CN116983283A - Preparation method of nucleic acid-loaded and tracer-engineered extracellular vesicles - Google Patents
Preparation method of nucleic acid-loaded and tracer-engineered extracellular vesicles Download PDFInfo
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- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 52
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 52
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 52
- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- 239000000700 radioactive tracer Substances 0.000 claims abstract description 45
- 239000006228 supernatant Substances 0.000 claims abstract description 30
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- 238000000034 method Methods 0.000 claims abstract description 22
- ZPWOOKQUDFIEIX-UHFFFAOYSA-N cyclooctyne Chemical compound C1CCCC#CCC1 ZPWOOKQUDFIEIX-UHFFFAOYSA-N 0.000 claims abstract description 20
- 238000004520 electroporation Methods 0.000 claims abstract description 20
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- 206010061535 Ovarian neoplasm Diseases 0.000 claims abstract description 12
- 238000000108 ultra-filtration Methods 0.000 claims abstract description 11
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- 150000001540 azides Chemical class 0.000 claims abstract description 8
- 230000007717 exclusion Effects 0.000 claims abstract description 8
- IVRMZWNICZWHMI-UHFFFAOYSA-N azide group Chemical group [N-]=[N+]=[N-] IVRMZWNICZWHMI-UHFFFAOYSA-N 0.000 claims abstract description 7
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- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 8
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- 230000004888 barrier function Effects 0.000 description 3
- AQCDIIAORKRFCD-UHFFFAOYSA-N cadmium selenide Chemical compound [Cd]=[Se] AQCDIIAORKRFCD-UHFFFAOYSA-N 0.000 description 3
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- 210000000170 cell membrane Anatomy 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 238000002591 computed tomography Methods 0.000 description 3
- -1 cyclooctyne modified gold Chemical class 0.000 description 3
- MARUHZGHZWCEQU-UHFFFAOYSA-N 5-phenyl-2h-tetrazole Chemical compound C1=CC=CC=C1C1=NNN=N1 MARUHZGHZWCEQU-UHFFFAOYSA-N 0.000 description 2
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- JWZVIRNOHHPTHQ-UHFFFAOYSA-N cyclooctyne;azide Chemical compound [N-]=[N+]=[N-].C1CCCC#CCC1 JWZVIRNOHHPTHQ-UHFFFAOYSA-N 0.000 description 1
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- WTFXARWRTYJXII-UHFFFAOYSA-N iron(2+);iron(3+);oxygen(2-) Chemical compound [O-2].[O-2].[O-2].[O-2].[Fe+2].[Fe+3].[Fe+3] WTFXARWRTYJXII-UHFFFAOYSA-N 0.000 description 1
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- 108091027963 non-coding RNA Proteins 0.000 description 1
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- 229910052723 transition metal Inorganic materials 0.000 description 1
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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Abstract
The invention provides a preparation method of an extracellular vesicle loaded with nucleic acid and tracer engineering, which adopts an azide metabolism glycoprotein marker Ac4ManNAZ to co-culture with cells, and generates azide groups on the surface of the released extracellular vesicle; obtaining extracellular vesicles from cell supernatant by ultracentrifugation, size chromatography exclusion, ultrafiltration and other methods; introducing nucleic acid drugs into extracellular vesicles by electroporation; and (3) preparing a cyclooctyne modified nano tracer and incubating the extracellular vesicles, and chemically connecting the tracer to the surfaces of the extracellular vesicles through copper-free click to obtain the functionalized extracellular vesicles for treating ovarian tumors. The preparation method loads the nucleic acid drug into the extracellular vesicles through electroporation technology and metabolic labeling technology and modifies the extracellular vesicles with the tracer, is high in efficiency, and provides a treatment scheme for ovarian epithelial tumors.
Description
Technical Field
The invention relates to a method in the field of biotechnology, in particular to a preparation method of an engineering extracellular vesicle loaded with nucleic acid and a tracer.
Background
Extracellular vesicles (Extracellular vesicles, EVs) are membrane vesicles released by most cell types and are of nanoscale size, and current research indicates that there are different types of extracellular vesicles, including exosomes, microvesicles and apoptotic bodies. Exosomes are considered the smallest vesicle type, varying in size from 40-100 nm, originating from endoluminal budding of a multivesicular body (MVB), which releases exosomes upon fusion with the plasma membrane. While microbubbles are non-uniform in size and originate from direct budding of the plasma membrane. Cells also release a heterogeneous vesicle that becomes an apoptotic body when apoptosis occurs. However, due to the overlapping of vesicle sizes and the lack of subtype-specific markers, it is currently difficult to purify the three, and all vesicle subtypes are collectively referred to as EV.
EV is a natural nucleic acid delivery vehicle, cargo including small and long RNAs, coding and non-coding RNAs. And thus is a medium for information exchange between cells. Cells can package nucleic acids into extracellular vesicles by endogenous sorting mechanisms and release EVs either constitutively or upon stimulation. And may then be internalized by the target cell, resulting in the transfer of mRNA and miRNA, thereby resulting in the production or silencing of the target protein. The use of EV as a novel nucleic acid drug delivery platform has therefore attracted considerable interest to researchers. EV has demonstrated many characteristics of ideal drug carriers, for example. EV has inherent cell targeting property and can overcome natural barriers such as blood brain barrier, and the like, the goods are not easily degraded naturally in the circulation process, and the EV has almost no immunogenicity, and clinical experiments prove that the EV is safe in human body.
Despite the tremendous promise of electric vehicles in nucleic acid drug delivery, clinical transformation of EVs requires the definition and optimization of their in vivo biodistribution, an important determinant of their therapeutic efficacy, as drugs can be delivered more specifically to the tissues of interest. Advances in molecular imaging technology have enabled high resolution tracking of EVs in vivo, providing valuable information for transportation, biodistribution, cellular uptake, and molecular mechanisms of electric vehicles. Tomographic techniques are efficient, noninvasive imaging techniques with excellent potential for clinical transformations. Common tomographic techniques are Computed Tomography (CT) and Magnetic Resonance Imaging (MRI). Gold nanoparticles and superparamagnetic iron oxide nanoparticles are common contrast agents for CT and MRI, respectively. The living body fluorescence imaging technology can perform noninvasive quantitative tracing, the detection time is faster, and the substrate injection is not needed. The quantum dots are nanocrystalline or semiconductor nanocrystalline formed by wrapping a layer of ZnS or CdS on cores of cadmium selenide (CdSe), cadmium telluride (CdTe), cadmium sulfide (CdS) and the like. The quantum dots have high fluorescence intensity, good stability and light bleaching resistance, and the luminous color is regulated by changing the particle size of the quantum dots or the composition of the nanocrystals.
The copper-free click reaction can be performed at a lower active energy barrier and without a cytotoxic transition metal catalyst. And thus are well suited for use in biomarkers. According to the invention, an azide group is firstly introduced into a cytoplasmic membrane by adopting a glycometabolism labeling method and is transferred to EV (electric vehicle) through an EV secretion mechanism, and simultaneously, cyclooctyne modification is carried out on a tracer (gold nanoparticle, superparamagnetic ferroferric oxide and fluorescent quantum dot). The tracer is covalently linked to the extracellular vesicles by a 1,3 dipolar cycloaddition reaction of cyclooctyne with azide. In addition, the drug loading uses electroporation technology to load specific nucleic acid drugs (ASO, siRNA, miRNA, mRNA) into the EV, and the obtained engineering EV can be used for the treatment of ovarian tumor and EV tracing.
Disclosure of Invention
The invention aims to provide a preparation method of a nucleic acid-loaded and tracer-engineered extracellular vesicle.
It is a further object of the present invention to provide such nucleic acid-loaded and tracer-engineered extracellular vesicle products.
It is a further object of the present invention to provide the use of the loaded nucleic acid and tracer-engineered extracellular vesicle products.
The invention aims at realizing the following scheme: preparation method and application of load nucleic acid and tracer engineering extracellular vesicles, and azide metabolism glycoprotein marker Ac is adopted 4 Co-culturing ManNAZ and cells to generate azide groups on the released EV surface; harvesting extracellular vesicles from the cell supernatant and introducing nucleic acid drugs into the extracellular vesicles by electroporation; preparing a cyclooctyne modified nano tracer and carrying out co-incubation on EV, chemically connecting the tracer to the surface of EV through copper-free click to obtain a functionalized extracellular vesicle for treating ovarian tumor, comprising the following steps of:
(1) Cell culture and glycometabolism markers: ovarian tumor cell lines were fully cultured with 1640 based on 5% CO at 37% 2 Is cultured under the condition of (1) until the logarithmic phase, and is replaced by the culture medium containing 25-50 mu M glycoprotein marker Ac 4 ManNAZ EV-free conditioned medium was cultured 48 h, and cell supernatants were collected.
(2) Pretreatment of cell supernatant: the collected cell supernatants were sequentially subjected to differential centrifugation of 500 g 10 min,2000 g 20 min,15000g 30 min to remove cell debris and large vesicles from the cell supernatants, followed by filtration through a 0.22 μm sterile filter.
(3) Extraction of EV: the EV in the supernatant was collected by ultracentrifugation, size exclusion chromatography, ultrafiltration, etc. Particle count, morphology and marker proteins of EV were detected by NTA, TEM and Western Bolt.
(4) Loading of nucleic acid drug: will be about 4-16 x 10 11 The EV was diluted to 200. Mu.L with dilution, one set per 100. Mu.L, mixed with nucleic acid, and the mixture was transferred to an electroporation cuvette for electroporation using the appropriate procedure.
(5) Preparation of the nano tracer: 1-30 nm nanometer tracing nanometer materials (such as gold nanometer particles for subsequent CT imaging, fluorescent quantum dots for fluorescent imaging, superparamagnetic ferroferric oxide nanometer particles for MRI imaging and the like) are prepared and modified by PEGylated cyclooctyne.
(6) Connection of nanotracers to EV: the azido group modified EV was mixed with cyclooctyne modified nanotracers, incubated at 37 ℃ for 2 h, and the mixture was passed through a size exclusion column and a 50K ultrafiltration tube to remove free nanotracers.
Application of engineering EV: will be 5X 10 11 The individual engineered EVs were intraperitoneally injected in the rat epithelial ovarian cancer in situ model 2 times per week. Tumor volume changes were recorded daily, tissue distribution of engineered EVs was analyzed using CT imaging, MRI imaging, or fluorescence imaging, and tumor tissues were harvested for treatment mechanism analysis.
Wherein the tracer is selected from gold nanoparticles, fluorescent quantum dots and superparamagnetic ferroferric oxide inorganic nano materials.
Preferably, the particle size of the tracer is between 1 and 30 and nm.
Preferably, the attachment of the nanotracers to extracellular vesicles is copper-free click chemistry between azide-cyclooctyne.
The blast cells of the extracellular vesicles are human or mouse ovarian tumor cells.
Preferably, the extraction method of extracellular vesicles is ultracentrifugation, size exclusion chromatography, ultrafiltration.
The nucleic acid drug comprises antisense oligonucleotide (ASO), small interfering RNA (siRNA), micro RNA (miRNA), messenger RNA (mRNA) and the like.
Preferably, the method for exosome loading with nucleic acid drug is electroporation.
The invention also provides a product loaded with the nucleic acid and the tracer-engineered extracellular vesicles.
The invention also provides application of the product loaded with the nucleic acid and the tracer-engineered extracellular vesicles in a tracing technology, including CT imaging, MRI imaging and fluorescence imaging.
The invention has the advantages of combining the delivery of nucleic acid medicine with EV tracing, having relatively simple operation and low cost, providing a treatment scheme for ovarian epithelial tumors and providing an effective research tool for the in vivo distribution of EV.
The extracellular vesicle is a natural nucleic acid carrier, is stable in blood circulation, can overcome natural barriers in vivo and has cell targeting capability, so that the extracellular vesicle has obvious advantages as a carrier of nucleic acid medicaments. The preparation method loads the nucleic acid drug into the extracellular vesicles through electroporation technology and metabolic labeling technology and modifies the extracellular vesicles with the tracer, is high in efficiency, and provides a treatment scheme for ovarian epithelial tumors.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The following examples are given with the invention as defined by the detailed description and the specific operation, but the scope of the invention is not limited to the following examples.
Example 1
An extracellular vesicle loaded with nucleic acid and tracer engineering and prepared from azide metabolism glycoprotein marker Ac 4 Co-culturing ManNAZ and cells to generate azide groups on the surface of the released extracellular vesicles; collecting extracellular vesicles from the cell supernatant; introducing nucleic acid drugs into extracellular vesicles by electroporation; the preparation method comprises the steps of preparing a cyclooctyne modified nano tracer agent and incubating the extracellular vesicles, connecting the tracer agent to the surfaces of the extracellular vesicles to obtain functionalized extracellular vesicles for ovarian tumor treatment, and preparing the functional extracellular vesicles according to the following steps:
(1) Thin and fineCell culture and glycometabolism markers: SKOV-3 ovarian tumor cell line was completely cultured with 1640 based on 5% CO at 37 ℃ 2 Is cultured under the condition of (1) until the logarithmic phase, and is replaced by the culture medium containing 50 mu M glycoprotein marker Ac 4 Culturing 48 h on ManNAZ Extracellular Vesicle (EV) -free conditioned medium, and collecting cell supernatant;
(2) Pretreatment of cell supernatant: sequentially removing cell fragments and large vesicles in the collected cell supernatant by 500 g 10 min,2000 g 20 min,15000g 30 min differential centrifugation, and then filtering by a sterile filter membrane of 0.22 mu m;
(3) Extraction of extracellular vesicles: collecting the supernatant by ultracentrifugation to collect azide-modified extracellular vesicles from the supernatant; particle number, morphology and marker proteins of EV were detected by NTA, TEM and Western Bolt;
(4) Loading of nucleic acid drug: will be about 4-16 x 10 11 The EV was diluted to 200. Mu.L with a dilution, and each 100. Mu.L was mixed with PLK1 small interfering RNA, and the mixture was transferred to an electroporation cuvette for electroporation using a suitable procedure;
(5) Preparation of the nano tracer: preparing gold nanoparticles of 15 nm by a citric acid reduction method, and performing cyclooctyne modification on the gold nanoparticles by using SH-PEG-BCN to obtain cyclooctyne modified gold nanoparticles;
(6) Attachment of nanotracers to extracellular vesicles: mixing the azido group modified EV and cyclooctyne modified nano tracer, incubating at 37 ℃ for 2 h, and removing the free nano tracer by the mixture through a size chromatographic exclusion column and a 50K ultrafiltration tube to obtain the loaded nucleic acid and the tracer engineering extracellular vesicles.
Will be 5X 10 11 The individual engineered EVs were intraperitoneally injected in the rat epithelial ovarian cancer in situ model 2 times per week. Tumor volume changes were recorded daily, tissue distribution of the engineered EV was determined using CT imaging, and tumor tissue was harvested for treatment mechanism analysis.
Example 2
An engineered extracellular vesicle loaded with nucleic acid and tracer, prepared by the steps of:
(1) Cell culture and glycometabolism markers: caov-3 ovarian tumor cell lineComplete culture with 1640 based on 37℃and 5% CO 2 Is cultured under the condition of (1) until the logarithmic phase, and is replaced by the culture medium containing 25-50 mu M glycoprotein marker Ac 4 Culturing 48 h on ManNAZ Extracellular Vesicle (EV) -free conditioned medium, and collecting cell supernatant;
(2) Pretreatment of cell supernatant: sequentially removing cell fragments and large vesicles in the collected cell supernatant by 500 g 10 min,2000 g 20 min,15000g 30 min differential centrifugation, and then filtering by a sterile filter membrane of 0.22 mu m;
(3) Extraction of extracellular vesicles: collecting the azide group-modified EV in the supernatant by an ultracentrifugation method; particle number, morphology and marker proteins of EV were detected by NTA, TEM and Western Bolt;
(4) Loading of nucleic acid drug: will be about 4-16 x 10 11 Individual extracellular vesicles were diluted to 200 μl with a dilution, each 100 μl being a set of small interfering RNAs mixed with elF3c, and the mixture was transferred to an electroporation cuvette for electroporation using appropriate procedures;
(5) Preparation of the nano tracer: preparation of 5 nm carboxylated superparamagnetic ferroferric oxide by precipitation method using NH 2 Performing cyclooctyne modification on the superparamagnetic ferroferric oxide particles by PEG-BCN to obtain a cyclooctyne modified nano tracer;
(6) Attachment of nanotracers to extracellular vesicles: mixing the azido group modified EV with cyclooctyne modified superparamagnetic ferroferric oxide particles, incubating at 37 ℃ for 2 h, and removing free superparamagnetic ferroferric oxide from the mixture through a size chromatographic exclusion column and a 50K ultrafiltration tube to obtain the load nucleic acid and tracer-engineered extracellular vesicles.
Will be 5X 10 11 The individual engineered EVs were intraperitoneally injected in the rat epithelial ovarian cancer in situ model 2 times per week. Tumor volume changes were recorded daily, tissue distribution of the engineered EV was determined using MRI imaging, and tumor tissue was harvested for treatment mechanism analysis.
Example 3
An engineered extracellular vesicle loaded with nucleic acid and tracer, prepared by the steps of:
(1) Cell culture and glycometabolism markers: PA-1 ovarian tumorCell line was completely cultured with 1640 based on 37℃and 5% CO 2 Is cultured under the condition of (1) until the logarithmic phase, and is replaced by the culture medium containing 25-50 mu M glycoprotein marker Ac 4 Culturing 48 h on ManNAZ Extracellular Vesicle (EV) -free conditioned medium, and collecting cell supernatant;
(2) Pretreatment of cell supernatant: sequentially removing cell fragments and large vesicles in the collected cell supernatant by 500 g 10 min,2000 g 20 min,15000g 30 min differential centrifugation, and then filtering by a sterile filter membrane of 0.22 mu m;
(3) Extraction of extracellular vesicles: collecting the azide group-modified EV in the supernatant by an ultracentrifugation method; detecting the particle number, morphology and marker proteins of extracellular vesicles by NTA, TEM and Western Bolt;
(4) Loading of nucleic acid drug: will be about 4-16 x 10 11 The individual EV was diluted to 200. Mu.L with dilution, one set per 100. Mu.L mixed with small interfering RNA of elF3c, and the mixture was transferred to an electroporation cuvette for electroporation using the appropriate procedure;
(5) Preparation of the nano tracer: carboxylated modified CdSe/ZnS quantum dots using NH 2 Performing cyclooctyne modification on PEG-BCN to obtain a cyclooctyne modified nano tracer;
(6) Attachment of nanotracers to extracellular vesicles: mixing the azido group modified EV and cyclooctyne modified CdSe/ZnS quantum dots, incubating at 37 ℃ for 2 h, and removing free quantum dots from the mixture through a size chromatographic exclusion column and a 50K ultrafiltration tube to obtain the extracellular vesicles carrying nucleic acid and tracer engineering.
Will be 5X 10 11 The individual engineered EVs were intraperitoneally injected in the rat epithelial ovarian cancer in situ model 2 times per week. Tumor volume changes were recorded daily, tissue distribution of engineered EVs was determined using in vivo fluorescence imaging of small animals, and tumor tissues were harvested for therapeutic mechanism analysis.
Example 4
An engineered extracellular vesicle loaded with nucleic acid and tracer, prepared by the steps of:
(1) Cell culture and glycometabolism markers: a2780 ovarian tumor cell line was completely cultured with 1640 based on 5% CO at 37% 2 Conditions of (2)Culturing until logarithmic phase, and changing the cell containing 25-50 μm glycoprotein marker Ac 4 Culturing 48 h on ManNAZ Extracellular Vesicle (EV) -free conditioned medium, and collecting cell supernatant;
(2) Pretreatment of cell supernatant: sequentially removing cell fragments and large vesicles in the collected cell supernatant by 500 g 10 min,2000 g 20 min,15000g 30 min differential centrifugation, and then filtering by a sterile filter membrane of 0.22 mu m;
(3) Extraction of extracellular vesicles: collecting the azide group-modified EV in the supernatant by an ultracentrifugation method; detecting the particle number, morphology and marker proteins of extracellular vesicles by NTA, TEM and Western Bolt;
(4) Loading of nucleic acid drug: will be about 4-16 x 10 11 Individual extracellular vesicles were diluted to 200 μl with a dilution, each 100 μl being a set of small interfering RNAs mixed with elF3c, and the mixture was transferred to an electroporation cuvette for electroporation using appropriate procedures;
(5) Preparation of the nano tracer: carboxylated modified CdSe/ZnS quantum dots using NH 2 Performing cyclooctyne modification on PEG-BCN to obtain a cyclooctyne modified nano tracer;
(6) Attachment of nanotracers to extracellular vesicles: mixing the azido group modified EV and cyclooctyne modified CdSe/ZnS quantum dots, incubating at 37 ℃ for 2 h, and removing free quantum dots from the mixture through a size chromatographic exclusion column and a 50K ultrafiltration tube to obtain the extracellular vesicles carrying nucleic acid and tracer engineering.
Will be 5X 10 11 The individual engineered EVs were intraperitoneally injected in the rat epithelial ovarian cancer in situ model 2 times per week. Tumor volume changes were recorded daily, tissue distribution of engineered EVs was determined using in vivo fluorescence imaging of small animals, and tumor tissues were harvested for therapeutic mechanism analysis.
Claims (10)
1. A method for preparing an extracellular vesicle engineering by loading nucleic acid and a tracer is characterized in that an azide metabolic glycoprotein marker Ac is adopted 4 Co-culturing ManNAZ and cells to generate azide groups on the surface of the released extracellular vesicles; collecting extracellular vesicles from the cell supernatant; by electroporationThe technology introduces nucleic acid drugs into extracellular vesicles; preparing a cyclooctyne modified nano tracer and incubating the nano tracer with an extracellular vesicle, connecting the tracer to the surface of the extracellular vesicle to obtain a functionalized extracellular vesicle for treating ovarian tumor, comprising the following steps of:
(1) Cell culture and glycometabolism markers: ovarian tumor cell lines were fully cultured with 1640 based on 5% CO at 37% 2 Is cultured under the condition of (1) until the logarithmic phase, and is replaced by the culture medium containing 25-50 mu M glycoprotein marker Ac 4 Culturing the ManNAZ extracellular vesicle-free conditioned medium for 48 h, and collecting cell supernatant;
(2) Pretreatment of cell supernatant: sequentially removing cell fragments and large vesicles in the collected cell supernatant by 500 g 10 min,2000 g 20 min,15000g 30 min differential centrifugation, and then filtering by a sterile filter membrane of 0.22 mu m;
(3) Extraction of extracellular vesicles: collecting the azide group modified extracellular vesicles in the supernatant by an ultracentrifugation, size chromatography exclusion and ultrafiltration method; detecting the particle number, morphology and marker proteins of extracellular vesicles by NTA, TEM and Western Bolt;
(4) Loading of nucleic acid drug: will be 4-16×10 11 The extracellular vesicles were diluted to 200. Mu.L with a dilution, each 100. Mu.L of the extracellular vesicles were mixed with nucleic acid, and the mixture was transferred to an electroporation cuvette for electroporation;
(5) Preparation of the nano tracer: preparing a nano-tracer nanomaterial of 1-30 nm, wherein the nano-tracer nanomaterial comprises gold nanoparticles for subsequent CT imaging, fluorescent quantum dots for fluorescent imaging, or superparamagnetic ferroferric oxide nanoparticles for MRI imaging, and modifying the gold nanoparticles with polyethylene glycol cyclooctyne to obtain a cyclooctyne modified nano-tracer;
(6) Attachment of nanotracers to extracellular vesicles: mixing the extracellular vesicles modified by the azide groups with a cyclooctyne modified nano tracer, incubating at 37 ℃ for 2 h, and removing the free nano tracer from the mixture through a size chromatographic exclusion column and a 50K ultrafiltration tube to obtain the nucleic acid-loaded extracellular vesicles and the tracer-engineered extracellular vesicles.
2. The method for preparing the nucleic acid-loaded and tracer-engineered extracellular vesicles according to claim 1, wherein the particle size of the tracer is between 1 and 30 and nm.
3. The method for preparing the extracellular vesicles loaded with nucleic acid and tracer engineering according to claim 1, wherein the connection mode of the nano tracer and the extracellular vesicles is copper-free click chemistry between azido-cyclooctyne.
4. The method of claim 1, wherein the extracellular vesicle-carrying parent cell is a human or mouse ovarian tumor cell.
5. The method for preparing the nucleic acid-loaded and tracer-engineered extracellular vesicles according to claim 1, wherein the extraction method of the extracellular vesicles is ultracentrifugation, size exclusion chromatography or ultrafiltration.
6. The method of claim 1, wherein the nucleic acid drug comprises antisense oligonucleotide (ASO), small interfering RNA (siRNA), microrna (miRNA), messenger RNA (mRNA).
7. The method for preparing the nucleic acid-loaded and tracer-engineered extracellular vesicles and the application thereof according to claim 1, wherein the method for loading the nucleic acid drug on exosomes is electroporation.
8. A nucleic acid-loaded and tracer-engineered extracellular vesicle obtainable by the method of any one of claims 1 to 7.
9. Use of the loaded nucleic acid and tracer-engineered extracellular vesicles of claim 8 in CT imaging, MRI imaging, fluorescence imaging.
10. The use according to claim 9, characterized in that 5 x 10 11 The individual engineered extracellular vesicles were intraperitoneally injected into the rat epithelial ovarian cancer in situ model, tumor volume changes were recorded 2 times per week, tissue distribution of the engineered extracellular vesicles was analyzed using CT imaging, MRI imaging, or fluorescence imaging, and tumor tissue was harvested for therapeutic mechanism analysis.
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