that the polydopamine nanoparticles are also suitable for loading into spherical extracellular vesicles. [Figure 3]The polydopamine nanoparticle-loaded extracellular vesicles and their manufacturing method of the present invention are obtained by sequential size exclusion and sequential extrusion of a mixture of polydopamine nanoparticles co-cultured with M2 macrophages. Transmission electron microscopy demonstrates that the polydopamine nanoparticle-loaded extracellular vesicles are uniform in size. Transmission electron microscopy reveals that polydopamine nanoparticles are surrounded by a transparent bilayer lipid membrane structure, i.e., extracellular vesicles with a bilayer membrane structure, and polydopamine nanoparticles are loaded within the vesicles. [Figure 4] 1 is a confocal image showing the antioxidant ability of polydopamine nanoparticle-loaded extracellular vesicles in the polydopamine nanoparticle-loaded extracellular vesicles and the method for producing the same of the present invention. In a cell intervention experiment in which macrophages, i.e., foam cells, were treated with 1 mM H2O2 for 24 hours, polydopamine nanoparticle-loaded extracellular vesicles (PDA@M2 NVs) were found to have excellent antioxidant properties. Confocal microscopy of DCFH-DA, JC-1, and MitoSox experiments showed that the green signal value of the DCFH-DA probe in cells treated with PDA@M2 NVs was significantly reduced compared to single polydopamine nanoparticles (PDA NPs), indicating a reduction in intracellular ROS content after intervention. In the JC-1 probe experiment, the red fluorescence signal after PDA@M2 NVs treatment was significantly higher than that after PDA NPs treatment. At the same time, the red fluorescence of MitoSox in the PDA@M2 NVs group was lower than that in the PDA NPs group, indicating a greater reduction in intracellular ROS content after PDA@M2 NVs treatment. Therefore, it can be concluded that treatment with polydopamine nanoparticle-loaded extracellular vesicles can reduce reactive oxygen species in H2O2-treated macrophages and exert antioxidant function. [Figure 5]Figure 1 shows fluorescence images of polydopamine nanoparticle-loaded extracellular vesicles (PDA@M2 NVs) actively targeting areas of atherosclerotic plaques. Fluorescence imaging of ex vivo blood vessels revealed that, when polydopamine nanoparticle-loaded extracellular vesicles (PDA@M2 NVs) were injected into the tail vein, they actively accumulated in the inflamed areas of atherosclerotic plaques, exhibiting a stronger fluorescent signal than single polydopamine nanoparticles (PDA NPs) that abundantly accumulated in ex vivo atherosclerotic blood vessels. (To ensure comparability between the two groups, both experiments used DiD-labeled PDA NPs at a dose of 20 mg / kg injected via tail vein for consistent signal levels.) [Figure 6] 1 shows tissue section images of the biological toxicity of polydopamine nanoparticle-loaded extracellular vesicles according to the present invention and the method for producing the same. HE staining showed that the polydopamine nanoparticle-loaded extracellular vesicles had no obvious toxic effects on the internal organs of mice. DETAILED DESCRIPTION OF THE INVENTION

[0022] Reference will now be made in detail to exemplary embodiments illustrated in the accompanying drawings. When referring to the drawings in the following description, unless otherwise noted, the same numbers in different drawings represent the same or similar elements. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with the present application.

[0023] Rather, they are merely examples of apparatus and methods consistent with aspects of the present application as set forth in the claims below.

[0024] The terminology used in this application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used in this application and the appended claims, the singular forms "a," "the," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It should also be understood that the term "and / or," as used herein, includes any and all possible combinations of one or more of the associated listed items.

[0025] In this application, terms such as "first," "second," and "third" may be used to describe various pieces of information, but it should be understood that such information is not limited to these terms. These terms are used only to distinguish between pieces of information of the same type. For example, first information may also be referred to as second information, and similarly, second information may also be referred to as first information, without departing from the scope of this application.

[0026] Through extensive research, the applicant has discovered that extracellular vesicles (EVs), nanovesicles secreted by various types of cells, possess excellent natural properties, such as excellent biocompatibility, low cytotoxicity, immunological inactivity, specific targeting, and long-term circulation, making them effective drug delivery vehicles. Exosomes derived from immune cells and mesenchymal stem cells have been effectively used to encapsulate and deliver chemotherapeutic drugs, nucleic acid drugs, neurotransmitters, and even nanoparticles for the treatment of various diseases, including cancer and neurological disorders, but have rarely been used for cardiovascular diseases. Furthermore, the specific accumulation of EV-based drug delivery systems remains limited, and even when they are modified for targeting and possess targeting functions, they still provoke some adverse reactions due to different resistances, such as the formation of a protein corona.

[0027] In addition to their unique properties, extracellular vesicles can inherit specific contents (e.g., RNA, DNA, proteins, and small molecules) from parent cells, allowing them to directly utilize the natural biological functions of the original cells for disease treatment. M2 macrophages can secrete anti-inflammatory cytokines, and even after sequential extrusion and encapsulation of anti-inflammatory polydopamine nanoparticles, M2 macrophages maintain their ability to target inflamed areas. The synergistic regulatory effect of M2 macrophage-derived extracellular vesicles and the intracellular release of pre-loaded cargo dually promote M2 polarization of macrophages at lesion sites and regulate inflammation. Macrophage phenotype conversion induced by M2 macrophage-derived extracellular vesicles, with its excellent cell reprogramming ability and inherent biocompatibility, may be a promising approach for the treatment of various inflammation-related diseases by modulating the balance between pro-inflammatory and anti-inflammatory macrophages. This study combines the advantages of both naturally derived extracellular vesicles and synthetic materials to provide a method for custom-designing modular extracellular vesicles for a variety of applications.

[0028] Through extensive research, the applicant has discovered that inflammation plays a key role in the development and progression of atherosclerotic plaques. The early stages of atherosclerosis are caused by endothelial damage, abnormal lipid metabolism, and hemodynamic disturbances. The atherosclerotic process is believed to be driven by inflammatory changes in endothelial cells via the bloodstream. When endothelial cells are activated, they express inflammatory factors, such as monocyte chemoattractant protein-1, intercellular adhesion molecule-1 (ICAM-1), vascular adhesion molecule-1 (VCAM-1), e-selectin, and p-selectin, which bind to the endothelium and attract lymphocytes and monocytes that infiltrate the arterial wall, initiating inflammation. Many cells and cytokines, including macrophages, lymphocytes (T cells and B cells), dendritic cells (DCs), endothelial cells (ECs), vascular smooth muscle cells (VSMCs), and tumor necrosis factor-α (TNF-α), are involved in this process. Therefore, to improve the therapeutic effect of the anti-inflammatory nanoparticle polydopamine, enhance its targeting to the atherosclerotic inflammatory lesion area, increase its circulation time in the body, and avoid its rapid elimination by the body's immune system, polarized M2 macrophage-derived extracellular vesicles could be used to carry polydopamine nanoparticles, inhibit inflammatory factors, promote macrophage polarization in the lesion area, and exert a dual anti-inflammatory effect, which may be key to improving atherosclerosis.

[0029] The applicant also discovered that extracellular vesicles (EVs), natural microvesicles obtained from various cell types, have excellent biocompatibility, low cytotoxicity, and immunological inactivity. Compared to commonly used synthetic carriers, EVs possess an appropriate nanosize and excellent biocompatibility, making them considered natural carriers for delivering drugs and nanomaterials without eliciting harmful pro-inflammatory or immune responses.

[0030] In addition to their unique properties, EVs are rich in specific components, such as proteins, mRNAs, and miRNAs, derived from parent cells. Therefore, they play important roles in various physiological and pathological processes, such as cell proliferation, differentiation, and viral transmission, by transferring the natural biological functions of the original cells. They can be directly used in the treatment of various diseases. The M2 macrophages used in this invention are derived from extracellular vesicles (EVs), which can inherit the abundant anti-inflammatory substances, such as various cytokines and nucleic acids, that M2 macrophages themselves possess while maintaining their natural chemotactic ability to target inflamed areas. However, current methods for extracting, purifying, and producing EVs have certain limitations. EVs, which mimic nanovesicles (NVs), are similar in size and composition to EVs and can be obtained by continuously extruding cells through microfilters. EVs obtained by cell extrusion not only retain the biological factors of stem cells themselves, but also significantly increase the vesicle yield.

[0031] Based on the above findings, the applicant has invented polydopamine-loaded extracellular vesicles for the treatment of atherosclerosis and a method for producing the same, specifically as follows:

[0032] The method for producing polydopamine-loaded extracellular vesicles includes the steps of: polarizing RAW264.7 macrophages into M2 macrophages by treating them with IL-4 and IL-10 cytokines; Polydopamine nanoparticles were co-cultured with M2 macrophages (100 μg / ml polydopamine nanoparticles at least 1 × 10 7 and adding the polydopamine nanoparticles to a culture system containing M2 macrophages at 1000 cells / ml and culturing for 12 hours in a cell culture incubator, followed by obtaining extracellular vesicles derived from the polydopamine nanoparticle-loaded M2 macrophages that target the inflamed area.

[0033] The method for producing melatonin-loaded extracellular vesicles includes the steps of: Obtaining M2 macrophages by treating RAW264.7 macrophages with IL-4 and IL-10 cytokines; co-culturing polydopamine nanoparticles with M2 macrophages; obtaining size-controllable and uniformly sized polydopamine nanoparticle-loaded extracellular vesicles by size exclusion, i.e., sequential extrusion; and said nano-extracellular vesicles are derived from M2 macrophages; After forming the polydopamine-loaded extracellular vesicles, the free polydopamine nanoparticles are washed with PBS and removed by centrifugation.

[0034] In some embodiments of the present invention, the size-controllable nano-extracellular vesicles are obtained by sequential extrusion of a co-culture mixture of polydopamine and polarized M2 macrophages by size exclusion.

[0035] In some embodiments, a method for producing polydopamine nanoparticle-loaded extracellular vesicles includes: RAW264.7 macrophage culture step (1); Step 2: Obtain M2 macrophages by co-treating RAW264.7 macrophages with IL-4 (20 ng / mL) and IL-10 (10 ng / mL) cytokines for 24 hours. 100ug / ml polydopamine nanoparticles at least 1x10 7 The cells were added to the M2 macrophage culture system containing 1 × 10 / ml of polydopamine nanoparticles and cultured in a cell culture incubator for 12 hours. The supernatant was discarded, and the M2 macrophages were washed three times with cold PBS to remove polydopamine nanoparticles that had not been taken up by the macrophages. The cells were then collected with a cell scraper and collected at a concentration of at least 1 × 10 / ml. 7The cells were suspended in PBS at a concentration of 1000 cells / ml. The cell suspension was extruded 5–6 times using a small extruder (Avanti lipid extruder) onto 1 μm, 400 nm, and 200 nm polycarbonate membranes (Whatman Inc, USA). Here, the extruded vesicles are defined as nanovesicles (NVs). The collected NVs were diluted with PBS and stored at -80°C. This step (3) yields polydopamine nanoparticle-loaded M2 macrophage nanovesicles.

[0036] Polydopamine nanoparticle-loaded M2 macrophage nanovesicles, wherein the polydopamine nanoparticle-loaded extracellular vesicles are produced by the above-mentioned production method.

[0037] In the vesicle preparation step of the present invention, nanoparticles are directly cultured with cells, and the suspension is continuously size-excluded. This means that the mixed suspension of raw cells and nanoparticles is continuously extruded through different pores to directly obtain nanoparticle-loaded extracellular vesicles. This continuous extrusion process can be completed on a cell manipulation table. The extrusion device is lightweight and easy to carry, and the resulting exosome yield reaches 1 microgram per microliter, which is up to 100 times the amount obtained by ultracentrifugation. At the same time, size exclusion ensures that the size of the resulting vesicles is primarily concentrated between 100 and 200 nm, allowing for size control and ensuring a consistent volume for nanoparticle loading and maintaining long-term circulation in the body. The loading process is further simplified, eliminating the need for sonication for loading, and polydopamine nanoparticle-loaded extracellular vesicles can be obtained in a single step. In summary, the technical problem addressed by this invention is to provide large-quantity vesicles with controllable size and inflammation-targeting function that can be used for anti-inflammatory treatment of inflamed areas of atherosclerotic plaques.

[0038] In combination with Figure 1, Western blot experiments showed that after treating RAW264.7 cells with IL-4 and IL-10 cytokines, the protein expression of the resulting M2 macrophages showed a significant increase in the expression of Arg1, a protein marker for M2 macrophages, and a significant decrease in the expression of iNOS, a protein marker for M1 macrophages, indicating that M2 macrophages were successfully obtained.

[0039] In combination with Figure 2, observation of polydopamine nanoparticles under a transmission electron microscope revealed that they were spherical, uniform in size, and evenly distributed, further demonstrating that polydopamine nanoparticles are suitable for loading into extracellular vesicles.

[0040] In combination with Figure 3, 100 μg / mL polydopamine nanoparticles were co-cultured with M2 macrophages and then continuously extruded using continuous size exclusion, enabling direct loading of polydopamine nanoparticles into M2 macrophage-derived extracellular vesicles. Size uniformity was ensured by slit extrusion of 1 μm, 400 nm, and 200 nm polycarbonate films, and the manufacturing process was simple. Transmission electron microscopy revealed that the polydopamine nanoparticles were surrounded by a transparent bilayer lipid membrane structure, i.e., extracellular vesicles with a bilayer membrane structure, and the polydopamine nanoparticles were loaded within the vesicles.

[0041] Combined with Figure 4, it was observed through cell experiments that polydopamine nanoparticle-loaded extracellular vesicles have excellent antioxidant properties. Confocal microscopy observations of DCFH-DA, JC-1, and MitoSox experiments clearly conclude that treatment with polydopamine nanoparticle-loaded extracellular vesicles reduces reactive oxygen species in H2O2-treated macrophages, thereby exerting antioxidant function.

[0042] Combined with Figure 5, the observation of in vitro vascular fluorescence imaging showed that when polydopamine nanoparticle-loaded extracellular vesicles, i.e., PDA@M2 NVs, were injected into the tail vein, PDA@M2 NVs could actively accumulate in the inflamed areas of atherosclerotic plaques, far superior to the ability of polydopamine nanoparticles alone, i.e., PDA NPs, to actively target inflammation.

[0043] In combination with Figure 6, after PDA@M2 NVs and PDA NPs were injected into mice via the tail vein, the toxic effects on the internal organs of the mice were observed, and HE staining showed no obvious toxic effects.

[0044] The above description is merely a preferred specific embodiment of the present invention, and the scope of protection of the present invention is not limited thereto. Any modifications or alternatives that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

[0045] The above is merely an example of the present application and is not intended to limit the present application. Those skilled in the art can make various modifications and changes to the present application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application should be included in the scope of the claims of the present application.

[0046] Industrial Practicality The polydopamine nanoparticle-loaded extracellular vesicles and their manufacturing method provided by the present invention can be used as carriers for delivering antioxidant nanoparticles, polydopamine, to inflammatory lesions. Not only do they extend circulation time in the body, but M2 macrophage-derived nanovesicles also have anti-inflammatory properties. By combining them with the delivery of anti-inflammatory particles, two therapeutic effects can be achieved with a single delivery. The manufacturing method of the present invention is simple and effective, allowing for easy acquisition of M2 macrophages. The continuous extrusion physical method is simple to operate and allows for large-scale production. The resulting product can be mass-produced and used in industry.

Claims

1. A method for producing polydopamine nanoparticle-loaded extracellular vesicles, comprising the steps of: treating RAW264.7 macrophages with IL-4 and IL-10 cytokines to polarize them into M2 macrophages; and co-culturing the polydopamine nanoparticles with the M2 macrophages, followed by continuous extrusion by a size-series extrusion method to obtain M2 macrophage-derived polydopamine nanoparticle-loaded extracellular vesicles that target inflamed areas.

2. The M2 macrophages were obtained by treating RAW264.7 macrophages with IL-4 and IL-10 cytokines and were cultured in a cell culture incubator at a concentration of at least 1 × 10 ７ The method for producing polydopamine nanoparticle-loaded extracellular vesicles according to claim 1, characterized in that the cells are co-cultured at 100 μg / mL with 100 μg / mL of polydopamine nanoparticles.

3. 2. The method for preparing polydopamine nanoparticle-loaded extracellular vesicles according to claim 1, wherein the step of loading the polydopamine nanoparticles into the nano-extracellular vesicles comprises the steps of: adding polydopamine nanoparticles to an M2 macrophage culture system; co-culturing the cells; collecting the cells using a cell scraper; extruding the cell suspension through 1 μm, 400 nm, and 200 nm polycarbonate membranes in turn using a microextruder; performing 5 to 6 consecutive extrusions; washing with PBS; and centrifuging the membranes to remove unloaded polydopamine nanoparticles; finally obtaining polydopamine nanoparticle-loaded extracellular vesicles.

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