Application and method of magnetic nanoparticles in preparation of exosome-like body

Abstract

The invention relates to the technical field of biology, in particular to application and a method of magnetic nanoparticles in preparation of exosomes. The method comprises the following steps: 1) co-culturing cells and magnetic nanoparticles, so that the cells endocytose the magnetic nanoparticles into endosomes; (2) removing liquid in a culture system in the step (1), resuspending the cells by using a hypotonic solution, and homogenizing; 3) performing magnetic separation and collecting the inner body wrapped with the magnetic nanoparticles; 4) dispersing the endosome collected in the step 3) so as to release the magnetic nanoparticles wrapped in the endosome, and regrouping the exosome in the endosome into a monodispersed exosome; and 5) performing magnetic separation on the exosome-like body obtained in the step 4), and collecting liquid to obtain the exosome-like body. According to the method, the yield and efficiency of the exosome are greatly improved, pollution is avoided, the biological complexity of the natural extracellular vesicles can be reproduced more truly, and the method can be widely applied to various types of cells.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to the use and method of magnetic nanoparticles in preparing exosome-like vesicles. Background Art

[0002] Extracellular vesicles (EVs) are a general term for various membrane-structured vesicles released by cells. Due to their different diameters and occurrence modes, they are divided into four subgroups: exosomes (diameter 30-150nm), microvesicles (diameter 100-1000nm) and apoptotic bodies (diameter 100-5000nm). Extracellular vesicles, especially exosomes, have attracted much attention in physiological and pathological research, disease diagnosis and treatment. Almost all cells secrete extracellular vesicles, which contain rich bioactive substances. Due to different source cells, extracellular vesicles show high heterogeneity and have multiple components unique to parent cells. Among them, the formation process of exosomes is as follows: cells produce small vesicles through endocytosis, and the small vesicles fuse to form early endosomes, which gradually become late endosomes. With the entry of some "cargoes" such as miRNA, enzyme molecules, and heat shock proteins into the cytoplasm, the late endosomes will produce many small vesicles (ILVs) and gradually evolve into multivesicular bodies (MVBs); subsequently, these small vesicles will be released outside the cell to form exosomes. After being released into the body fluid, exosomes are transported to distant places, where they are taken up by target cells and exert their effects. Therefore, they can be widely used to deliver bioactive molecules and drugs for tissue repair, tumor diagnosis and treatment, and immune regulation. However, the separation and purification of exosomes are inevitably affected by other biological nanoparticles (such as lipoproteins) and nanovesicles (such as extracellular vesicles from non-endosomal pathways) with similar biophysical properties in body fluids. The bottleneck that currently restricts the clinical transformation and commercial development of exosome therapy is the serious lack of technology that can extract exosomes on a large scale and with high reproducibility. Traditional exosome separation techniques (such as ultracentrifugation and size exclusion chromatography) have low yields (10 7 ~10 8 / 1 million cells), long production cycle (24-48 hours), poor product quality repeatability, and the need for expensive and energy-intensive production equipment cannot meet the current clinical demand for large-scale production of exosomes.

[0003] Since exosomes are limited in clinical translation, various types of exosome mimics (EMs) have been developed into extracellular vesicle analogs with similar biological complexity. Exosomes, especially endosome-derived exosomes, as substitutes for natural extracellular vesicles, have received extensive attention in production due to their highly similar structure and function and strong scalability; for example, exosomes derived from human umbilical cord mesenchymal stem cells (hUC MSCs) have the wound healing effect of natural extracellular vesicles and are richer in protein composition. Although whole-cell-derived exosomes have the biological complexity of extracellular vesicles, they are heterogeneous due to contamination by various organelles and nucleic acids. Protein mass spectrometry analysis shows that compared with extracellular vesicle-specific proteins, exosome-specific proteins are more located in mitochondria and endoplasmic reticulum. To date, the most commonly used exosome-like is a cell plasma membrane-derived nanovesicle. The preparation of such nanovesicles is relatively simple, requiring only direct extrusion of the entire parent cell. However, these nanovesicles cannot reproduce natural extracellular vesicles due to the following two shortcomings: first, the biological source of these nanovesicles is the plasma membrane, rather than the endosomal membrane with different lipid and protein compositions like natural extracellular vesicles; second, the entire cell extrusion process will produce a large number of contaminants such as proteins, nucleic acids and lipids in non-EV organelles and cell debris, which inevitably leads to the heterogeneity of exosome-like vesicles. Therefore, it is urgent to develop a method for preparing exosome-like vesicles that can overcome the above shortcomings. Summary of the invention

[0004] In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a use and method of magnetic nanoparticles in preparing exosome-like vesicles, so as to solve the problems in the prior art.

[0005] To achieve the above objectives and other related objectives, the present invention provides the use of magnetic nanoparticles in preparing exosome-like vesicles or in preparing and producing exosome-like products.

[0006] The magnetic nanoparticles are selected from iron oxide magnetic nanoparticles. The iron oxide magnetic nanoparticles are iron oxide magnetic nanoparticles. The iron oxide magnetic nanoparticles are surface-modified iron oxide magnetic nanoparticles. In certain preferred embodiments of the present invention, the iron oxide magnetic nanoparticles are poly-lysine-modified iron oxide magnetic nanoparticles.

[0007] The present invention provides a method for preparing exosome-like vesicles, the method comprising the following steps:

[0008] 1) co-culturing cells with magnetic nanoparticles so that the cells internalize the magnetic nanoparticles into endosomes;

[0009] 2) removing the liquid in the culture system of step 1), resuspending the cells with a hypotonic solution and homogenizing to release the endosomes that have wrapped the magnetic nanoparticles;

[0010] 3) magnetic separation to collect the endosomes that have been wrapped with magnetic nanoparticles;

[0011] 4) dispersing the endosomes collected in step 3) to release the magnetic nanoparticles encapsulated in the endosomes, and causing the exosome-like vesicles in the endosomes to spontaneously reorganize into monodispersed exosome-like vesicles;

[0012] 5) The exosome-like particles obtained in step 4) are subjected to magnetic separation to remove the magnetic nanoparticles, and the liquid is collected to obtain the exosome-like particles.

[0013] In certain embodiments of the present invention, the magnetic nanoparticles in step 1) are selected from iron oxide magnetic nanoparticles. The iron oxide magnetic nanoparticles are iron oxide magnetic nanoparticles. The iron oxide magnetic nanoparticles are surface-modified iron oxide magnetic nanoparticles. In certain preferred embodiments of the present invention, the iron oxide magnetic nanoparticles are poly-lysine-modified iron oxide magnetic nanoparticles.

[0014] In certain embodiments of the present invention, the mixing ratio of cells and magnetic nanoparticles is 10 6 The cells were mixed with 50-200 μg of magnetic nanoparticles.

[0015] In certain embodiments of the present invention, the dispersing conditions in step 4) further include any one or more of the following: a dispersing speed of 100 to 300×g, a dispersing time of 3 to 10 minutes, and dispersing in an ice bath.

[0016] The present invention also provides exosome-like particles obtained by the preparation method.

[0017] As described above, the present invention uses a magnetic separation-assisted high-speed dispersion method to prepare a new type of exosome-like (EMs) with the same biological source and similar structure, morphology and protein composition as natural extracellular vesicles (EVs) on a large scale, which has the following beneficial effects: the endosomes wrapped with magnetic nanoparticles are easily collected by magnetic separation, avoiding the cumbersome steps of the traditional parent cell extrusion method, saving time, and not requiring expensive equipment; the yield and efficiency of EMs have been greatly improved, greatly reducing costs and manpower. No non-EV contamination will be generated, and the exosome-like prepared by these purified endosomes can more realistically reproduce the biological complexity of natural EVs. Under the condition of the same number of cells, the number of exosome-like homologous to natural EVs obtained is an exponential multiple of the natural EVs extracted by ultracentrifugation, and this method can be widely applied to various types of cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The figure shows a schematic diagram of the process of preparing exosome-like particles by the magnetic-assisted high-speed dispersion method of the present invention. The general process is as follows: Step 1: endocytosis of magnetic nanoparticles into endosomes; Step 2: release endosomes encapsulating magnetic nanoparticles after hypotonic treatment and low-speed homogenization. Step 3: magnetic separation to collect endosomes encapsulating magnetic nanoparticles; Step 4: endosomes are dispersed by a high-speed disperser and spontaneously reassembled into monodisperse nanovesicles; Step 5: magnetic separation to remove free IONPs and nanovesicles encapsulating magnetic nanoparticles, and collect nanovesicles without IONPs, which are the desired exosome-like particles.

[0019] Figure 2 The results of characterization of the exosome-like exosomes of the present invention are shown, wherein: (A) The morphology of BMSC cells endocytosing IONPs, endosomes and EMs photographed by transmission electron microscopy. The scale bar in the figure represents 500nm. (B) NTA characterization of the hydrated particle size of BMSC-EM, BMSC-EV, 293T-EM and 293T-EV. (C) Western blot analysis results of EMs, EVs and cell lysates.

[0020] Figure 3 Shown are the yield and total protein concentration of exosome-like vesicles, including: (A) the yield of EMs and EVs prepared from BMSCs. (B) the total protein concentration of EMs and EVs prepared from BMSCs, ***p<0.001.

[0021] Figure 4 Representative fluorescence microscopy images showing the uptake of PKH26-labeled EVs and EMs by BMSCs and ID8.

[0022] Figure 5 Shown is the exploration of EM preparation conditions. Among them: (A) Set the dispersion time to 5min, change the dispersion speed, and use DLS to detect the diameter and PDI of BMSC-EMs. (B) When the dispersion speed is set to 140x g, change the dispersion time, and DLS detect the changes in the diameter and PDI of BMSC-EMs with the dispersion time. (C) DLS detects the diameter and PDI of BMSCs-EMs at different storage times at 4°C after preparation. (D) When the time is set to 5min, change the dispersion speed, and NTA detects the changes in the diameter and concentration of BMSC-EMs with the dispersion speed. (E) When the dispersion speed is 140x g, change the dispersion time, and NTA detects the diameter and concentration of BMSC-EMs. (F) BMSC cells were co-incubated with different concentrations of IONPs, and the diameter and yield of BMSC-EMs were detected by NTA, ****p<0.0001. DETAILED DESCRIPTION

[0023] The inventors of the present application unexpectedly discovered an interesting biological phenomenon in the experiment, that is, magnetic nanoparticles can be effectively internalized by almost all cells through endocytosis, and only accumulate in endosomes, rather than other organelles. Based on this phenomenon, the present invention provides the use of magnetic nanoparticles in preparing exosome-like vesicles or in preparing and producing exosome-like products.

[0024] The term endosome, also known as endosome, refers to a membrane-enclosed organelle in eukaryotic cells. It is a vesicle structure whose function is to transport substances taken up by endocytosis to lysosomes for degradation, or to recycle them back to the cytoplasmic membrane.

[0025] In the present invention, the exosome-like nanovesicles refer to nanovesicles that have the same biological origin, and similar structure, morphology and protein composition as naturally produced exosomes.

[0026] The magnetic nanoparticles are selected from iron oxide magnetic nanoparticles. The iron oxide magnetic nanoparticles are iron oxide magnetic nanoparticles. The iron oxide magnetic nanoparticles are surface-modified iron oxide magnetic nanoparticles. In certain preferred embodiments of the present invention, the iron oxide magnetic nanoparticles are poly-lysine-modified iron oxide magnetic nanoparticles.

[0027] In certain embodiments of the present invention, the iron oxide magnetic nanoparticles further include any one or more of the following features: a particle size ranging from 1 to 1000 nm, and a zeta potential ranging from -50 to 100 mV.

[0028] In certain embodiments of the present invention, the particle size of the iron oxide magnetic nanoparticles (IONPs) can be 1-5nm, 1-10nm, 1-50nm, 1-100nm, 1-200nm, 5-50nm, 5-100nm, 5-200nm, 10-50nm, 10-100nm, 10-200nm, 10-20nm, 20-30nm, 30-40nm, 40-50nm, 50-60nm, 60-70nm, 70-80nm, 80-90nm, 90-100nm, 100-110nm, 110-120nm, 120-130nm, 130-140nm, 140-150nm, 150-200nm, 200-250nm, 250-300nm, 350-400nm, 450-500nm, 550-600nm, 650-700nm, 750-800nm, 850-900nm, 900-950nm, 950-1000nm. Preferably, it is 1 to 200nm. More preferably, it is 1 to 100nm. More preferably, it is 1-30nm. More preferably, it is 10-30nm. More preferably, it is 10-20nm. More preferably, it is 10nm.

[0029] In certain embodiments of the present invention, the zeta-potential range of the iron oxide magnetic nanoparticles (IONPs) may be -50 to -40 mV, -40 to -30 mV, -30 to -20 mV, -20 to -10 mV, -10 to 0 mV, 0 to 10 mV, 10 to 20 mV, 20 to 30 mV, 30 to 40 mV, 40 to 50 mV, 50 to 70 mV, 70 to 90 mV, 90 to 10 mV; preferably, 0 to 30 mV; further preferably, 0 to 20 mV; further preferably, 10 mV.

[0030] The present invention provides a method for preparing exosome-like vesicles, the method comprising the following steps:

[0031] 1) co-culturing cells with magnetic nanoparticles so that the cells internalize the magnetic nanoparticles into endosomes;

[0032] 2) removing the liquid in the culture system of step 1), resuspending the cells with a hypotonic solution and homogenizing to release the endosomes that have wrapped the magnetic nanoparticles;

[0033] 3) magnetic separation to collect the endosomes that have been wrapped with magnetic nanoparticles;

[0034] 4) dispersing the endosomes collected in step 3) to release the magnetic nanoparticles encapsulated in the endosomes, and causing the exosome-like vesicles in the endosomes to spontaneously reorganize into monodispersed exosome-like vesicles;

[0035] 5) The exosome-like particles obtained in step 4) are subjected to magnetic separation to remove the magnetic nanoparticles, and the liquid is collected to obtain the exosome-like particles.

[0036] In step 1), the cells are selected from any cells that can secrete exosomes. In certain embodiments of the present invention, in step 1), the cells are selected from stem cells. The stem cells are, for example, mesenchymal stem cells, hematopoietic stem cells, or neural stem cells. The mesenchymal stem cells are, for example, umbilical cord mesenchymal stem cells or bone marrow mesenchymal stem cells.

[0037] In certain embodiments of the present invention, the magnetic nanoparticles in step 1) are selected from iron oxide magnetic nanoparticles. The iron oxide magnetic nanoparticles are iron oxide magnetic nanoparticles. The iron oxide magnetic nanoparticles are surface-modified iron oxide magnetic nanoparticles. In certain preferred embodiments of the present invention, the iron oxide magnetic nanoparticles are poly-lysine-modified iron oxide magnetic nanoparticles.

[0038] In certain embodiments of the present invention, the iron oxide magnetic nanoparticles have a particle size range of 1-1000 nm, a polydispersity index <0.3, and a zeta potential range of -50 to 100 mV.

[0039] In certain embodiments of the present invention, the mixing ratio of cells and magnetic nanoparticles is 10 6 10 cells are mixed with 50-200 μg of magnetic nanoparticles. 6 The cells are mixed with 50-70 μg, 70-90 μg, 90-110 μg, 110-130 μg, 130-150 μg, 150-170 μg or 170-200 μg of magnetic nanoparticles.

[0040] In certain embodiments of the present invention, in step 1), based on the total volume of the co-culture system, the final concentration of the magnetic nanoparticles is 50-400 μg / mL. For example, the final concentration of the magnetic nanoparticles is 50-70 μg / mL, 70-90 μg / mL, 90-110 μg / mL, 110-130 μg / mL, 130-150 μg / mL, 150-170 μg / mL, 170-200 μg / mL, 200-250 μg / mL, 250-300 μg / mL, 300-350 μg / mL or 350-400 μg / mL.

[0041] In step 1), the co-culture time varies according to the cell type, as long as the cells fully internalize the magnetic nanoparticles into the endosomes. In certain embodiments of the present invention, the co-culture time is 4 to 48 hours. For example, the co-culture time is 4 to 8 hours, 8 to 12 hours, 12 to 16 hours, 16 to 20 hours, 20 to 24 hours, 24 to 32 hours, 32 to 40 hours, and 40 to 48 hours.

[0042] In certain embodiments of the present invention, in step 2), after removing the liquid in the culture system in step 1), the cells are washed and then resuspended in a hypotonic solution.

[0043] In certain embodiments of the present invention, if the cells are adherent cells, they need to be digested after removing the liquid in the culture system in step 1) in step 2); if the cells are suspended cells, they do not need to be digested and can be directly washed and resuspended in a hypotonic solution, which is a routine operation in the art.

[0044] The hypotonic solution refers to a solution whose osmotic pressure and ionic strength are lower than those of normal cell physiological conditions, such as water, hypotonic sodium citrate or sodium chloride, potassium glycerophosphate (0.65M), potassium chloride (0.075M), etc.

[0045] In certain embodiments of the present invention, the hypotonic solution comprises 70-80 mmol / L potassium chloride, 1.0-1.5 mmol / L sodium citrate, and a pH of 7.2-7.4.

[0046] In certain embodiments of the present invention, the hypotonic solution is used in step 2) for 10 to 20 minutes, for example, for 10 to 12 minutes, 12 to 14 minutes, 14 to 16 minutes, 16 to 18 minutes, or 18 to 20 minutes.

[0047] In certain embodiments of the present invention, the homogenization in step 2) is specifically performed by using a homogenizer to impact the mixture 20 to 40 times at a rotation speed of 300 to 1500 rpm.

[0048] In certain embodiments of the present invention, the magnetic separation in step 3) and step 5) may be performed by placing the container containing the endosomes on a magnetic rack for separation.

[0049] In certain embodiments of the present invention, a high-speed disperser is used for dispersion in step 4).

[0050] In certain embodiments of the present invention, the conditions for dispersion in step 4) also include any one or more of the following: a dispersion speed of 100 to 300 × g, a dispersion time of 3 to 10 minutes, and dispersion in an ice bath. The dispersion speed is, for example, 100 to 120 × g, 120 to 140 × g, 140 to 160 × g, 160 to 180 × g, 180 to 200 × g, 200 to 220 × g, 220 to 240 × g, 240 to 260 × g, 260 to 280 × g, 280 to 300 × g. The dispersion time is 3 to 4 minutes, 4 to 5 minutes, 5 to 6 minutes, 6 to 7 minutes, 7 to 8 minutes, 8 to 9 minutes, 9 to 10 minutes. In a preferred embodiment, the dispersion speed is 100 to 180 × g, and the dispersion time is 3 to 7 minutes.

[0051] After the dispersion treatment in step 4), the endosomes are sheared and broken, and the magnetic nanoparticles encapsulated therein are released.

[0052] In certain embodiments of the present invention, the magnetic separation time in step 5) is 0.5 to 2 hours, for example 0.5 to 1 hour, 1 to 1.5 hours or 1.5 to 2 hours.

[0053] In step 5), magnetic separation can remove free magnetic nanoparticles and exosome-like vesicles that have not released or have not completely released magnetic nanoparticles.

[0054] The magnetic separation-assisted high-speed dispersion method of the present invention can mass-produce endosomal-derived nanovesicles as a new type of exosome mimics (EMs) with the same biological source. In this method, parent cells internalize magnetic nanoparticles (MNPs) by endocytosis, and accumulate MNPs in endosomes. Endosomes containing MNPs are then collected by hypotonic treatment and magnetic separation purification. The endosomes encapsulating MNPs are then dispersed into individual nanovesicles using a high-speed disperser. The endosomal-derived vesicles thus produced have the same biological source and similar structure, morphology and protein composition as naturally occurring vesicles. EMs may become potential low-cost and high-yield alternative vesicles for clinical transformation.

[0055] The present invention also provides exosome-like particles obtained by the preparation method.

[0056] The exosome-like vesicles (EMs) prepared by the present invention can also be modified, for example, by selecting specific parent cells (such as stem cells) or genetic modification to integrate biologically active substances (such as proteins and nucleic acids) into EMs.

[0057] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0058] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing the specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0059] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0060] The experimental materials and equipment used in the examples are as follows

[0061]

[0062]

[0063] Example 1 Preparation of exosomes

[0064] The process of preparing exosome-like vesicles by the magnetic separation-assisted high-speed dispersion method of the present invention is as follows Figure 1 As shown. Cells internalize 10nm poly-lysine-modified iron oxide nanoparticles, which specifically aggregate in endosomes through endocytosis. After treatment with hypotonic buffer and low-speed homogenization, IONPs-encapsulated endosomes are released from the cells and can be collected by magnetic separation. The collected endosomes are further decomposed and reorganized into monodisperse nanovesicles by a high-speed disperser, which are called exosome-like nanovesicles. Through a second round of magnetic separation, free IONPs and IONPs-loaded exosomes are removed from the solution to obtain IONP-free exosome-like nanovesicles. This method prepares highly uniform and monodisperse nanovesicles from parent cell endosomes, which have the same biological source as natural vesicles. The specific experimental steps are as follows ( Figure 1 ):

[0065] 1. Cellular internalization of magnetic nanoparticles

[0066] 1) Inoculation of cells: 10 6 Rat bone marrow mesenchymal stem cells (BMSC) or 293T cells were resuspended in DMEM complete medium supplemented with 10% serum and 5% double antibody in a six-well plate, with 2 mL of complete medium per six-well plate. The plate was placed in a 5% CO2, 37°C incubator for 24 hours to allow the cells to adhere to the wall. The number of cells was about 10 6 indivual.

[0067] 2) Cell endocytosis of nanoparticles: The volume of culture medium in each six-well plate was reduced to 1 mL, and 10 nm poly-lysine-modified iron oxide magnetic nanoparticles (IONP) were added at 100 μg / 10 6 The cells were co-cultured with the cells at a concentration of 10 cells / mL and placed in a 5% CO2, 37°C incubator overnight to allow the cells to fully internalize the nanoparticles.

[0068] 2. Collection and dispersion of endosomes

[0069] 1) Cell hypotonic treatment: 500 μL of trypsin was added to each well of BMSC cells that had fully internalized IONPs for digestion for 2 min, and 1 mL of complete culture medium was added to terminate the digestion. The cells were centrifuged at 1000 x g for 5 min to the bottom of the tube, the supernatant was aspirated, PBS was added to resuspend and centrifuged again to wash away the residual culture medium, and finally a pre-prepared hypotonic solution (potassium chloride 71.4 mM / L, sodium citrate 1.3 mM / L, pH 7.2-7.4) was used at a concentration of no more than 8 × 10 5 Resuspend at a concentration of cells / mL and treat the cells with hypotonicity

[0070] 15min.

[0071] 2) Low-speed homogenization to release organelles: Transfer the hypotonic solution containing cells to the glass test tube equipped with the glass homogenizer.

[0072] The organelles were released by impacting the cell at 1000 rpm for 30 times.

[0073] 3) Magnetic separation to collect endosomes: Transfer the broken cell solution to a 1.5 mL microcentrifuge tube (EP tube) and

[0074] The tube was inserted into the hole of the magnetic rack and magnetic sorting was performed for one hour to fully separate the IONPs-encapsulated endosomes from other organelles (such as nuclei and mitochondria) and cell fragments. It can be seen that brown substances aggregated on the surface of the EP tube and the magnetic rack, which is the encapsulation.

[0075] For IONPs endosomes, remove the liquid in the EP tube and add PBS to resuspend the endosomes.

[0076] 4) High-speed dispersion: Transfer the solution containing the endosomes into a centrifuge tube with a liquid volume between 3 and 20 mL. The endonuclease probe should extend to the bottom of the centrifuge tube without touching the bottom. Set the speed to 140 x g and the time to 5 min. Disperse in an ice bath.

[0077] 5) Magnetic separation of vesicles without IONPs: The liquid containing nanovesicles obtained by high-speed dispersion is divided into EP tubes and placed on a magnetic rack for separation for 1 hour. Light brown substances, i.e. IONPs-containing vesicles, can be seen adhering to the wall of the EP tube. The liquid from which IONPs have been removed is collected, i.e. the desired exosome-like vesicles.

[0078] Steps for preparing extracellular vesicles by ultracentrifugation:

[0079] When the cell confluence reaches about 80%, replace the medium containing 10% exosome-free FBS, incubate for 24-48h, and collect the cell culture supernatant. Centrifuge at 300x g for 10 minutes at 4°C, collect the supernatant, and discard the cell debris. Centrifuge at 2000x g for 20 minutes at 4°C, collect the supernatant, and discard the larger vesicles (apoptotic bodies and some larger microvesicles). Centrifuge the supernatant again at 10000x g for 30 minutes at 4°C, collect the supernatant, and discard the larger vesicles. Ultracentrifuge at 120,000xg for 70 minutes at 4°C, discard the supernatant, leave less than 5ml of liquid, and add PBS to resuspend the attached exosomes. Centrifuge the resuspended exosomes at 120000xg for 70min, and resuspend with 200μl PBS to obtain extracellular vesicles.

[0080] The protein concentration was detected according to the instructions of the BCA protein detection kit. The results showed that the total protein concentration of BMSC-EMs and BMSC-EVs was similar, which was 11.15 μg / 10 9 particles and 14.71μg / 10 9 The total protein concentrations of 293T-EMs and 293T-EVs were approximately 31.8 μg / 10 9 particles and 9.95μg / 10 9 particles( Figure 3 B).

[0081] Example 2 Characterization of exosomes

[0082] 1. DLS analysis

[0083] 1) Inject 1 mL of sample into the sample pool along the wall and place it into the instrument sample slot.

[0084] 2) Open the Zetasize Nano software, select particle size test, and start the test.

[0085] 2.NTA Analysis

[0086] 1) Dilute the freshly prepared exosome-like sample with sterile 1x PBS to contain 10 7 -10 8 1 mL of liquid containing 100 μL / mL of particles was injected into the sample cell using a 1 mL syringe at a pushing speed of 500-1000 μL / min.

[0087] 2) Ensure that the exosome particles in the software interface are within 100-300, visualize the particles using laser scattering, and capture their Brownian motion in a digital video.

[0088] 3) Select the corresponding SOP (EV-488) to ensure that the sensitivity of the instrument is suitable for exosome detection.

[0089] 3. Transmission Electron Microscopy

[0090] 1) 10 μL of 5% glutaraldehyde was added to 10 μL of exosomes (1 μg / 1 μL) and fixed for 30 min.

[0091] 2) Take 10 μL of EMs and drop it on the carbon surface of the copper mesh. Let it adsorb for 10 minutes. Use filter paper to absorb the excess liquid from the side. Add 10 μL of PBS and then absorb it with filter paper. Repeat twice to wash away the glutaraldehyde.

[0092] 3) Add 5 μL of uranyl acetate, stain for 1 min, wash three times with deionized water, and dry at room temperature.

[0093] 4) The exosome samples were imaged using a transmission electron microscope at an accelerating voltage of 100 kV.

[0094] The results are as follows Figure 2 and 3 As shown in Figure 2, the diameter and morphology of EMs were analyzed by NTA and TEM. The morphology of BMSC-EMs has typical bowl-shaped vesicle-like structural characteristics and is separated by lipid bilayers ( Figure 2 A). Based on NTA analysis, the hydrated particle size of BMSC-EMs and 293T-EMs was similar to that of native EVs (BMSC-EVs and 293T-EVs) collected by ultracentrifugation ( Figure 2 B).

[0095] According to the number of nanovesicles detected by NTA, the yield of BMSC-EMs obtained by high-speed dispersion method was 1.42×10 9 / 10 6 cells, and the yield of natural BMSC-EVs prepared by ultracentrifugation was only 7.2×107 / 10 6 Similarly, the yield of 293T-EMs obtained by high-speed dispersion was 7.58×10 8 / 10 6 cells, which is much higher than the yield of natural 293T-EVs prepared by traditional ultracentrifugation (≈5.5×10 6 / 10 6 cells) is about 100 times higher ( Figure 3 A).

[0096] 4. Western Blot

[0097] 1) Add 100 μL of cell lysis buffer and 5 μL of 50x protease inhibitor cocktail to the exosome sample. Mix by gently pipetting and lyse on ice for 30 minutes.

[0098] 2) Centrifuge the mixture at 1000 x g for 3 minutes at 4°C and collect the supernatant. Measure the protein concentration using a protein assay kit. Add 25 μL of 5x protein loading buffer and heat in a 100°C metal bath for 10 minutes.

[0099] 3) Prepare gel according to the reagent instructions, add 15 μL protein to each well, the concentration is 10 mg / mL, set the gel electrophoresis at 80V, change to 100V electrophoresis when the sample runs to the separation gel, and then transfer to the membrane (100V, 4°C, 60min). Incubate the primary antibody overnight at 4°C, wash the membrane 3 times with TBST, 10min each time, incubate the secondary antibody at room temperature for 1h, wash the membrane with TBST for 10min x 3 times, prepare the luminescent solution for development, and detect non-exosome specific markers (calnexin) and exosome biomarkers (CD63 and Annexin).

[0100] The results are as follows Figure 2 As shown in C, BMSC-EMs contain the same protein markers as EVs (CD63 and Annexin). Both EMs and EVs were negative for Calnexin expression, indicating that EMs generated by this method have little cytoplasmic membrane contamination.

[0101] Example 3 Cell endocytosis experiment

[0102] 1. Exosome Fluorescence Labeling

[0103] 1) 1 μL PKH26 was mixed with 250 μL diluent C (1:250) to prepare 2x staining solution (4x 10 -6 M).

[0104] 2) Mix 250 μL of exosome-like vesicles with 250 μL of 2x staining solution and mix quickly and evenly using a pipette.

[0105] 3) Incubate at room temperature for 2-5 minutes, gently inverting the tube during the process.

[0106] 4) Add an equal volume of serum or 1% bovine serum albumin and incubate for 1 min to terminate the digestion.

[0107] 5) Remove excess dye by ultrafiltration: centrifuge at 3000 x g for 30 min in a 1000KD ultrafiltration tube, add PBS and repeat centrifugation twice. Filter the remaining liquid with a 0.22 μm filter and resuspend it in PBS to 500 μL.

[0108] 2. Seeding Cells

[0109] Take 10 6 The cells were seeded into confocal dishes at a concentration of 1:1 / mL and cultured in an incubator overnight.

[0110] 3. Endocytosis

[0111] 5×10 fluorescently labeled 8 Each type of exosome was added to the confocal dish and co-cultured with cells for 8 hours.

[0112] 4. Fixing and staining nuclei

[0113] Remove the culture medium, wash twice with PBS, add 1 mL of paraformaldehyde and fix at room temperature for 10 min, remove the paraformaldehyde

[0114] Wash twice with PBS, add 1 ml of DAPI, incubate at 37°C for 10 min, and wash twice with PBS.

[0115] 5. Single-photon confocal imaging

[0116] Select DAPI and TRIT fluorescence channels and scan and take pictures of the samples.

[0117] The results are as follows Figure 4 As shown, in this example, pkh26-labeled EMs and EVs were co-incubated with BMSCs and mouse ovarian epithelial cancer cells (ID8) at a concentration of 10 mg / mL for 8 h, and the cells were observed under a confocal fluorescence microscope, showing that both EMs and EVs could be taken up by cells and exert their effects.

[0118] Example 5 Exploration of different preparation parameters

[0119] 1) The method is the same as in Example 1, except that the concentration of IONPs was changed. BMSC cells were co-incubated with IONPs of different concentrations, and the particle size and yield of BMSC-EMs were analyzed by NTA. The results are as follows: Figure 5As shown in F, the results show that when every 10^6 cells were co-cultured with 100ugIONPs, the cell uptake of IONPs reached saturation, and the EM production was the highest at this time. Therefore, a co-culture concentration of 100ug / 10^6cells was selected for subsequent experiments.

[0120] 2) Investigate the effect of different dispersion speeds under fixed dispersion time. The other steps are the same as Example 1: adjust the time button at the bottom of the high-speed disperser screen to set the time to 5 minutes, adjust the speed button to set the speed to 50 / 140 / 270 / 555×g, and then press the OK button. After placing an ice box under the sample (the collected solution containing endosomes), press the start button for high-speed dispersion. The sample was then tested using NTA / DLS. The results are shown in Figure 5 As shown in A and 5D, the results show that when the dispersion time is 5 minutes, there is no significant difference in the particle size of the vesicles produced at different rotation speeds. There is a certain trend change in the yield, reaching a peak at 270×g; when the particle size and PDI are measured by DLS, when the dispersion time is 5 minutes, the particle size of the vesicles under the conditions of 50×g and 140×g is below 150nm, and the particle size of 140×g is the smallest, which is in line with the natural exosome particle size range. As the rotation speed increases, the PDI decreases, that is, the size uniformity is better. Therefore, a dispersion speed of 140×g was selected for subsequent experiments.

[0121] 3) Investigate the effect of different dispersion times at a fixed dispersion speed. The other steps are the same as in Example 1: adjust the speed button at the bottom of the high-speed disperser screen to set the speed to 140×g, adjust the time button to set the time to 1 / 3 / 4 / 5 / 7 / 10 minutes, and then press the OK button. Place an ice box under the sample (the collected solution containing endosomes), and press the start button for high-speed dispersion. The sample was then tested using NTA / DLS. The results are shown in Figure 1. Figure 5 As shown in Figures B and 5E, the results show that when the speed is 5000 rpm, there is no significant difference in the particle size of the vesicles produced at different dispersion times, and there is no significant difference in the yield after the dispersion time exceeds 4 minutes; when the speed is constant, there is no significant difference in the particle size of the vesicles produced at dispersion times of 5 minutes and 7 minutes, and within the range of natural exosome particle size, PDI decreases with the increase of dispersion time. Therefore, a dispersion time of 5 minutes was selected for subsequent experiments.

[0122] 4) The BMSC-EMs prepared in Example 1 were stored at 4°C for 120 hours, and the diameter and PDI of BMSCs-EMs at different storage times were detected by DLS. The results showed that under the low temperature storage condition of 4°C, the particle size of EMs prepared by this method was stable within 5 days.

[0123] The results of the above examples indicate that the method of the present invention can be used to prepare endosome-derived EMs on a large scale and reproducibly, and the prepared EMs have a protein composition similar to that of natural EVs and the same biological source.

[0124] The above examples are for the purpose of illustrating the embodiments disclosed by the present invention and are not to be construed as limiting the present invention. In addition, the various modifications listed herein and the variations of the methods in the invention are obvious to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of the present invention.

Claims

1. Use of magnetic nanoparticles in the preparation of exosome-like vesicles or in the preparation of exosome-like products.

2. The use according to claim 1, characterized in that The magnetic nanoparticles are selected from iron oxide magnetic nanoparticles; preferably, the iron oxide magnetic nanoparticles are iron oxide magnetic nanoparticles; more preferably, the iron oxide magnetic nanoparticles are surface-modified iron oxide magnetic nanoparticles; more preferably, the iron oxide magnetic nanoparticles are poly-lysine-modified iron oxide magnetic nanoparticles.

3. The use according to claim 2, characterized in that: The iron oxide magnetic nanoparticles have a particle size range of 1-1000 nm, and / or a zeta potential range of -50 to 100 mV.

4. A method for preparing exosome-like vesicles, characterized in that: The method comprises the following steps: 1) co-culturing cells with magnetic nanoparticles so that the cells internalize the magnetic nanoparticles into endosomes; 2) removing the liquid in the culture system of step 1), resuspending the cells with a hypotonic solution and homogenizing to release the endosomes that have wrapped the magnetic nanoparticles; 3) magnetic separation to collect the endosomes that have been wrapped with magnetic nanoparticles; 4) dispersing the endosomes collected in step 3) to release the magnetic nanoparticles encapsulated in the endosomes, and causing the exosome-like vesicles in the endosomes to spontaneously reorganize into monodispersed exosome-like vesicles; 5) The exosome-like particles obtained in step 4) are subjected to magnetic separation to remove the magnetic nanoparticles, and the liquid is collected to obtain the exosome-like particles.

5. The method according to claim 4, characterized in that In step 1), the cells are selected from cells that can secrete exosomes; preferably, the cells in step 1) are selected from stem cells; more preferably, the stem cells are mesenchymal stem cells, hematopoietic stem cells or neural stem cells; more preferably, the mesenchymal stem cells are umbilical cord mesenchymal stem cells or bone marrow mesenchymal stem cells.

6. The method according to claim 4, characterized in that In step 1), the magnetic nanoparticles are selected from iron oxide magnetic nanoparticles; more preferably, the iron oxide magnetic nanoparticles are iron oxide magnetic nanoparticles; more preferably, the iron oxide magnetic nanoparticles are surface-modified iron oxide magnetic nanoparticles; more preferably, the iron oxide magnetic nanoparticles are poly-lysine-modified iron oxide magnetic nanoparticles; more preferably, the iron oxide magnetic nanoparticles have a particle size range of 1-1000 nm.

7. The method according to claim 4, characterized in that In step 1), any one or more of the following features are also included: 1a) The mixing ratio of the cells and magnetic nanoparticles is 10 6 Each cell was mixed with 50-200 μg of magnetic nanoparticles; 1b) based on the total volume of the co-culture system, the final concentration of the magnetic nanoparticles is 50-400 μg / mL; 1c) The co-cultivation time is 5 to 24 hours.

8. The method according to claim 4, characterized in that In step 2), any one or more of the following features are also included: 2a) after removing the liquid in the culture system in step 1), washing and then resuspending with a hypotonic solution; 2b) The hypotonic solution comprises 70-80 mmol / L potassium chloride, 1.0-1.5 mmol / L sodium citrate, and a pH of 7.2-7.4; 2c) treating with a hypotonic solution in step 2) for 10 to 20 minutes; 2d) The homogenization is performed by using a homogenizer at a rotation speed of 300 to 1500 rpm for 20 to 40 times.

9. The method according to claim 4, characterized in that The dispersion conditions in step 4) may further include any one or more of the following: a dispersion speed of 100 to 300×g, a dispersion time of 3 to 10 minutes, and dispersion in an ice bath.

10. The method according to claim 4, characterized in that The magnetic separation time in step 5) is 0.5 to 2 hours.

11. The exosome-like substance obtained by the preparation method according to any one of claims 4 to 10.

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