Method for preparing exosome by simulating low-density lipoprotein cholesterol standard-exceeding environment

Abstract

The invention discloses a method for preparing exosomes by simulating a low-density lipoprotein cholesterol standard-exceeding environment, and relates to the technical field of exosomes, and the method comprises the following steps: inoculating umbilical cord mesenchymal stem cells into a culture plate, after the cell fusion degree reaches 85%, adding chloroquine-liposome, and culturing for 24-48 hours; collecting cell supernate, and extracting exosomes by adopting an ultracentrifugation method; the chloroquine lipidosome is obtained by fusing lipidosome and chloroquine and then performing freeze thawing treatment, the lipidosome is prepared from phospholipid, low-density lipoprotein, low-density protein cholesterol and mesoporous silica nanoparticles through a film dispersion method, and vitamin E is further loaded in the mesoporous silica nanoparticles; the problem of liposome burst release caused by ph change can be solved, a more stable chloroquine release process is realized, and the problem that the exosome yield and the treatment effect cannot be considered at the same time is solved.

Description

Technical Field

[0001] The present invention relates to the field of exosome preparation, and particularly to a method for preparing exosomes by simulating an environment with elevated low-density lipoprotein cholesterol. Background Art

[0002] Atherosclerosis (AS) is the main pathological basis of cardiovascular diseases, and its occurrence and development are closely related to the elevation of low-density lipoprotein cholesterol (LDL-C) levels. In recent years, exosomes derived from mesenchymal stem cells (MSCs) have shown great potential in the treatment of AS due to their immunomodulatory, angiogenesis-promoting, apoptosis-inhibiting and other effects. However, the therapeutic effect of MSCs exosomes obtained under conventional culture conditions on AS is limited.

[0003] Studies have shown that elevated LDL-C levels can induce functional disorders in vascular endothelial cells, smooth muscle cells, etc., and promote the occurrence and development of AS. Therefore, culturing MSCs by simulating an environment with elevated LDL-C may induce the secretion of exosomes with stronger anti-AS activity.

[0004] For example, in Chinese Patent with application number 202311058158.X, there is a lipidosome loaded with drugs, its preparation method and a method for increasing the exosome secretion amount. The lipidosome is composed of chloroquine, phospholipids and cholesterol; wherein, the mass ratio of chloroquine to phospholipids is 1:20 - 120, and the mass ratio of phospholipids to cholesterol is 2.5 - 7.5:1. It can not only reduce the drug dosage but also effectively increase the exosome secretion amount of cells, achieving both reducing cell toxicity and increasing the exosome secretion amount of cells, and can reduce the drug dosage and production cost.

[0005] However, although the exosomes produced using lipidosomes also use cholesterol, they do not have the pathogenic factor effect of low-density lipoprotein cholesterol, making it difficult for the exosomes to have specific therapeutic effects. And in the lysosomal environment with a lower pH, it is easy to increase the membrane permeability of the lipidosome, resulting in a large amount of chloroquine release, significant changes in the intracellular environment, and still a relatively high cell mortality rate. Summary of the Invention

[0006] By providing a method for preparing exosomes by simulating an environment with elevated low-density lipoprotein cholesterol in the embodiments of the present application, the problem of sudden release of lipidosomes caused by pH changes in the prior art is solved, and a more stable chloroquine release process is achieved.

[0007] An embodiment of the present application provides a method for preparing exosomes by simulating an environment with elevated low-density lipoprotein cholesterol. The steps include: inoculating umbilical cord mesenchymal stem cells in a culture plate, and after the cell confluence reaches 85%, adding chloroquine-liposomes and culturing for 24-48 hours; collecting the cell supernatant and extracting exosomes by ultracentrifugation; the chloroquine-liposomes are obtained by fusing liposomes with chloroquine and then performing freeze-thaw treatment. The liposomes are prepared by the thin-film dispersion method from phospholipids, low-density lipoprotein, low-density lipoprotein cholesterol, and mesoporous silica nanoparticles, and vitamin E is loaded in the mesoporous silica nanoparticles.

[0008] Further, the chloroquine-liposomes account for 1-20% of the total weight of the basal medium in the entire culture system.

[0009] Further, the mass ratio of low-density lipoprotein cholesterol to phospholipids is 1:2, and the content of oxidized low-density lipoprotein in the low-density lipoprotein is 20%.

[0010] Further, the mass ratio of low-density lipoprotein to low-density lipoprotein cholesterol is 1:10.

[0011] Further, the mass ratio of mesoporous silica nanoparticles to low-density lipoprotein cholesterol is (2-8):4.

[0012] Further, the mass ratio of chloroquine to liposomes is 1:250.

[0013] Further, the mass ratio of vitamin E to mesoporous silica is 1:(1-4); the specific method for loading vitamin E is: dissolving vitamin E and mesoporous silica in ethanol together, stirring overnight, and then removing the solvent by reduced pressure evaporation to obtain mesoporous silica loaded with vitamin E.

[0014] Further, the specific preparation method of the chloroquine-liposomes is as follows:

[0015] S1: Disperse low-density lipoprotein cholesterol, mesoporous silica nanoparticles, phospholipids, and low-density lipoprotein in absolute ethanol, and after mixing evenly; then rotate and evaporate ethanol under reduced pressure at 30-40 °C until a film is formed, that is, a lipid film is obtained;

[0016] S2: Add PBS buffer solution with pH 7.0 to the lipid film, and perform rotary hydration at 50-60 °C for 40-50 min to obtain a crude liposome product;

[0017] S3: Perform intermittent ultrasonic treatment on the crude liposome product for 3 min with ultrasonic waves of 50 kHz for 20 s each time and a pause of 20 s each time. After ultrasonic treatment, place it at 50-60 °C for 1-2 h to obtain a liposome suspension;

[0018] S4: Add chloroquine to the liposome suspension for fusion. After rapid freezing at -40°C for 50 - 60 min, slowly thaw it at room temperature to obtain chloroquine-liposomes.

[0019] Furthermore, there are multiple combinations of chloroquine-liposomes, and one or more of them are used in combination when in use.

[0020] Furthermore, after the cell confluence reaches 85%, the chloroquine-liposomes are added in batches. When the cell confluence reaches 85%, add them for the first time, and then add them for the second time after continuing to culture for 12 h.

[0021] One or more technical solutions provided in the embodiments of the present application have at least the following technical effects or advantages:

[0022] The exosomes prepared by using LDL and LDL-c to prepare liposomes can treat atherosclerosis. By adding mesoporous silica, the release rate of chloroquine is controlled, the release conditions are specialized, and the time is extended, solving the problem of cell damage caused by the burst release and extracellular release of chloroquine.

[0023] By adding vitamin E, the oxidation degree and position of LDL and cells can be controlled, thereby optimizing the yield and therapeutic effect of exosomes and also regulating the release rate of chloroquine, solving the problem that the release of chloroquine is difficult to control and the therapeutic effect and yield of exosomes cannot be taken into account at the same time.

[0024] The combined use of chloroquine-liposomes can further increase the yield and therapeutic effect of exosomes on the basis of reducing cell mortality. And due to the decrease in cell mortality, the purity of exosomes increases, solving the problems of low exosome purity and poor therapeutic effect.

[0025] Using the staged addition method to achieve precise regulation of "stimulate first and then repair", breaking through the contradiction between "yield - survival rate". Specific Embodiments

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this invention belongs; the terms used in the specification of this invention herein are only for the purpose of describing specific embodiments, and are not intended to limit the present invention; the term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0027] Example 1: A method for preparing exosomes by simulating an environment with excessive low-density lipoprotein cholesterol, the steps include:

[0028] Umbilical cord mesenchymal stem cells were seeded in a culture plate. When the cell confluence reached 85%, chloroquine-liposomes were added and the cells were cultured for another 24 - 48 hours. The cell supernatant was collected and exosomes were extracted by ultracentrifugation;

[0029] The chloroquine-liposomes accounted for 1 - 20% of the total weight of the basal medium in the entire culture system. The basal medium is a commonly used basal medium for cell culture after seeding. In this application, Thermo Fisher's M9 basal medium was selected;

[0030] The liposomes were prepared by the thin film dispersion method from phospholipids, low-density lipoprotein (LDL), low-density lipoprotein cholesterol (LDL-c), and mesoporous silica nanoparticles (MSN). Then the liposomes were fused with chloroquine and subjected to freeze-thaw treatment to obtain chloroquine-liposomes. The content of oxidized LDL in the low-density lipoprotein was 20%.

[0031] The mass ratio of low-density lipoprotein cholesterol to phospholipids was 1:2; the mass ratio of low-density lipoprotein to low-density lipoprotein cholesterol was 1:10; the mass ratio of mesoporous silica nanoparticles to low-density lipoprotein cholesterol was (2 - 8):4;

[0032] The mass ratio of chloroquine to liposomes was 1:250.

[0033] The specific preparation method of the chloroquine-liposomes was as follows:

[0034] S1: Low-density lipoprotein cholesterol, mesoporous silica nanoparticles, phospholipids, and low-density lipoprotein were dispersed in absolute ethanol. After mixing evenly, ethanol was removed by rotary evaporation under reduced pressure at 30 - 40 °C until a film was formed, i.e., a lipid film was obtained;

[0035] S2: PBS buffer solution with pH 7.0 was added to the lipid film and rotary hydrated at 50 - 60 °C for 40 - 50 min to obtain the initial liposome product;

[0036] S3: The initial liposome product was subjected to intermittent ultrasonic treatment with 20 s of ultrasonic treatment followed by 20 s of rest for 3 min. The ultrasonic frequency was 50 kHz. After ultrasonic treatment, it was placed at 50 - 60 °C for 1 - 2 h to obtain a liposome suspension;

[0037] S4: Chloroquine was added to the liposome suspension for fusion. It was rapidly frozen at -40 °C for 50 - 60 min and then slowly thawed at room temperature to obtain chloroquine-liposomes;

[0038] The technical solutions in the embodiments of the present application at least have the following technical effects or advantages:

[0039] In Example 1, exosomes prepared by using LDL and LDL-c to prepare liposomes can treat atherosclerosis. By adding mesoporous silica, the release rate of chloroquine is controlled, the release conditions are specialized, and the time is extended, solving the problem of sudden release of chloroquine and extracellular release leading to cell damage.

[0040] Phospholipid molecules are amphiphilic and spontaneously form a bilayer membrane structure through the thin film dispersion method, constituting the basic framework of liposomes and enabling liposomes to fuse with cell membranes to achieve the delivery of chloroquine.

[0041] Low-density lipoprotein (LDL) and low-density lipoprotein cholesterol (LDL-c) specifically bind to LDL receptors on the cell membrane through their surface apolipoproteins, can simulate a high-cholesterol environment, and induce intracellular cholesterol accumulation, making the secreted exosomes have the potential to treat atherosclerosis; LDL-c binds to LDL receptors on the cell surface (such as highly expressed in hepatocytes and tumor cells), realizes receptor-mediated endocytosis, improves the targeting and biocompatibility of liposomes, and the low rigidity of LDL-c combined with the strongly rigid mesoporous silica can prevent the leakage of chloroquine.

[0042] Chloroquine (CQ) is a weakly basic drug that accumulates after protonation in the acidic environment of lysosomes, inhibits the activity of lysosomal enzymes, thereby preventing the degradation of exosomes by lysosomes and increasing the fusion rate of exosomes with the plasma membrane.

[0043] The mesopore diameter of mesoporous silica nanoparticles (MSN) (2 - 8 nm) matches the size of chloroquine molecules (about 1 nm). The drug molecules need to overcome the pore diffusion resistance to be released, so that in a non-lysosomal environment, chloroquine needs to overcome the pore diffusion resistance to be released, reducing sudden release and further reducing the cytotoxicity caused by sudden release; the Si-OH on the surface of MSN forms hydrogen bonds with the amino groups of chloroquine, delaying the desorption of the drug; MSN is embedded in the liposome bilayer membrane to form a double barrier (lipid membrane + silicon pore), further extending the release path; MSN partially dissolves in the acidic environment (pH 4.5 - 5.5) of lysosomes, enabling part of the chloroquine to be rapidly released; and because MSN is electrostatically adsorbed and anchored on the surface of liposomes to form "liposome-MSN hybrid particles", the liposomes simultaneously have the cell membrane affinity of liposomes and the drug controlled-release ability of MSN. The rigid structure of MSN reduces the fluidity of the liposome membrane and reduces the risk of drug leakage.

[0044] It can maintain the effective concentration of chloroquine in cells for a longer time, more persistently inhibit lysosomal function; reduce the cytotoxicity of high-concentration chloroquine, improve the exosome yield, combine the cholesterol overload of LDL / LDL-c and the slow release stress of chloroquine, and more realistically simulate the atherosclerotic plaque microenvironment; the content of inflammatory factors (such as IL-6, TNF-α) and oxidative stress markers (MDA) in exosomes is significantly increased, which is highly correlated with the pathological state.

[0045] To verify the effect of LDL / LDL-c on exosomes, experiments were conducted. UCMSCs were isolated from Wharton's jelly of umbilical cords by enzymatic digestion method and cultured in α-MEM medium containing 10% fetal bovine serum. The medium was changed every two days. Passage was carried out when the cell confluence reached 90%. The third-generation umbilical cord mesenchymal stem cells were seeded in 6-well plates at a density of 1×10 5 cells / well. When the cell confluence reached 85%, a medium containing liposomes (the liposomes in this experiment did not contain mesoporous silica and were not loaded with chloroquine) was added, and the cells were cultured for another 48 hours. The cell supernatant was collected, and exosomes were extracted by ultracentrifugation (first centrifuged at 300g for 10 min, then at 2000g for 20 min, and finally at 10000g for two times of 30 min and then filtered through a 0.22 μm filter membrane). The secretion amount of UCMSC exosomes was detected by nanoparticle tracking analyzer. The changes in the expression levels of TSG101, CD63, and Alix proteins were detected by Western blot method, and the values were calculated by normalization method. The protective effect of 50 μg / mL UCMSC exosomes on vascular endothelial cell injury after 24 hours was detected by CCK-8 method. The effect of 50 μg / mL UCMSC exosomes on ox-LDL-induced macrophage polarization was detected by flow cytometry. The results are shown in Table 1;

[0046] Table 1

[0047]

[0048] To verify the effect of different amounts of MSN on the release of chloroquine, a release test was conducted on chloroquine-liposomes. The release rate of chloroquine was tested in different media for 24 hours. The media were physiological saline with two pH values, pH 7.2 and pH 5.0. The results are shown in Table 2;

[0049] Table 2

[0050]

[0051] To verify the effect of different amounts of MSN on exosomes, the exosome concentration was tested on the basis of adding 10% of chloroquine-liposomes. The protective effect of 50 μg / mL UCMSC exosomes on vascular endothelial cell injury after 24 hours was detected by CCK-8 method. The results are shown in Table 3;

[0052] Table 3

[0053]

[0054] Example 2: The exosomes prepared by using LDL and LDL-c to prepare liposomes in the above example can treat atherosclerosis. By adding mesoporous silica, the release rate of chloroquine is controlled, the release conditions are specialized, and the time is extended. On the basis of Example 1, it is further improved to increase the stability and pH responsiveness of liposomes.

[0055] Vitamin E is also added to the mesoporous silica particles, and the mesoporous silica nanoparticles and vitamin E are combined by the solvent evaporation method. Specifically, vitamin E and mesoporous silica are dissolved in ethanol together, stirred overnight, and then the solvent is removed by reduced pressure evaporation to obtain mesoporous silica loaded with vitamin E; the mass ratio of vitamin E to mesoporous silica is 1:(1-4).

[0056] The technical solutions in the embodiments of the present application at least have the following technical effects or advantages:

[0057] By adding vitamin E, the oxidation degree and position of LDL and cells can be controlled, thereby optimizing the yield and therapeutic effect of exosomes and also regulating the release rate of chloroquine, solving the problem that the release of chloroquine is difficult to control and the therapeutic effect and yield of exosomes cannot be taken into account at the same time.

[0058] Vitamin E is a fat-soluble antioxidant. Its long-chain alkyl structure (such as the hydrophobic tail of tocopherol) can bind to chloroquine (weakly basic, with a hydrophobic aromatic ring) through hydrophobic interaction to form a "vitamin E-chloroquine complex". And vitamin E, as a "molecular glue", adsorbs on the surface of MSN, anchors chloroquine in the pores or on the surface through hydrophobic interaction, reduces drug leakage, and makes chloroquine not only encapsulated inside the chloroquine-liposome but also distributed on the surface of the chloroquine-liposome along with vitamin E and mesoporous silica, optimizing the release mechanism of chloroquine; and vitamin E can also be embedded in the lipid bilayer to increase membrane stability and prevent the shedding of mesoporous silica;

[0059] The antioxidant effect of vitamin E can reduce cellular oxidative stress and lower LDL oxidation, decreasing cell mortality. However, LDL's ability to simulate the atherosclerotic environment is far inferior to that of oxidized low-density lipoprotein (OX-LDL). When chloroquine-liposomes are in the extracellular environment, vitamin E adsorbs chloroquine, providing antioxidant capacity. When chloroquine-liposomes are in the lysosomal environment, the decrease in pH leads to a decline in the binding ability of MSN, chloroquine, and vitamin E. The chloroquine adsorbed by vitamin E is preferentially released, followed by the slow release of chloroquine inside the liposomes. The controlled release of chloroquine can reduce cytotoxicity and minimize the dissolution of exosomes as much as possible. Vitamin E is also released, and its ability to provide antioxidant capacity decreases, enabling the production of oxidized low-density lipoprotein. Cells experience stress in the atherosclerotic environment simulated by oxidized low-density lipoprotein and produce a large number of exosomes. Through intercellular transmission of cells, the overall cells can produce specific exosomes, thereby obtaining a high therapeutic effect of cells while maintaining a low cell mortality rate.

[0060] To verify the effect of different amounts of vitamin E on the release of chloroquine, a release test was conducted on chloroquine-liposomes. Under the conditions of a mass ratio of mesoporous silica nanoparticles to low-density lipoprotein cholesterol of 4:4 and a chloroquine-liposome dosage of 10%, the release rate of chloroquine was tested in different media. The test times were 12 h and 48 h, and the medium was physiological saline at pH 5.0. The results are shown in Table 4.

[0061] Table 4

[0062]

[0063] To verify the effect of different amounts of vitamin E on exosomes, under the conditions of a mass ratio of mesoporous silica nanoparticles to low-density lipoprotein cholesterol of 4:4 and a chloroquine-liposome dosage of 10%, the exosome concentration was tested. The protective effect of 50 μg / mL of UCMSC exosomes on vascular endothelial cell injury after 24 h was detected using the CCK-8 method. The results are shown in Table 5.

[0064] Table 5

[0065]

[0066] In the absence of added vitamin E, there may be more free radicals in cells, leading to more exosomes produced by cell stress, but with a high cell mortality rate and insufficient therapeutic effect of exosomes. After adding a small amount of vitamin E, cell death is less, the exosome secretion amount is normal, and the antioxidant effect decreases in a low-pH environment, resulting in partial LDL oxidation. Eventually, some cells experience stress and produce a large number of exosomes, increasing the number of exosomes and improving the therapeutic effect. However, after adding a large amount of vitamin E, the stress behavior of cells is reduced, the exosome secretion amount decreases, and the therapeutic effect decreases.

[0067] The DCFH-DA method was used to detect cellular free radicals. At the end of cell culture, a sample was taken and 10 μM DCFH-DA was added. The fluorescence intensity at Ex / Em = 488 / 525 nm was detected using a fluorescence microscope. The amount of free radicals was detected under the addition of different amounts of vitamin E. The results are shown in Table 6;

[0068] Table 6

[0069]

[0070]

[0071] Example 3: In Example 2, the oxidation degree and position of LDL were made controllable by adding vitamin E, the controllability of chloroquine release was improved, and the therapeutic effect and yield of exosomes were increased; to further improve the therapeutic effect and yield of exosomes, different chloroquine-liposomes were used in combination, and it was improved on the basis of Example 2.

[0072] When chloroquine-liposomes are used for cell culture to prepare exosomes, different combinations of chloroquine-liposomes are used;

[0073] The combined use of chloroquine-liposomes is to use one or more chloroquine-liposomes for the preparation of exosomes. The mass ratio of mesoporous silica nanoparticles to low-density lipoprotein cholesterol in the chloroquine-liposome is (2-8):4, and the mass ratio of vitamin E to mesoporous silica is 1:(1-4). The total addition amount of chloroquine-liposomes accounts for 1-20% of the total weight of the basal medium in the cell culture system.

[0074] The technical solutions in the embodiments of the present application at least have the following technical effects or advantages:

[0075] By using the combined use of chloroquine-liposomes, it is possible to further increase the yield and therapeutic effect of exosomes on the basis of reducing cell mortality. And due to the decrease in cell mortality, the purity of exosomes increases, solving the problems of low exosome purity and poor therapeutic effect.

[0076] Different liposome combinations complement each other through mechanisms such as targeting, slow release, antioxidant, and secretion promotion, breaking through the limitations of a single formulation; when the mass ratio of mesoporous silica nanoparticles (MSN) to low-density lipoprotein cholesterol (LDL-c) is 2:4, the MSN content is low, the liposome structure has high flexibility, the chloroquine release rate is fast, but the cell uptake efficiency is high. When the mass ratio is 8:4, the MSN rigidity increases, forming a dense pore barrier, and the chloroquine slow release effect is significant, but it may inhibit the lysosomal escape efficiency;

[0077] When the mass ratio of vitamin E (VE) to mesoporous silica (MSN) is 1:4, VE anchors chloroquine through hydrophobic interaction, reducing the burst release. However, its antioxidant ability is weak, the cell survival rate is high but the exosome function is limited. When the mass ratio is 1:1, VE adsorbs too much chloroquine, inhibits the release of lysosomes, and the excessive antioxidant effect weakens the pathological simulation effect.

[0078] By using the above different combinations and different ratios of chloroquine-liposomes, more optimized exosome preparation effects and sustained release effects of chloroquine can be achieved.

[0079] For example, when the mass ratio of mesoporous silica nanoparticles (MSN) to low-density lipoprotein cholesterol (LDL-c) is 2:4, the mass ratio of vitamin E (VE) to mesoporous silica (MSN) is 1:4, and the addition amount of chloroquine-liposome is 3%, and when the mass ratio of mesoporous silica nanoparticles (MSN) to low-density lipoprotein cholesterol (LDL-c) is 8:4, the mass ratio of vitamin E (VE) to mesoporous silica (MSN) is 1:1, and the addition amount of chloroquine-liposome is 15% are used in combination. After some cells take up liposomes, a large number of exosomes with therapeutic effects are produced. After the exosomes are utilized by other cells, the exosome secretion effect of the overall cells is improved, and the remaining cells can obtain a more stable living environment, with a low apoptosis rate, an increased exosome release amount, and the exosomes have the ability to treat atherosclerosis.

[0080] To verify the effect of the chloroquine-liposome combination on exosomes, a combined experiment was carried out to measure the exosome concentration, and the CCK-8 method was used to detect the protective effect of 50 μg / mL of UCMSC exosomes on vascular endothelial cell injury after 24 hours. The results are shown in Table 7;

[0081] Table 7

[0082]

[0083] Example 4: In Example 3, different liposomes were used in combination to obtain stronger therapeutic effects and yields. To further improve the performance of liposomes, it was further improved on the basis of Example 3.

[0084] During the exosome preparation process, chloroquine-liposomes were also added in portions during cell culture. Specifically:

[0085] When the cell confluence reaches 85%, the culture medium containing chloroquine-liposomes is added for the first time. After 12 hours, chloroquine-liposomes are added for the second time. Among the chloroquine-liposomes added for the first time, a chloroquine-liposome combination with high LDL-c and a high proportion of low VE is used. Among the chloroquine-liposomes added for the second time, a chloroquine-liposome combination with low LDL-c and a high proportion of high VE is used.

[0086] The technical solutions in the embodiments of the present application at least have the following technical effects or advantages:

[0087] Use a staged addition method to achieve precise regulation of "stimulate first and then repair", and break through the contradiction between "yield - survival rate".

[0088] The first addition simulates a hypercholesterolemic environment of atherosclerosis and induces cell stress; high LDL-c promotes intracellular cholesterol accumulation by binding to cell surface receptors, simulates the plaque microenvironment, triggers the cell stress response, and stimulates exosome secretion; low VE reduces antioxidant protection, allows moderate oxidative stress, and enhances the exosome secretion signaling pathway; the initial high LDL-c and low VE act synergistically to maximize exosome production through dual stimulation of oxidative stress and cholesterol overload.

[0089] The second addition protects cells and maintains exosome function; low LDL-c reduces further cholesterol load and avoids excessive cell death, high VE scavenges excessive free radicals, inhibits lipid peroxidation, maintains cell survival rate, and extends the exosome secretion cycle; in the later stage, VE repairs oxidative damage through antioxidant effects, while low LDL-c reduces the intensity of pathological simulation and balances yield and cell health.

[0090] To verify the effect of the chloroquine-liposome combination on exosomes, a combined experiment was conducted to test the exosome concentration, and the protective effect of 50 μg / mL of UCMSC exosomes on vascular endothelial cell injury after 24 hours was detected by the CCK-8 method. The results are shown in Table 8; among them, high LDL-c means MSN: LDL-c = 2:4, low VE means VE: MSN = 1:4, low LDL-c means MSN: LDL-c = 8:4, and high VE means VE: MSN = 1:1;

[0091] Table 8

[0092]

[0093] The above is only the preferred embodiment of the present invention and is not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for preparing exosomes by simulating an environment with elevated low-density lipoprotein cholesterol, characterized in that the steps Including: Inoculate umbilical cord mesenchymal stem cells in a culture plate. After the cell confluence reaches 85%, add chloroquine-liposome and culture for 24 - 48 hours; collect the cell supernatant and extract exosomes by ultracentrifugation method; chloroquine-liposome is obtained by fusing liposome with chloroquine and then performing freeze-thaw treatment. The liposome is prepared by the thin film dispersion method from phospholipids, low-density lipoprotein, low-density lipoprotein cholesterol, and mesoporous silica nanoparticles. Vitamin E is also loaded in the mesoporous silica nanoparticles.

2. The method for preparing exosomes by simulating an environment with elevated low-density lipoprotein cholesterol as described in claim 1, wherein, Chloroquine-liposome accounts for 1 - 20% of the total weight of the basal medium in the whole culture system.

3. The method for preparing exosomes by simulating an environment with elevated low-density lipoprotein cholesterol as described in claim 1, wherein, The mass ratio of low-density lipoprotein cholesterol to phospholipids is 1:2; the content of oxidized low-density lipoprotein in low-density lipoprotein is 20%.

4. The method for preparing exosomes by simulating an environment with elevated low-density lipoprotein cholesterol as claimed in claim 1, wherein, The mass ratio of low-density lipoprotein to low-density lipoprotein cholesterol is 1:

10.

5. A method for preparing exosomes by simulating an environment with elevated low-density lipoprotein cholesterol as described in claim 1, wherein The mass ratio of mesoporous silica nanoparticles to low-density lipoprotein cholesterol is (2 - 8):

4.

6. A method for preparing exosomes by simulating an environment with elevated low-density lipoprotein cholesterol as described in claim 1, characterized in that, The mass ratio of chloroquine to liposome is 1:

250.

7. A method for preparing exosomes by simulating an environment with elevated low-density lipoprotein cholesterol as described in claim 1, characterized in that, The mass ratio of vitamin E to mesoporous silica is 1:(1 - 4); the specific method for loading vitamin E is: dissolve vitamin E and mesoporous silica in ethanol together, stir overnight, and then remove the solvent by rotary evaporation under reduced pressure to obtain mesoporous silica loaded with vitamin E.

8. A method for preparing exosomes by simulating an environment with elevated low-density lipoprotein cholesterol as claimed in claim 1, characterized in that, The specific preparation method of chloroquine-liposome is as follows: S1: Disperse low-density lipoprotein cholesterol, mesoporous silica nanoparticles, phospholipids, and low-density lipoprotein in absolute ethanol. After mixing evenly, rotary evaporate to remove ethanol at 30 - 40 °C under reduced pressure until a film is formed, thus obtaining a lipid film. S2: Add PBS buffer solution with pH 7.0 to the lipid film and perform rotary hydration at 50 - 60 °C for 40 - 50 min to obtain a crude liposome. S3: Perform intermittent ultrasonic treatment on the crude liposome with 20 s of ultrasonic treatment followed by 20 s of pause for 3 min. The ultrasonic frequency is 50 kHz. After ultrasonic treatment, place it at 50 - 60 °C for 1 - 2 h to obtain a liposome suspension. S4: Add chloroquine to the liposome suspension for fusion. Rapidly freeze at -40 °C for 50 - 60 min and then slowly thaw at room temperature to obtain chloroquine-liposome.

9. A method for preparing exosomes by simulating an environment with elevated low-density lipoprotein cholesterol as claimed in claim 1, wherein, Chloroquine-liposome has multiple combinations, and one or more of them can be used in combination when in use.

10. A method for preparing exosomes by simulating an environment with elevated low-density lipoprotein cholesterol as described in claim 1, wherein, After the cell confluence reaches 85%, chloroquine-liposome is added in batches. When the cell confluence reaches 85%, it is added for the first time, and then added for the second time after continuing to culture for 12 h.

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