Extracellular vesicle nucleic acid nanodrug delivery system, preparation method and application thereof

Abstract

The application provides an extracellular vesicle nucleic acid nanomedicine delivery system, and also provides a preparation method and application thereof. The extracellular vesicle surface highly expresses CD47 protein, can be combined with SIRP alpha on the surface of mononuclear macrophages, avoids phagocytosis of the mononuclear macrophage system, and prolongs the circulation time. Meanwhile, the extracellular vesicle is modified by an engineering modification method to realize tumor tissue targeted delivery and nuclear magnetic imaging tracking by using homing peptide and nuclear magnetic contrast agent modification. The engineered extracellular vesicle can also be loaded with siYTHDF1 capable of knocking down YTHDF1 expression by an electroporation method, and is effectively delivered to gastric cancer cells to realize efficient and low-toxicity gastric cancer treatment strategies by targeting and regulating epigenetic regulatory factors in the gastric cancer cells. The engineered extracellular vesicle nucleic acid nanomedicine delivery system constructed in the application provides a feasible method and technology for targeted and regulated m 6 A epigenetic factor gastric cancer treatment.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of nanomedicine, and particularly relates to an extracellular vesicle nucleic acid nanomedicine delivery system, a preparation method and application thereof. BACKGROUND

[0002] Gastric cancer is a disease that seriously threatens human health, and its morbidity and mortality are among the highest. Gastric cancer has the characteristics of early non-recurrence and poor prognosis, and has potential biological and genetic heterogeneity at the molecular level. Existing studies have shown that epigenetic regulation may play an important role in the cause and development of gastric cancer. 6 - methylation of methyladenine nucleotides (m 6 A) is the most common post-transcriptional modification of RNA in eukaryotes, and is involved in basic life activities such as DNA repair, cell differentiation, and cell cycle apoptosis. Existing studies have shown that m 6 A recognition protein YTHDF1 is overexpressed in gastric cancer tissues, and is closely related to abnormal proliferation, distant metastasis and progression of gastric tumor cells. Therefore, the use of siRNA and other gene therapy methods to knock down the expression of YTHDF1 protein can achieve inhibition and treatment of gastric cancer through m 6 A epigenetic regulation. The commonly used siRNA drug carriers (mainly including viruses, polyethyleneimine nanoparticles, and liposomes) have poor selectivity, low tumor targeting ability, can cause serious systemic toxicity and immunogenicity, and other characteristics, which limit their clinical application. Therefore, it is of great significance to use a suitable drug carrier to specifically deliver nucleic acid drugs that can knock down the expression of YTHDF1 protein to gastric cancer cells.

[0003] Extracellular vesicles are vesicular structures produced by cells with a phospholipid bilayer structure, and have good biocompatibility, low immunogenicity, and low toxicity. Extracellular vesicles with high expression of CD47 on the surface (RsEVs) can bind to SIRPα on the surface of mononuclear macrophages, avoid phagocytosis by the mononuclear macrophage system, and achieve long circulation. Further modification of the extracellular vesicles with tumor targeting peptides and magnetic resonance contrast agents can achieve targeted delivery to tumor tissues and MRI tracking. By using engineered extracellular vesicles, siYTHDF1 genes that can knock down the expression of YTHDF1 can be effectively delivered to gastric cancer cells, target and regulate epigenetic regulators in gastric cancer to achieve a high-efficiency and low-toxicity treatment strategy for gastric cancer. There is no research on the combination of nanomedicine technology based on extracellular vesicles and m 6 A epigenetic regulation to achieve treatment of gastric cancer. SUMMARY

[0004] Therefore, the present application aims to overcome the defects in the prior art, and provides an extracellular vesicle nucleic acid nanomedicine delivery system, a preparation method and application thereof. 6 A epigenetic regulation treatment related to high systemic toxicity, low tumor targeting and drug efficiency, etc. problems, for through the way of targeting and regulating m 6 A epigenetic factor to achieve tumor treatment provides a new idea, with great clinical application potential.

[0005] Before describing the present application, the terms used herein are defined as follows:

[0006] The term "RsEVs" refers to cell-derived extracellular vesicles with high expression of CD47.

[0007] The term "c(RGDyC)" refers to a cyclic RGD polypeptide.

[0008] The term "DSPE-DOTA" refers to a compound formed by linking distearoyl phosphatidyl ethanolamine (DSPE) with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid (DOTA).

[0009] The term "PGRsEVs" refers to c(RGDyC) polypeptide and Gd nuclear magnetic contrast agent engineered modified extracellular vesicles.

[0010] The term "PGRsEVs-siYTHDF1" refers to siYTHDF1 loaded engineered modified extracellular vesicles.

[0011] The term "siYTHDF1" refers to an siRNA capable of knocking down YTHDF1 protein expression, and its sequence is 5'-GGAACAACATCTATCAGCA-3.

[0012] The term "m 6 A" refers to N 6 - methylated adenine nucleotide.

[0013] The term "MRI" refers to magnetic resonance imaging.

[0014] The term "DMEM medium" refers to a medium containing various amino acids and glucose.

[0015] The term "PBS" refers to a phosphate buffered saline solution.

[0016] The term "HEPES" refers to 4-hydroxyethylpiperazine ethanesulfonic acid.

[0017] The term "DAPI" refers to 4',6-diamidino-2-phenylindole.

[0018] The term "RAW264.7" refers to mouse monocyte macrophage leukemia cells.

[0019] The term "YTHDF1" refers to YTH domain N 6 - methyladenine (m 6 A) nucleotide binding protein 1.

[0020] The term "CD47" refers to a class Ig membrane protein widely expressed on various cells, which can interact with a variety of cell surface receptors.

[0021] The term "SIRPα" refers to signal regulatory protein alpha (SIRPα).

[0022] The term "DSPE-PEG2000-c(RGDyC)" refers to a c(RGDyC) polypeptide linked to distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000.

[0023] The term "Optiprep" refers to a separation solution.

[0024] The term "RsVEs-NC-siYTHDF1" refers to an extracellular vesicle loaded with negative control (NC) siYTHDF1.

[0025] The term "RsEVs-siYTHDF1" refers to an extracellular vesicle loaded with siYTHDF1.

[0026] The term "NC-siYTHDF1" refers to a negative control (NC) siYTHDF1.

[0027] The term "Lipo" refers to siRNA transfection reagent Lipofectamine RNAiMAX.

[0028] The term "Lipo-NC-siYTHDF1" refers to a negative control siRNA sequence transfected with siYTHDF1 using Lipofectamine RNAiMAX.

[0029] To achieve the above-mentioned purpose, the first aspect of the present application provides an extracellular vesicle nucleic acid nanomedicine delivery system, which comprises an extracellular vesicle and siRNA; wherein the siRNA is loaded into the extracellular vesicle modified by a tumor targeting polypeptide and a nuclear magnetic contrast agent on the surface;

[0030] Preferably, the extracellular vesicle surface highly expresses CD47 protein.

[0031] More preferably, the source of the extracellular vesicle is selected from one or more cells of the following: macrophages, macrophage stable strains with high expression of CD47, and induced pluripotent stem cells, further preferably macrophages, and most preferably Raw264.7 cells.

[0032] The extracellular vesicle nucleic acid nanodrug delivery system according to the first aspect of the present application, wherein,

[0033] The particle size of the nanoparticles in the extracellular vesicle nucleic acid nanodrug delivery system is 150-250 nm, preferably 150-200 nm;

[0034] The mass ratio of the siRNA to the extracellular vesicle is 0.1-5:0.5-10, preferably 0.5-1.5:0.5-4, and most preferably 1:1; and / or

[0035] The siRNA is an RNA sequence siYTHDF1 capable of knocking down the expression of YTHDF1 protein;

[0036] Preferably, the RNA sequence siYTHDF1 is selected from one or more of the following: siYTHDF1-01, siYTHDF1-02, siYTHDF1-03, further preferably siYTHDF1;

[0037] More preferably, the target sequence of siYTHDF1-01 is 5'-GGAACAACATCTATCAGCA-3'; the target sequence of siYTHDF1-02 is 5'-GGAAACGTCCAGCCTAATT-3'; and / or the target sequence of siYTHDF1-03 is 5'-GCTCAACCGCAGTATCAGA-3'.

[0038] The extracellular vesicle nucleic acid nanodrug delivery system according to the first aspect of the present application, wherein the tumor-targeting polypeptide is an RGD polypeptide or a derivative sequence thereof, preferably a cyclic RGD polypeptide;

[0039] Preferably, the cyclic RGD polypeptide is selected from one or more of the following: c(RGDyC), c(RGDyK), c(RGDfC), and c(RGDfK), and most preferably c(RGDyC).

[0040] The extracellular vesicle nucleic acid nanodrug delivery system according to the first aspect of the present application, wherein the nuclear magnetic resonance contrast agent is a nuclear magnetic resonance contrast agent containing a nuclear magnetic resonance imaging functional metal element;

[0041] Preferably, the nuclear magnetic resonance contrast agent is selected from one or more of the following: a nuclear magnetic resonance contrast agent containing a gadolinium element, a nuclear magnetic resonance contrast agent containing a manganese element, and a nuclear magnetic resonance contrast agent containing an iron element.

[0042] The second aspect of the present application provides a method for preparing the extracellular vesicle nucleic acid nanomedicine delivery system of the first aspect, the method comprising the following steps:

[0043] (1) extracting extracellular vesicles;

[0044] (2) modifying the extracellular vesicles prepared in step (1) with a tumor-targeting polypeptide and a nuclear magnetic contrast agent to obtain engineered extracellular vesicles;

[0045] (3) loading siRNA into the engineered extracellular vesicles prepared in step (3) to obtain the engineered extracellular vesicle nucleic acid nanomedicine delivery system.

[0046] According to the method of the second aspect of the present application, wherein the step (1) comprises the following steps:

[0047] (A) preparing extracellular vesicle-free medium;

[0048] (B) isolating and extracting extracellular vesicles.

[0049] According to the method of the second aspect of the present application, wherein the step (1) comprises the following steps:

[0050] The step (A) further comprises: after centrifuging the medium, filtering the supernatant to obtain the extracellular vesicle-free medium; the centrifugal force is preferably 90,000-130,000g, and further preferably 100,000-120,000g; and / or the filter membrane diameter for filtering is preferably 0.2-0.4μm, and further preferably 0.22μm; and / or

[0051] The step (B) further comprises: when the density of the source cells of the extracellular vesicles reaches 50-60%, the medium prepared in step (A) is used for culture and the supernatant is collected, and then the extracellular vesicles are isolated and stored in a refrigerator; the culture time is preferably 18-36 hours, and further preferably 24 hours; and / or the temperature of the refrigerator is preferably -80℃.

[0052] According to the method of the second aspect of the present application, wherein the step (2) further comprises the following steps:

[0053] (C) synthesizing DSPE-DOTA-Gd;

[0054] (D) preparing engineered extracellular vesicles modified with a tumor-targeting polypeptide and a nuclear magnetic contrast agent on the surface;

[0055] Preferably, the step (C) further comprises centrifuging and purifying the obtained product; and / or

[0056] Preferably, the step (D) further comprises centrifuging the prepared end product, purifying to remove unreacted reagents, to obtain the engineered extracellular vesicle.

[0057] According to the method of the second aspect of the present application, wherein in the step (3), the entrapment method is selected from one or more of the following: electroporation, ultrasound, room temperature incubation, most preferably electroporation.

[0058] Preferably, when the entrapment method is electroporation, the method further comprises the following steps after electroporation: culturing and centrifuging to obtain the extracellular vesicle nucleic acid nanomedicine delivery system.

[0059] The third aspect of the present application provides the use of the extracellular vesicle nucleic acid nanomedicine delivery system of the first aspect or the extracellular vesicle nucleic acid nanomedicine delivery system prepared according to the method of the second aspect in the preparation of a pharmaceutical product for the treatment of gastric cancer.

[0060] Preferably, the treatment of gastric cancer is in the form of epigenetic regulation.

[0061] More preferably, the treatment of gastric cancer is in the form of targeting and regulating N 6 - methylated methylenetetrahydrofolate.

[0062] The present application provides a siYTHDF1, the target sequence of which is as follows:

[0063] The target sequence of siYTHDF1-01 is 5'-GGAACAACATCTATCAGCA-3';

[0064] The target sequence of siYTHDF1-02 is 5'-GGAAACGTCCAGCCTAATT-3';

[0065] The target sequence of siYTHDF1-03 is 5'-GCTCAACCGCAGTATCAGA-3'; (Since the computer-readable carrier of the RNA sequence table cannot recognize the marks 5'- and -3', the sequence of siYTHDF1 is as recorded in the sequence of the specification here).

[0066] According to one specific embodiment of the present application, the first aspect of the present application provides an engineered extracellular vesicle nanomedicine delivery system, which utilizes an engineered modification method to modify CD47 high-expression cell-derived extracellular vesicles with c(RGDyC) tumor-targeting peptides and a nuclear magnetic contrast agent, and encapsulates siYTHDF1 capable of knocking down YTHDF1 protein expression by an electroporation method. The engineered nucleic acid nanomedicine delivery system can avoid phagocytosis by mononuclear macrophages when circulating in the body, prolonging the in vivo circulation time. At the same time, the nucleic acid nanomedicine delivery system also has the characteristics of enhanced tumor targeting and nuclear magnetic imaging, and can target and regulate epigenetic regulatory factors in gastric cancer to achieve efficient and low-toxicity gastric cancer diagnosis and treatment integration research.

[0067] The second aspect of the present application provides a preparation method of the engineered extracellular vesicle nanomedicine delivery system of the first aspect, which comprises the following steps:

[0068] (1) extracting CD47 high-expression cell-derived extracellular vesicles (RsEVs);

[0069] (2) modifying the RsEVs with c(RGDyC) tumor-targeting peptides and a nuclear magnetic contrast agent to obtain engineered extracellular vesicles (PGRsEVs);

[0070] (3) encapsulating siYTHDF1 into the engineered extracellular vesicles (PGRsEVs) by an electroporation method to obtain an engineered extracellular vesicle nucleic acid nanomedicine delivery system (PGRsEVs-siYTHDF1).

[0071] According to the preparation method of the second aspect of the present application, wherein the step (1) comprises the following steps:

[0072] (A) preparing an extracellular vesicle-free medium.

[0073] DMEM medium containing 20%-50% fetal bovine serum is placed in an ultracentrifuge and centrifuged for 12-14 hours, and the supernatant is collected. The obtained supernatant is filtered with a filter membrane, and DMEM medium is added for dilution to obtain a DMEM cell culture medium containing 10% fetal bovine serum without extracellular vesicles.

[0074] Preferably, the centrifugal force of the ultracentrifugation in step (A) is 100,000-120,000g, and the filter membrane used is a 0.22μm filter membrane.

[0075] (B) extracting extracellular vesicles of CD47 high-expression cells.

[0076] When the cell density reaches 50-60%, the culture medium without extracellular vesicles is used for 24 hours and the supernatant is collected, and then the following steps are carried out to isolate the extracellular vesicles:

[0077] a) The collected cell supernatant is removed from the necrotic cells and cell debris by programmed centrifugation at 4°C, and filtered with a 0.22 μm filter.

[0078] Preferably, the programmed centrifugation method of step a) is preferably 800g centrifugation for 5 minutes, and after collecting the supernatant, 2,000g centrifugation is continued for 15-20 minutes.

[0079] b) The cell supernatant obtained in step a) is divided into ultracentrifuge tubes, and after balancing, it is uniformly placed in an ultracentrifuge at 4°C, and ultracentrifuged for 1.5-2 hours to remove the supernatant and collect the extracellular vesicle sample on the wall of the centrifuge tube.

[0080] Preferably, the ultracentrifugation of step b) has a centrifugal force of 100,000-150,000g, and the balancing should control the mass difference of each centrifuge tube to be within 0.05g.

[0081] c) Resuspend the extracellular vesicles obtained in step b) with PBS, continue ultracentrifugation at 4°C for 1.5-2 hours, remove the supernatant after centrifugation, add PBS to the ultracentrifuge tube to disperse and collect the extracellular vesicle sample on the wall of the centrifuge tube, and store it in a -80°C refrigerator after division.

[0082] Preferably, the ultracentrifugation of step c) has a centrifugal force of 100,000-150,000g.

[0083] According to the preparation method of the second aspect of the present application, wherein the step (2) comprises the following steps:

[0084] (A) Synthesis of DSPE-DOTA-Gd:

[0085] 80-100 mg of DSPE-DOTA (Xi'an Rixi) is mixed with 30-50 ml of acetic acid buffer, and then 0.8 mmol-1.2 mmol of Gd(OAc)3 solution is added at 45-55°C for 10-14 hours. After the temperature of the mixture drops to room temperature, centrifugal purification is carried out, and the obtained product is washed with acetic acid buffer for 3-5 times to remove non-chelated Gd 3+ .

[0086] (B) Preparation of PGRsEVs:

[0087] 15-25mg of DSPE-PEG2000-c(RGDyC) (Xi'an Ruihi) and 60-100mg of DSPE-DOTA-Gd are dissolved in 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) at 55-65℃, and RsEVs are added when the system temperature drops to about 37℃, and the reaction is continued for 1.5-2.5 hours to obtain the final product PGRsEVs, and then the product is purified by ultracentrifugation to remove unreacted reagents.

[0088] According to the preparation method of the second aspect of the present application, wherein the step (3) comprises the following steps:

[0089] PGRsEVs and siYTHDF1 are dissolved in the electroporation buffer solution at the same weight ratio, and then the system is added to a 4mm electroporation cuvette, and the cuvette is placed on ice at 4℃ for half an hour. Then the cuvette is placed in an electroporator for electroporation, and after the end of the electroporation, the system is placed in a 37℃ incubator for recovery of the RsEVs, and then ultracentrifugation is continued at 4℃ to separate the free siYTHDF1.

[0090] Preferably, the electroporation buffer solution is a phosphate buffer containing 1-1.5mM potassium phosphate (pH 7.2), 20-30mM potassium chloride and 18%-25% Optiprep. The electroporation method uses the following electroporation parameters: volume, 400μL; voltage, 350-450V; capacitance, 100-150μF.

[0091] The third aspect of the present application provides the engineered extracellular vesicle nucleic acid nanomedicine delivery system of the first aspect and / or the extracellular vesicle nucleic acid nanomedicine delivery system prepared according to the preparation method of the second aspect, which targets and regulates m 6 A gastric cancer diagnosis and treatment integration research is realized in the manner of an epigenetic factor.

[0092] The present application provides an engineered extracellular vesicle nucleic acid nanomedicine delivery system, the surface of which highly expresses CD47 protein, can combine with SIRPα on the surface of mononuclear macrophages, avoid phagocytosis by the mononuclear macrophage system, and prolong the circulation time. At the same time, the extracellular vesicle is modified by an engineering modification method to realize the targeted delivery and MRI tracking of tumor tissues. The engineered extracellular vesicle can also encapsulate siYTHDF1 capable of knocking down YTHDF1 expression by an electroporation method, and effectively deliver it to gastric cancer cells to target and regulate epigenetic regulatory factors in gastric cancer cells to achieve a high-efficiency and low-toxicity gastric cancer treatment strategy. The engineered extracellular vesicle nucleic acid nanomedicine delivery system constructed by the present application targets and regulates m 6The gastric cancer treatment of the epigenetic factor provides feasible methods and techniques.

[0093] The extracellular vesicle nucleic acid nanomedicine delivery system of the present application can have, but is not limited to, the following beneficial effects:

[0094] 1. The present application selects CD47 high expression cell derived extracellular vesicles, and verifies that the surface of the extracellular vesicles highly expresses CD47 protein, can combine with SIRP alpha on the surface of mononuclear macrophages, and avoid phagocytosis by the mononuclear macrophage system in the body circulation;

[0095] 2. The present application uses an engineered modification method to modify the CD47 high expression cell derived extracellular vesicles with homing peptide c(RGDyC) and nuclear magnetic resonance contrast agent, which can realize the targeted delivery and MRI tracking of tumor tissues;

[0096] 3. The nucleic acid extracellular vesicle nanomedicine delivery of the present application can effectively deliver the siYTHDF1 gene for knocking down YTHDF1 expression to gastric cancer cells, target and regulate the epigenetic regulatory factor m 6 A realizes the treatment of gastric cancer;

[0097] 4. The nucleic acid nanomedicine delivery system of the present application has low immunogenicity, low toxicity and high siRNA delivery efficiency. BRIEF DESCRIPTION OF DRAWINGS

[0098] Hereinafter, embodiments of the present application will be described in detail with reference to the accompanying drawings, in which:

[0099] Figure 1 The morphology of the CD47 high expression cell derived extracellular vesicles in Example 1 is shown.

[0100] Figure 2 The particle size distribution of the PGRsEVs-siYTHDF1 nanoparticles in Example 1 is shown.

[0101] Figure 3 The expression of Hsp70, Flotillin-1, CD47 and CD81 proteins of the RsEVs and PGRsEVs-siYTHDF1 nanoparticles in Example 3 is shown.

[0102] Figure 4 The expression of CD47 of the Raw264.7, CD47 high expression Raw264.7 stable strain and induced pluripotent stem cell derived extracellular vesicles in Example 4 is shown.

[0103] Figure 5The uptake of the two nanoparticles, RsEVs-siYTHDF1 and PGRsEVs-siYTHDF1, by the two gastric cancer cells, MGC-803 and HGC-27, in Example 5 is shown. DAPI is used to stain the cell nucleus; siYTHDF1 is Cy5 fluorescently labeled and can be used to indicate the distribution of the engineered extracellular vesicle drug delivery system in the gastric cancer cells; Merge is the combined analysis of DAPI fluorescence indicating the cell nucleus and Cy5 fluorescence indicating the distribution of the engineered extracellular vesicle drug delivery system in the cells.

[0104] Figure 6 The delivery efficiency of siYTHDF1 by the three nanoparticles, Lipo-siYTHDF1, RsEVs-siYTHDF1 and PGRsEVs-siYTHDF1, in MGC-803 and HGC-27 gastric cancer cells in Example 6 (PBS, RsEVs and siYTHDF1 as controls) is shown.

[0105] Figure 7 The expression of the target YTHDF1 protein after the administration of PBS, RsEVs, RsVEs-NC-siYTHDF1, Lipo-siYTHDF1, RsEVs-siYTHDF1 and PGRsEVs-siYTHDF1 to the two gastric cancer cells, MGC-803 and HGC-27, in Example 7 is shown.

[0106] Figure 8 The expression of the target YTHDF1 protein after the administration of the control group and the three siYTHDF1 corresponding RsVEs-siYTHDF1-01, RsVEs-siYTHDF1-02 and RsVEs-siYTHDF1-03 nanoparticles to MGC-803 gastric cancer cells in Examples 1 and 2 is shown.

[0107] Figure 9 The toxic effects of PBS, RsEVs, RsVEs-NC-siYTHDF1, RsEVs-siYTHDF1 and PGRsEVs-siYTHDF1 on the two gastric cancer cells, MGC-803 and HGC-27, in Example 8 are shown.

[0108] Figure 10 The nuclear magnetic imaging (MRI) of the tumor site after the tail vein injection of PGRsEVs-siYTHDF1 nanoparticles to MGC-803 tumor cell-bearing mice in Example 9 at 0h, 6h, 12h, 24h, 48h and 72h is shown.

[0109] Figure 11The diagram shows the changes in tumor volume in mice with MGC-803 tumor cells after intravenous injection of saline, RsEVs, RsVEs-NC-siYTHDF1, RsEVs-siYTHDF1 and PGRsEVs-siYTHDF1 nanoparticles via the tail vein in Example 10. Detailed Implementation

[0110] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for more detailed and specific illustration and should not be construed as limiting the present invention in any way.

[0111] This section provides a general description of the materials and testing methods used in the experiments of this invention. While many of the materials and methods of operation used to achieve the objectives of this invention are well known in the art, the invention is still described in as much detail as possible herein. It will be apparent to those skilled in the art that, unless otherwise stated in the context, the materials and methods of operation used in this invention are well known in the art.

[0112] The reagents and instruments used in the following examples are as follows:

[0113] Reagents:

[0114] DSPE-DOTA and DSPE-PEG2000-c(RGDyC) were purchased from Xi'an Ruixi Co., Ltd.

[0115] 5'-GGAACAACATCTATCAGCA-3'(siYTHDF1-01), 5'-GGAAACGTCCAGCCTAATT-3'(siYTHDF1-02) and 5'-GCTCAACCGCAGTATCAGA-3'(siYTHDF1-03) were all purchased from Guangzhou Ruibo Biotechnology Co., Ltd.

[0116] Acetic acid was purchased from Aladdin Company;

[0117] Gd(OAc)3 was purchased from Hines Ltd.;

[0118] 4-Hydroxyethylpiperazine ethanesulfonic acid (HEPES) was purchased from Shanghai Mairui Chemical Technology Co., Ltd.

[0119] 4′,6-Diamidinyl-2-phenylindole (DAPI), MTS cell proliferation kit, and BCA protein assay kit were purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0120] The protease inhibitor (87785), phosphatase inhibitor (A32957), and protein lysis buffer (87787) were all purchased from ThermoFisher (USA) Ltd.

[0121] Fetal bovine serum (FBS) and Dulbecco's Modified Eagle's medium (DMEM) cell culture medium were purchased from Gibco (USA).

[0122] Instrument:

[0123] Bio-transmission electron microscope, purchased from Hitachi, Ltd. (Japan), model HT7700;

[0124] Nanoparticle tracking analyzer, purchased from Malvern Instruments Ltd. (UK), model NS300;

[0125] Single-photon laser confocal imaging system, purchased from Carl Zeiss AG (Germany), model Zeiss 710;

[0126] Flow cytometer, purchased from BD (USA), model C6;

[0127] Microplate reader, purchased from Molecular Devices (China) Co., Ltd. (China), model Molecular Devices I3;

[0128] Small animal nuclear magnetic resonance imaging instrument, purchased from Bruker (Germany), model BioSpec 70 / 20USR;

[0129] X-cell electroporator, purchased from Bio-Rad (USA), model 165-2081.

[0130] Example 1

[0131] This example is used to illustrate the preparation method of the extracellular vesicle nucleic acid nanodrug delivery system of the application, which comprises the following steps:

[0132] (1) Extraction of Raw 264.7 cell-derived extracellular vesicles (RsEVs):

[0133] (A) Preparation of extracellular vesicle-free medium.

[0134] DMEM medium containing 40% fetal bovine serum was placed in an ultracentrifuge and centrifuged at a centrifugal force of 120,000g for 12 hours, and the supernatant was collected. The obtained supernatant was filtered with a 0.22μm filter membrane, and simple DMEM medium was added for dilution to obtain a DMEM cell culture medium containing 10% fetal bovine serum without vesicles, which was placed in a 4°C refrigerator for use.

[0135] (B) Extraction of extracellular vesicles of Raw264.7 cells.

[0136] When the density of Raw 264.7 cells reaches 50-60%, replace the culture medium without extracellular vesicles and culture for 24 hours and collect the supernatant, and then the following steps are carried out to isolate the extracellular vesicles of Raw 264.7 cells:

[0137] a) Centrifuge the collected supernatant of Raw 264.7 cells at 4°C at a centrifugal force of 800g for 5 minutes and collect the supernatant. Continue centrifuging the obtained supernatant at 4°C at a centrifugal force of 2,000g for 15 minutes, collect the supernatant again, and filter it using a 0.22μm filter membrane;

[0138] b) Divide the supernatant obtained in step a) into 26mL centrifuge tubes, ensure that there are no air bubbles, balance the weight so that the weight difference of all centrifuge tubes is within 0.05g, and then place them in a Beckman ultracentrifuge. After centrifuging at 4°C at a centrifugal force of 150,000g for 1.5 hours, remove the supernatant and retain the precipitate sample on the wall of the centrifuge tube;

[0139] c) Add PBS to each centrifuge tube, wash the precipitate, and combine the precipitate samples of eight centrifuge tubes into one centrifuge tube, resuspend the precipitate in 26mL of PBS, and continue centrifuging at 4°C at a centrifugal force of 150,000g for 1.5 hours;

[0140] d) After the ultracentrifugation is completed, remove the supernatant, add 200μL of PBS to the ultracentrifugation tube to disperse and collect the extracellular vesicle sample on the wall of the centrifuge tube, and store it in a -80°C refrigerator after being divided.

[0141] (2) Modify the RsEVs obtained in step (1) with tumor-targeting polypeptide c(RGDyC) and nuclear magnetic resonance contrast agent DEPE-DOTA-Gd to obtain PGRsEVs:

[0142] (A) Synthesis of DSPE-DOTA-Gd.

[0143] Mix 100mg DSPE-DOTA (Xi'an Rixi) with 40mL acetic acid buffer, then add 1mmol Gd(OAc)3 at 50°C and react for 12 hours. After the temperature of the mixture drops to room temperature, centrifuge at 4500g for 10 minutes for purification. The obtained product is washed with acetic acid buffer for 3 times to remove non-chelated Gd 3+ .

[0144] (B) Preparation of PGRsEVs.

[0145] PGRsEVs final product was obtained by adding RsEVs to the system at 37°C for 2 hours, followed by purification using ultracentrifugation to remove unreacted reagents.

[0146] (3) siYTHDF1 was loaded into PGRsEVs obtained from step (2) by electroporation to obtain PGRsEVs-siYTHDF1 nanoparticles.

[0147] PGRsEVs and siYTHDF1-01 (target sequence: 5'-GGAACAACATCTATCAGCA-3', Guangzhou RiboBio Biotechnology Co., Ltd.) were dissolved in 400 μL of electroporation buffer solution (1.15 mM potassium phosphate (pH 7.2), 25 mM potassium chloride, 21% Optiprep) at a weight ratio of 1:1, so that the final concentration of PGRsEVs was not more than 0.5 μg / μL. Then the system was placed in a 4 mm electroporation cuvette, and the cuvette was placed on ice at 4°C for half an hour. Subsequently, the cuvette was placed in a Gene Pulser X-cell electroporator (BioRad, 165-2081) for electroporation, and the electroporation parameters were set as follows: volume, 400 μL; voltage, 400 V; capacitance, 125 μF. The pulse time after electroporation was about 12 to 18 milliseconds. After electroporation, the system was placed in a 37°C incubator for half an hour to one hour to allow the RsEVs to recover, and then it was suspended in 26 mL of RNase-free PBS and subjected to ultracentrifugation (4°C, 150,000 g, 90 minutes) to separate free siYTHDF1 to obtain the final PGRsEVs-siYTHDF1.

[0148] Figure 1 The morphology of the extracellular vesicles from CD47 high expression cells in Example 1 is shown.

[0149] Figure 2 The particle size distribution of PGRsEVs-siYTHDF1 nanoparticles in Example 1 is shown.

[0150] Example 2

[0151] This example will synthesize the extracellular vesicle nucleic acid nanomedicine delivery system loaded with siYTHDF1-02 and siYTHDF1-03 respectively according to the method of Example 1. The siYTHDF1-02 target sequence used in this example is 5'-GGAAACGTCCAGCCTAATT-3'; the targeting sequence of siYTHDF1-03 is 5'-GCTCAACCGCAGTATCAGA-3', both purchased from Guangzhou RiboBio Biotechnology Co., Ltd.

[0152] Example 3

[0153] This example is used to test the expression of Hsp70, Flotillin-1, CD47 and CD81 proteins of two kinds of nanoparticles RsEVs and PGRsEVs-siYTHDF1.

[0154] First, the protein lysis concentration of RsEVs and PGRsEVs-siYTHDF1 extracellular vesicles was determined. The extracellular vesicle samples for immunoblotting analysis should be resuspended with as little PBS as possible to obtain a higher concentration. Add 20 μL of extracellular vesicle suspension to the protein lysis solution containing 1 / 100 (v / v) protease inhibitors and 1 / 10 phosphatase inhibitors. After vortexing thoroughly, the sample was placed on ice for about 30 minutes of lysis. Then the sample was placed in an ice water bath and ultrasonicated three times using an ultrasonic cleaner. The protein concentration was calculated using the BCA protein determination kit (Solarbio, PC0020) for protein quantification. The lysed extracellular vesicles were mixed with 5x loading buffer buffer (NuPAGE LDS Sample Buffer, 4X, NP0007), heated at 100°C for 5 minutes, and stored at -20°C for use. TM

[0155] The siYTHDF1 target sequence used in this example is 5'-GGAACAACATCTATCAGCA-3, purchased from Guangzhou RiboBio Biotechnology Co., Ltd.

[0156] ​Next, Western blotting was used to analyze the expression of different proteins in RsEVs and PGRsEVs-siYTHDF1 nanoparticles. 10 μg of total protein of extracellular vesicles was added to 10% Tris-HCl gel (Invitrogen), and the total protein was separated by electrophoresis and transferred from the gel to a polyvinylidene fluoride (PVDF) membrane (Invitrogen) using a wet transfer method. After the transfer was completed, the PVDF membrane was washed with 0.1% Tween 20-Tris (TBST) buffer solution, then blocked with 5% (w / v) skimmed milk at room temperature for 2 hours, and then 4 mL of primary antibody dilution buffer (antibody dilution is 5% skimmed milk powder prepared with TBST solution) was added and incubated at 4°C overnight. The membrane was washed with TBST three times (5 minutes each time), and then a secondary antibody dilution buffer (antibody dilution is the same as the primary antibody) connected to HRP was added and incubated for 2 hours, and the membrane was washed with TBST three times again (5 minutes each time). Finally, the PVDF membrane was placed in a gel imager (Bio-Rad, USA) and developed on the membrane (Solarbio) for exposure.

[0157] Figure 3 The expression of Hsp70, Flotillin-1, CD47 and CD81 proteins in RsEVs and PGRsEVs-siYTHDF1 nanoparticles in Example 3 is shown. As shown in Figure 3 RsEVs highly expressed Hsp70, Flottlin-1, CD81 extracellular vesicle characteristic proteins (GADPH as internal control), and also highly expressed CD47 protein on the surface. The PGRsEVs-siYTHDF1 nanoparticles obtained by engineering modification of ReEVs did not change the expression of Hsp70, Flottlin-1, CD81 and CD47 proteins on the surface. It is indicated that the process of obtaining PGRsEVs-siYTHDF1 nanoparticles by engineering modification of ReEVs does not change the expression level of RsEVs related proteins.

[0158] Example 4

[0159] In this example, the expression of CD47 on the surface of macrophages, CD47 highly expressed macrophage stable strains and induced pluripotent stem cell derived extracellular vesicles was determined according to the method of Example 3.

[0160] Figure 4 The expression of CD47 on the surface of Raw264.7, CD47 highly expressed Raw264.7 stable strains and induced pluripotent stem cell derived extracellular vesicles in Example 4 is shown.

[0161] Example 5 In this example, the expression of CD47 on the surface of macrophages, CD47 highly expressed macrophage stable strains and induced pluripotent stem cell derived extracellular vesicles was determined according to the method of Example 3.

[0162] This embodiment is used to test the uptake of RsEVs-siYTHDF1 and PGRsEVs-siYTHDF1 nanoparticles by MGC-803 and HGC-27 gastric cancer cells.

[0163] MGC-803 and HGC-27 cells were cultured in confocal dishes (4 x 10 5 cells / well) overnight, and then 10 μL of RsEVs and PGRsEVs-siYTHDF1 (same concentration of extracellular vesicles: 1 x 10 10 particles / mL) were added respectively for 1 hour and 12 hours (the siYTHDF1 used in this experiment was Cy5 fluorescently labeled and could be used to indicate the distribution of the engineered extracellular vesicle drug delivery system in gastric cancer cells). The MGC-803 and HGC-27 cells were washed with PBS, stained with DAPI for 15 minutes, and then observed for uptake of extracellular vesicles by single-photon confocal microscopy (Zeiss, Germany) after washing. When analyzing the uptake, the DAPI fluorescence indicating the cell nucleus and the Cy5 fluorescence indicating the distribution of the engineered extracellular vesicle drug delivery system in cells were analyzed by merging.

[0164] Figure 5 The uptake of RsEVs-siYTHDF1 and PGRsEVs-siYTHDF1 nanoparticles by MGC-803 and HGC-27 gastric cancer cells in Example 5 is shown. DAPI was used to stain the cell nucleus; siYTHDF1 was Cy5 fluorescently labeled and could be used to indicate the distribution of the engineered extracellular vesicle drug delivery system in gastric cancer cells; and Merge was the combined analysis of DAPI fluorescence indicating the cell nucleus and Cy5 fluorescence indicating the distribution of the engineered extracellular vesicle drug delivery system in cells.

[0165] As shown in Figure 5 , both MGC-803 and HGC-27 gastric cancer cells showed stronger Cy5 signals after incubation with PGRsEVs-siYTHDF1, indicating that more nanoparticles were taken up by the cells. The experimental results show that the nanoparticles modified with c(RGDyC) peptides can bind to the α v β3 integrin on the surface of tumor cells, enhancing the targeting ability of the nanoparticles to gastric cancer cells. The siYTHDF1 target sequence used in this embodiment is 5'-GGAACAACATCTATCAGCA-3', purchased from Guangzhou Ribobio Biotechnology Co., Ltd.

[0166] Example 6

[0167] This example is used to test the delivery efficiency of siYTHDF1 of three kinds of nanoparticles Lipo-siYTHDF1, RsEVs-siYTHDF1 and PGRsEVs-siYTHDF1 in MGC-803 and HGC-27 two strains of gastric cancer cells (PBS, RsEVs and siYTHDF1 as controls).

[0168] MGC-803 and HGC-27 cells were placed in CLSM culture dishes (4x10 5 cells per well), after 12 hours of culture, PBS, RsEVs, siYTHDF1, Lipo-siYTHDF1, RsEVs-siYTHDF1 and PGRsEVs-siYTHDF1 nanoparticles were added respectively (the volume of all samples was 80 μL, and the number of RsEVs contained was the same, 1x10 10 particles / mL), and incubated for 1 and 12 h respectively. Then the MGC-803 and HGC-27 cells were washed with PBS and stained with DAPI for 15 minutes, and finally the uptake of siYTHDF1 was detected by flow cytometry.

[0169] Figure 6 The delivery efficiency of siYTHDF1 of three kinds of nanoparticles Lipo-siYTHDF1, RsEVs-siYTHDF1 and PGRsEVs-siYTHDF1 in MGC-803 and HGC-27 two strains of gastric cancer cells (PBS, RsEVs and siYTHDF1 as controls) is shown in Example 6.

[0170] Figure 6 The flow cytometry results show that the uptake rate of free NC-siYTHDF1 by MGC-803 and HGC-27 two strains of gastric cancer cells is negligible, the delivery rate of siYTHDF1 by Lipo transfection reagent is 35.7%-40.3%, the delivery rate of siYTHDF1 by RsEVs is 64.4%-70.9%, which confirms that the extracellular vesicle as a delivery system can significantly improve the delivery efficiency of siYTHDF1. When the RsEVs are engineered and modified, the delivery efficiency is further enhanced to 88.6%-96.8%, which is due to the modification of homing peptide c(RGDyC) that enhances the uptake of nanoparticles by two strains of gastric cancer cells. The siYTHDF1 target sequence used in this example is 5'-GGAACAACATCTATCAGCA-3', purchased from Guangzhou Ribo Biotechnology Co., Ltd.

[0171] Example 7

[0172] This example is used to test the expression of target YTHDF1 protein after PBS, RsEVs, Lipo-siYTHDF1, RsVEs-NC-siYTHDF1, RsEVs-siYTHDF1 and PGRsEVs-siYTHDF1 are respectively administered to MGC-803 and HGC-27 two strains of gastric cancer cells.

[0173] MGC-803 and HGC-27 cells were placed in a six-well cell culture plate (4x10 5 cells per well), and after being cultured overnight, PBS, RsEVs, Lipo-siYTHDF1, RsVEs-NC-siYTHDF1, RsEVs-siYTHDF1 and PGRsEVs-siYTHDF1 nanoparticles (the volume of all samples was 80 μL, and the number of RsEVs contained was the same, 1x10 10 particles / mL) were added respectively, and incubated for 48 hours, and then the proteins of the gastric cancer cells in each group after incubation were extracted according to the steps of Test Example 1, and the expression level of YTHDF1 protein after Lipo and extracellular vesicle-mediated drug carrier delivery of siYTHDF1 to MGC-803 and HGC-27 cells was analyzed by Western blot.

[0174] Figure 7 The expression of target YTHDF1 protein after PBS, RsEVs, RsVEs-NC-siYTHDF1, Lipo-siYTHDF1, RsEVs-siYTHDF1 and PGRsEVs-siYTHDF1 are respectively administered to MGC-803 and HGC-27 two strains of gastric cancer cells in Example 7 is shown.

[0175] Figure 7 The Western blot results show that the YTHDF1 protein level of the RsEVs-siYTHDF1 and PGRsEVs-siYTHDF1 groups is significantly reduced, indicating that the constructed engineered extracellular vesicle drug delivery system can deliver siYTHDF1 to two strains of gastric cancer cells and successfully knock down the expression level of YTHDF1 protein. The siYTHDF1 target sequence used in this example is 5'-GGAACAACATCTATCAGCA-3', purchased from Guangzhou Ribo Bio-technology Co., Ltd.

[0176] Example 8

[0177] The expression of target YTHDF1 protein after treating MGC-803 gastric cancer cells with PBS, RsEVs, PGRsEVs, PGRsEVs-siYTHDF1-01, PGRsEVs-siYTHDF1-02 and PGRsEVs-siYTHDF1-03 respectively according to the method of Example 7 was tested in this example. The siYTHDF1-01 target sequence used in this example is 5'-GGAACAACATCTATCAGCA-3'; the siYTHDF1-02 target sequence is 5'-GGAAACGTCCAGCCTAATT-3'; and the siYTHDF1-03 target sequence is 5'-GCTCAACCGCAGTATCAGA-3', all purchased from Guangzhou Ribobio Biotechnology Co., Ltd.

[0178] Figure 8 The Western blot results show that siYTHDF1-01, siYTHDF1-02 and siYTHDF1-03 can successfully knock down the expression level of YTHDF1 protein.

[0179] Example 9

[0180] This example was used to test the toxic effects of PBS, RsEVs, RsEVs-NC-siYTHDF1, RsEVs-siYTHDF1 and PGRsEVs-siYTHDF1 on MGC-803 and HGC-27 gastric cancer cells.

[0181] MGC-803 and HGC-27 cells were seeded into 96-well plates at a density of 1 x 10 4 cells / well and cultured overnight. Then different samples (PBS, RsEVs, NC-siYTHDF1, Lipo-NC-siYTHDF1, RsEVs-NC-siYTHDF1 and PGRsEVs-NC-siYTHDF1) (the amount of siYTHDF1 was 2 pmol per well) were added to the two strains of gastric cancer cells, and incubated for 0, 24, 48, 72 and 96 hours. After incubation, 20 μL of CCK8 was added to each well and incubated for 2-3 hours, and the absorbance was measured at 490 nm using a microplate reader.

[0182] Figure 9 The toxic effects of PBS, RsEVs, RsEVs-NC-siYTHDF1, RsEVs-siYTHDF1 and PGRsEVs-siYTHDF1 on MGC-803 and HGC-27 gastric cancer cells are shown in Example 9.

[0183] Figure 9The results showed that RsEVs and RsEVs-NC-siYTHDF1 groups had no obvious toxic side effects on both MGC-803 and HGC-27 gastric cancer cells, while the RsEVs-siYTHDF1 nanoparticles could well inhibit the proliferation of the two gastric cancer cells, and the inhibition of PGRsEVs-siYTHDF1 nanoparticles on tumor cell proliferation was further enhanced. The siYTHDF1 target sequence used in this example is 5'-GGAACAACATCTATCAGCA-3', purchased from Guangzhou Ribo Biotechnology Co., Ltd.

[0184] Example 10

[0185] This example is used to test the nuclear magnetic imaging (MRI) of the tumor site of the MGC-803 tumor cell-bearing mice after intravenous injection of PGRsEVs-siYTHDF1 nanoparticles in the tail vein of the mice, at 0h, 6h, 12h, 24h, 48h and 72h after injection.

[0186] In order to detect the MR imaging of PGRsEVs-siYTHDF1 nanoparticles in mice in vivo, the tumor-bearing mice were intravenously injected with PGRsEVs-siYTHDF1 nanoparticles (Gd was 0.04 mmol / kg), and MR images were collected at different time points (0h, 6h, 12h, 24h, 48h and 72h) after injection using a 7.0T small animal nuclear magnetic resonance imaging instrument (BioSpec 70 / 20USR, Bruker, Germany).

[0187] Figure 10 The nuclear magnetic imaging (MRI) of the tumor site of the MGC-803 tumor cell-bearing mice after intravenous injection of PGRsEVs-siYTHDF1 nanoparticles in the tail vein of the mice, at 0h, 6h, 12h, 24h, 48h and 72h after injection is shown in Example 10.

[0188] Figure 10The results show the effect of PGRsEV-siYTHDF1 nanoparticles on magnetic resonance imaging (MRI) at different time points (0 h, 6 h, 12 h, 24 h, 48 h and 72 h) after intravenous injection. Among them, the MR image of T1 shows that the tumor outline is obvious, the boundary is clear, and the magnetic resonance imaging contrast is good. After injection of PGRsEVs-siYTHDF1 nanoparticles, the MRI signal value of PGRsEVs-siYTHDF1 at the tumor site gradually increased, reached a maximum at 24 h, and then slowly weakened, and at 72 h, it could still be detected at the tumor site, indicating that PGRsEVs-siYTHDF1 nanoparticles had good magnetic resonance imaging characteristics and tumor targeting effect. The siYTHDF1 target sequence used in this embodiment is 5'-GGAACAACATCTATCAGCA-3', purchased from Guangzhou Ribobio Biotechnology Co., Ltd.

[0189] Example 11

[0190] This embodiment is used to test the change in tumor volume of mice administered with physiological saline, RsEVs, RsVEs-NC-siYTHDF1, RsEVs-siYTHDF1 and PGRsEVs-siYTHDF1 nanoparticles via tail vein injection of MGC-803 tumor cells.

[0191] To study the anti-tumor effect of PGRsEVs-siYTHDF1, we first established a subcutaneous cell line xenotransplantation model on Balb / c nude mice using MGC-803 cells and randomly divided them into 5 groups (six in each group). Each group of mice was injected with physiological saline (Control), RsEVs, RsEVs-NC-siYTHDF1, RsEVs-siYTHDF1 and PGRsEVs-siYTHDF1 (the amount of extracellular vesicles injected in each group was 1 x 10 11 μL, 120 μL; the amount of siYTHDF1 used was 3 μg) via the tail vein. Drug treatment was performed every other day, and the long diameter (a) and short diameter (b) of the tumors of the nude mice in each group were measured using a vernier caliper, and the tumor volume was calculated. The volume calculation formula: V = ab 2 / 2. The siYTHDF1 target sequence used in this embodiment is 5'-GGAACAACATCTATCAGCA-3', purchased from Guangzhou Ribobio Biotechnology Co., Ltd.

[0192] Figure 11 A graph showing the change in tumor volume of mice administered with physiological saline, RsEVs, RsVEs-NC-siYTHDF1, RsEVs-siYTHDF1 and PGRsEVs-siYTHDF1 nanoparticles via tail vein injection of MGC-803 tumor cells in Example 11 is shown.

[0193] Figure 11 The results show that the tumor volume inhibition effect of PGRsEVs-siYTHDF1 is the most obvious compared with other treatment groups, and the tumor picture size and weight are significantly lower than other groups after 28 days of treatment, which proves the treatment effect of the extracellular vesicle nucleic acid nanodrug delivery system on gastric cancer.

[0194] Although the present application has been described to a certain extent, it is obvious that appropriate changes can be made to individual conditions without departing from the spirit and scope of the present application. It is understood that the present application is not limited to the described embodiments, but is subject to the scope of the claims, which includes equivalent substitutions of each factor described. SEQUENCE LISTING <110> National Center for Nanoscience <120> Extracellular vesicle nucleic acid nanodrug delivery system, its preparation method and application <160> 3 <170> SIPOSequenceListing 1.0 <210> 1 <211> 19 <212> DNA / RNA <213> Artificial Sequence <400> 1 ggaacaacat ctatcagca 19 <210> 2 <211> 19 <212> DNA / RNA <213> Artificial Sequence <400> 2 ggaaacgtcc agcctaatt 19 <210> 3 <211> 19 <212> DNA / RNA <213> Artificial Sequence <400> 3 gctcaaccgc agtatcaga 19

Claims

1. An extracellular vesicle nucleic acid nanomedicine delivery system, characterized in that, The extracellular vesicle nucleic acid nano-drug delivery system comprises an extracellular vesicle and siRNA; wherein the siRNA is loaded into the extracellular vesicle modified by a tumor-targeting polypeptide and a nuclear magnetic contrast agent; wherein: The extracellular vesicle surface highly expresses CD47 protein; The source of the extracellular vesicle is selected from one or more cells of macrophages, CD47 high expression macrophage stable cells and induced pluripotent stem cells; The siRNA is an RNA sequence siYTHDF1 capable of knocking down YTHDF1 protein expression; And the mass ratio of the siRNA to the extracellular vesicle is 0.1-5:0.5-10; The RNA sequence siYTHDF1 is selected from one or more of siYTHDF1-01, siYTHDF1-02 and siYTHDF1-03; and the target sequence of siYTHDF1-01 is 5'-GGAACAACATCTATCAGCA-3', the target sequence of siYTHDF1-02 is 5'-GGAAACGTCCAGCCTAATT-3', and the target sequence of siYTHDF1-03 is 5'-GCTCAACCGCAGTATCAGA-3'.

2. The extracellular vesicle nucleic acid nano-drug delivery system according to claim 1, wherein: The source of the extracellular vesicle is macrophages; The particle size of the nanoparticles in the extracellular vesicle nucleic acid nano-drug delivery system is 150-250 nm; and / or The mass ratio of the siRNA to the extracellular vesicle is 0.5-1.5:0.5-4.

3. The extracellular vesicles nucleic acid nanomedicine delivery system of claim 2, wherein, The particle size of the nanoparticles in the extracellular vesicle nucleic acid nano-drug delivery system is 150-200 nm.

4. The extracellular vesicle nucleic acid nano-drug delivery system according to claim 2, wherein: The source of the extracellular vesicle is Raw264.7 cells; and / or The mass ratio of the siRNA to the extracellular vesicle is 1:

1.

5. The extracellular vesicles nucleic acid nanomedicine delivery system of claim 1, wherein, The RNA sequence siYTHDF1 is siYTHDF-01.

6. The extracellular vesicles nucleic acid nanomedicine delivery system according to any one of claims 1 to 5, wherein: The tumor-targeting polypeptide is an RGD polypeptide.

7. The extracellular vesicles nucleic acid nanomedicine delivery system of claim 6, wherein: The tumor-targeting polypeptide is a cyclic RGD polypeptide.

8. The extracellular vesicles nucleic acid nanomedicine delivery system of claim 7, wherein: The cyclic RGD polypeptide is selected from one or more of c(RGDyC), c(RGDyK), c(RGDfC) and c(RGDfK).

9. The extracellular vesicles nucleic acid nanomedicine delivery system of claim 8, wherein: The cyclic RGD polypeptide is c(RGDyC).

10. The extracellular vesicles nucleic acid nanomedicine delivery system according to any one of claims 1 to 5, wherein: The nuclear magnetic contrast agent is a nuclear magnetic contrast agent containing a nuclear magnetic imaging functional metal element.

11. The extracellular vesicles nucleic acid nanomedicine delivery system of claim 10, wherein: The nuclear magnetic contrast agent is selected from one or more of a nuclear magnetic contrast agent containing a gadolinium element, a nuclear magnetic contrast agent containing a manganese element and a nuclear magnetic contrast agent containing an iron element.

12. A method of preparing the extracellular vesicles nucleic acid nanomedicine delivery system according to any one of claims 1 to 11, characterized in that, The method comprises the following steps: (1) extracting extracellular vesicles; (2) modifying the extracellular vesicles prepared in step (1) with a tumor-targeting polypeptide and a nuclear magnetic contrast agent to obtain engineered extracellular vesicles; (3) loading siRNA into the engineered extracellular vesicles prepared in step (3) to obtain the engineered extracellular vesicle nucleic acid nano-drug delivery system.

13. The method of claim 12, wherein, The step (1) comprises the following steps: (A) preparing a culture medium without extracellular vesicles; (B) isolating extracellular vesicles.

14. The method of claim 13, wherein, The step (1) comprises the following steps: The step (A) further comprises: after centrifuging the culture medium, filtering the supernatant to obtain the culture medium without extracellular vesicles; and / or The step (B) further comprises: when the density of the source cells of the extracellular vesicles reaches 50-60%, using the culture medium without extracellular vesicles prepared in step (A) to culture and collect the supernatant, and then isolating the extracellular vesicles and storing them in a refrigerator.

15. The method of claim 14, wherein, The step (1) comprises the following steps: In the step (A), the centrifugal force is 90,000-130,000g; and / or the filter membrane diameter for filtration is 0.2-0.4μm; and / or In the step (B), the culture medium is cultured for 18-36 hours; and / or the temperature of the refrigerator is -80℃.

16. The method of claim 15, wherein, The step (1) comprises the following steps: In the step (A), the centrifugal force is 100,000-120,000g; and / or the filter membrane diameter for filtration is 0.22μm; and / or In the step (B), the culture medium is cultured for 24 hours.

17. The method of claim 13, wherein, The step (2) further comprises the following steps: (C) synthesizing DSPE-DOTA-Gd; (D) preparing engineered extracellular vesicles modified with tumor-targeting polypeptides and a nuclear magnetic contrast agent on the surface.

18. The method of claim 17, wherein: The step (C) further comprises centrifuging and purifying the obtained product; and / or The step (D) further comprises centrifuging the final product prepared, purifying and removing unreacted reagents to obtain the engineered extracellular vesicles.

19. The method of claim 12, wherein, The step (3) comprises the following steps:

20. The method of claim 19, wherein, The step (3) comprises the following steps:

21. The method of claim 20, wherein, The step (3) further comprises the following steps after electroporation: culturing and centrifuging to obtain the extracellular vesicle nucleic acid nanomedicine delivery system.

22. Use of the extracellular vesicle nucleic acid nanomedicine delivery system of any one of claims 1 to 11 or prepared according to the method of any one of claims 12 to 21 in the preparation of a medicinal product for the treatment of gastric cancer.

23. The use according to claim 22, characterized in that, The treatment of gastric cancer is in the form of epigenetic regulation. The treatment of gastric cancer is in the form of epigenetic regulation.

24. The use according to claim 23, characterized in that, The mode of the gastric cancer treatment is targeting and regulating N 6 - methyladenine nucleotide methylation epigenetic factors.

Images