CN117530932B - Artificial exosome for inhibiting tumor growth and lung metastasis and application thereof - Google Patents
Artificial exosome for inhibiting tumor growth and lung metastasis and application thereof Download PDFInfo
- Publication number
- CN117530932B CN117530932B CN202410020375.8A CN202410020375A CN117530932B CN 117530932 B CN117530932 B CN 117530932B CN 202410020375 A CN202410020375 A CN 202410020375A CN 117530932 B CN117530932 B CN 117530932B
- Authority
- CN
- China
- Prior art keywords
- mirna
- anta
- exosome
- antagonist
- exo
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 210000001808 exosome Anatomy 0.000 title claims abstract description 152
- 206010027476 Metastases Diseases 0.000 title claims abstract description 48
- 230000009401 metastasis Effects 0.000 title claims abstract description 47
- 210000004072 lung Anatomy 0.000 title claims abstract description 18
- 230000002401 inhibitory effect Effects 0.000 title abstract description 18
- 230000004614 tumor growth Effects 0.000 title abstract description 4
- 239000002105 nanoparticle Substances 0.000 claims abstract description 116
- 239000005557 antagonist Substances 0.000 claims abstract description 53
- 239000012528 membrane Substances 0.000 claims abstract description 53
- 238000002360 preparation method Methods 0.000 claims abstract description 47
- 201000008968 osteosarcoma Diseases 0.000 claims abstract description 43
- 206010006187 Breast cancer Diseases 0.000 claims abstract description 17
- 208000026310 Breast neoplasm Diseases 0.000 claims abstract description 17
- 125000002091 cationic group Chemical group 0.000 claims abstract description 6
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims abstract description 4
- 229940098773 bovine serum albumin Drugs 0.000 claims abstract description 4
- 238000002156 mixing Methods 0.000 claims abstract description 4
- 201000010893 malignant breast melanoma Diseases 0.000 claims abstract description 3
- 230000004048 modification Effects 0.000 claims description 31
- 238000012986 modification Methods 0.000 claims description 31
- 206010028980 Neoplasm Diseases 0.000 claims description 22
- 125000000446 sulfanediyl group Chemical group *S* 0.000 claims description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
- 102000006382 Ribonucleases Human genes 0.000 claims description 11
- 108010083644 Ribonucleases Proteins 0.000 claims description 11
- 230000011987 methylation Effects 0.000 claims description 10
- 238000007069 methylation reaction Methods 0.000 claims description 10
- 108020004707 nucleic acids Proteins 0.000 claims description 10
- 102000039446 nucleic acids Human genes 0.000 claims description 10
- 150000007523 nucleic acids Chemical class 0.000 claims description 10
- 239000011248 coating agent Substances 0.000 claims description 8
- 238000000576 coating method Methods 0.000 claims description 8
- 239000003814 drug Substances 0.000 claims description 5
- 238000000034 method Methods 0.000 claims description 4
- 230000008878 coupling Effects 0.000 claims description 2
- 238000010168 coupling process Methods 0.000 claims description 2
- 238000005859 coupling reaction Methods 0.000 claims description 2
- 108010071390 Serum Albumin Proteins 0.000 claims 2
- 102000007562 Serum Albumin Human genes 0.000 claims 2
- 238000004519 manufacturing process Methods 0.000 claims 1
- 239000002679 microRNA Substances 0.000 abstract description 83
- 210000004881 tumor cell Anatomy 0.000 abstract description 5
- 206010061289 metastatic neoplasm Diseases 0.000 abstract description 4
- 239000002502 liposome Substances 0.000 abstract 1
- 230000035755 proliferation Effects 0.000 abstract 1
- 238000005119 centrifugation Methods 0.000 description 37
- 239000006228 supernatant Substances 0.000 description 35
- 210000004027 cell Anatomy 0.000 description 25
- 239000000243 solution Substances 0.000 description 21
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 18
- 229930006000 Sucrose Natural products 0.000 description 18
- 239000000872 buffer Substances 0.000 description 18
- 239000000203 mixture Substances 0.000 description 18
- 239000005720 sucrose Substances 0.000 description 18
- 230000005764 inhibitory process Effects 0.000 description 13
- 201000001441 melanoma Diseases 0.000 description 13
- 230000000694 effects Effects 0.000 description 12
- 108091070501 miRNA Proteins 0.000 description 10
- 241000699670 Mus sp. Species 0.000 description 9
- 238000001085 differential centrifugation Methods 0.000 description 9
- 238000011534 incubation Methods 0.000 description 9
- 238000002955 isolation Methods 0.000 description 9
- 239000002245 particle Substances 0.000 description 9
- 239000008188 pellet Substances 0.000 description 9
- 229920006289 polycarbonate film Polymers 0.000 description 9
- 239000007983 Tris buffer Substances 0.000 description 8
- 238000001125 extrusion Methods 0.000 description 8
- 238000011065 in-situ storage Methods 0.000 description 8
- 229910052749 magnesium Inorganic materials 0.000 description 8
- 239000011777 magnesium Substances 0.000 description 8
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 8
- 230000014509 gene expression Effects 0.000 description 7
- 238000002347 injection Methods 0.000 description 6
- 239000007924 injection Substances 0.000 description 6
- 201000011510 cancer Diseases 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- 230000007423 decrease Effects 0.000 description 4
- 238000004442 gravimetric analysis Methods 0.000 description 4
- 230000012010 growth Effects 0.000 description 4
- 108091080253 miR-194-1 stem-loop Proteins 0.000 description 4
- 108091089177 miR-194-2 stem-loop Proteins 0.000 description 4
- 238000010186 staining Methods 0.000 description 4
- 238000012546 transfer Methods 0.000 description 4
- 210000003462 vein Anatomy 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 108091088730 miR-215 stem-loop Proteins 0.000 description 3
- 208000018084 Bone neoplasm Diseases 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- KPKZJLCSROULON-QKGLWVMZSA-N Phalloidin Chemical compound N1C(=O)[C@@H]([C@@H](O)C)NC(=O)[C@H](C)NC(=O)[C@H](C[C@@](C)(O)CO)NC(=O)[C@H](C2)NC(=O)[C@H](C)NC(=O)[C@@H]3C[C@H](O)CN3C(=O)[C@@H]1CSC1=C2C2=CC=CC=C2N1 KPKZJLCSROULON-QKGLWVMZSA-N 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000000090 biomarker Substances 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 239000008187 granular material Substances 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 210000002303 tibia Anatomy 0.000 description 2
- NIDNOXCRFUCAKQ-UMRXKNAASA-N (1s,2r,3s,4r)-bicyclo[2.2.1]hept-5-ene-2,3-dicarboxylic acid Chemical compound C1[C@H]2C=C[C@@H]1[C@H](C(=O)O)[C@@H]2C(O)=O NIDNOXCRFUCAKQ-UMRXKNAASA-N 0.000 description 1
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 206010061309 Neoplasm progression Diseases 0.000 description 1
- 208000037273 Pathologic Processes Diseases 0.000 description 1
- 108010009711 Phalloidine Proteins 0.000 description 1
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 1
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 1
- 230000033115 angiogenesis Effects 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 230000005907 cancer growth Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 230000008094 contradictory effect Effects 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 208000035250 cutaneous malignant susceptibility to 1 melanoma Diseases 0.000 description 1
- 210000004292 cytoskeleton Anatomy 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 239000003596 drug target Substances 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 230000013931 endocrine signaling Effects 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000035992 intercellular communication Effects 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L magnesium chloride Substances [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 210000005075 mammary gland Anatomy 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 210000002901 mesenchymal stem cell Anatomy 0.000 description 1
- 230000014306 paracrine signaling Effects 0.000 description 1
- 230000009054 pathological process Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 230000002685 pulmonary effect Effects 0.000 description 1
- 230000022532 regulation of transcription, DNA-dependent Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000007634 remodeling Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000004627 transmission electron microscopy Methods 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- 230000005751 tumor progression Effects 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/5176—Compounds of unknown constitution, e.g. material from plants or animals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
- A61K47/643—Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/04—Antineoplastic agents specific for metastasis
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Epidemiology (AREA)
- Molecular Biology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Optics & Photonics (AREA)
- Botany (AREA)
- Zoology (AREA)
- Physics & Mathematics (AREA)
- Biomedical Technology (AREA)
- Nanotechnology (AREA)
- Oncology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses an artificial exosome for inhibiting tumor growth and lung metastasis and application thereof. An artificial exosome for use in the treatment of metastatic tumors comprising a cationic bovine serum albumin-coupled miRNA-194-5p/215-5p cluster antagonist and an exosome membrane. The invention also provides a preparation method of the artificial exosome, which comprises the following steps: (1) isolating tumor cell exosomes; (2) preparing an exosome membrane; (3) preparing CBSA-miRNA cluster antagonist nanoparticles; (4) And mixing and incubating the exosome membrane and the CBSA-miRNA cluster antagonist nano-particles, extruding the membrane by using a liposome extruder to form an artificial exosome, and obtaining the artificial exosome containing the miRNA-194-5p/215-5p cluster antagonist. The artificial exosomes can effectively inhibit proliferation and metastasis of osteosarcoma, breast cancer and melanoma.
Description
Technical Field
The invention relates to the field of biological medicine, in particular to an artificial exosome for inhibiting tumor growth and lung metastasis and application thereof.
Background
The tumor is an important factor threatening human health, and most of conventional antitumor drugs can only be aimed at in-situ tumors, and have limited effect on metastatic tumors, so that it is necessary to develop drugs capable of effectively inhibiting tumor metastasis. In addition to the inherent characteristics of cancer cells, interactions between the lung microenvironment and cancer cells have been found to be involved in the formation and development of lung lesions. Cancer cells can disrupt the lung remodeling process, initiate a malignant cycle, and support the development of disseminated tumor cells in the lung. Notably, the pulmonary microenvironment also reprograms tumor cells, imparting them characteristics different from the primary cancer, promoting secondary metastasis. These breakthroughs help elucidate the cancer lung metastasis cascade and alter the patient's treatment.
The exosome is an extracellular vesicle secreted by eukaryotic cells and having a particle size of 50-150 nm. For osteosarcoma, exosomes are promising biomarkers for early diagnosis and prognosis. Furthermore, during tumor progression, exosomes regulate cell growth, metastasis and angiogenesis by transferring bioactive cargo (including lipids, proteins and regulatory RNAs). Exosome miRNA-769-5p derived from bone mesenchymal stem cells promotes osteosarcoma metastasis by targeting bispecific phosphatase 16 (DUSP 16). In addition, decreasing miRNA-135a in osteosarcoma can also inhibit osteosarcoma progression and lung metastasis. However, studies report that single miRNA effects are ambiguous and contradictory, making them unavailable as effective therapeutic targets. A group of adjacent miRNA genes located on chromosome generate miRNA clusters; tissue clustered mirnas may share the same transcriptional regulation pattern, so they may be more reliable biomarkers and drug targets.
Exosomes serve as intercellular communication, and cargo molecules involved in physiological and pathological processes are transferred from parent cells to recipient cells. This intercellular trafficking may be directed to neighboring cells, as a form of paracrine signaling, and/or distant cells, as an endocrine signaling. This property gives the exosomes great potential as targeted drug delivery vehicles.
Disclosure of Invention
The invention aims to provide an application of an artificial exosome for inhibiting tumor lung metastasis miRNA cluster expression in treatment and medicine of metastatic tumors.
The invention also aims to provide a preparation method of the artificial exosome, which is simple, easy to operate and low in cost.
The invention also provides application of the exosome.
The invention adopts the following technical scheme:
An artificial exosome for treating metastatic tumors, wherein the artificial exosome is obtained by coating miRNA-194-5p/miRNA-215-5p cluster antagonist nano-particles with exosome membranes; the miRNA-194-5p/miRNA-215-5p cluster antagonist nano-particles are nano-particles containing an antagonist aiming at miRNA-194-5p and an antagonist aiming at miRNA-215-5 p; the sequence of miRNA-194-5p is shown as SEQ ID NO.1, and the sequence of miRNA-215-5p is shown as SEQ ID NO. 2.
As a preferred aspect of the present invention, the miRNA-194-5p/miRNA-215-5p cluster antagonist nanoparticle is obtained by coupling a Cationic Bovine Serum Albumin (CBSA) to an antagonist against miRNA-194-5p and an antagonist against miRNA-215-5 p.
As a preferred aspect of the present invention, the antagonist against miRNA-194-5p is a nucleic acid obtained by methylation modification of the entire 5'-UCCACAUGGAGUUGCUGUUACA-3' sequence, thio modification of 2 bases at the 5 '-end and thio modification of 4 bases at the 3' -end; the antagonist against miRNA-215-5p is nucleic acid obtained by methylation modification of 5'-GUCUGUCAAUUCAUAGGUCAU-3' sequence full chain, thio modification of 2 bases at the 5 'end and thio modification of 4 bases at the 3' end.
As one preferable aspect of the invention, the mass ratio of CBSA to miRNA-194-5p to miRNA-215-5p is 45-25:1.
As one preferable aspect of the invention, the miRNA-194-5p and miRNA-215-5p cluster nanoparticle is prepared by the following method: dissolving anta-miRNA-194-5p and anta-miRNA-215-5p in RNase-free water, mixing with CBSA solution according to a proportion, and incubating for 20-40 minutes at 0-4 ℃.
The preparation method of the artificial exosome comprises the following steps:
a) Preparing an exosome membrane;
b) Preparing miRNA-194-5p/miRNA-215-5p cluster antagonist nano-particles;
c) Coating miRNA-194-5p/miRNA-215-5p cluster antagonist nano particles by using an exosome membrane to form the artificial exosome.
As one preferable mode of the invention, the preparation method of the miRNA-194-5p/miRNA-215-5p cluster antagonist nano-particle comprises the following steps: antagonists for miR-194-5p and miR-215-5p are dissolved in RNase-free water, mixed with CBSA solution and incubated for 20-40 minutes at 0-4 ℃.
As one preferable aspect of the invention, the mass ratio of CBSA to the antagonist against miRNA-194-5p and miRNA-215-5p is 45-25:1; the antagonist for miRNA-194-5p is nucleic acid obtained by carrying out methylation modification on 5'-UCCACAUGGAGUUGCUGUUACA-3' sequence full chain, carrying out thio modification on 2 bases at the 5 'end and carrying out thio modification on 4 bases at the 3' end; the antagonist against miRNA-215-5p is nucleic acid obtained by methylation modification of 5'-GUCUGUCAAUUCAUAGGUCAU-3' sequence full chain, thio modification of 2 bases at the 5 'end and thio modification of 4 bases at the 3' end.
The invention also provides a preparation method of the artificial exosome, which comprises the following steps:
1. isolation of tumor cell exosomes:
a. Tumor cells (e.g., HOS-LuT 3) were cultured for 48 hours.
B. culture supernatants were collected and exosomes were isolated using differential centrifugation.
C. the supernatant was collected by first centrifugation (2000 g,10 minutes).
D. the second centrifugation (10000 g,30 minutes) and the supernatant collected again.
E. third centrifugation (110000 g,70 min), the pellet was collected and resuspended in PBS.
F. Fourth centrifugation (110000 g,70 min), the exosomes were collected and resuspended in an appropriate amount of PBS.
2. Preparation of exosome membrane:
a. the collected exosomes were resuspended in ice-cold Tris-MgCl2 buffer.
B. quick-freeze at-80 ℃ and then thaw at room temperature.
C. The freeze-thawing cycle was repeated five more times.
D. the mixture was mixed with 1M sucrose to a final concentration of 0.25M, and centrifuged at 2000 g min.
E. The supernatant was collected, RNase was added, and further centrifuged at 3000g for 30 minutes to collect the exosome membrane.
F. The exosome membrane was washed with 0.25M sucrose in low temperature TM buffer and centrifuged at 3000 g for 30 min.
3. Preparation of miRNA-194-5p/miRNA-215-5p cluster antagonist nanoparticles:
a. dissolving anta-miRNA-194-5p and anta-miRNA-215-5p in a mass ratio of 1:1 by using RNase-free water.
B. The anta-miRNA-194-5p and anta-miRNA-215-5p solutions were mixed with the CBSA solution in a ratio (anta-miRNA/cbsa=1/30, w/w) such that the final CBSA concentration was 1 mg/mL, followed by incubation at 4 ℃ for 30 minutes.
4. Preparation of artificial exosome nanoparticles:
a. The miRNA-194-5p/miRNA-215-5p cluster antagonist nano particles and the exosome membrane are mixed according to the mass ratio of 1:1, and incubating for 30 minutes at 4 ℃.
B. Incubate with shaking at 37℃for 1 hour.
C. The mixture was co-extruded 20 times through a 200 nm polycarbonate film to form anta-mirnas@exo nanoparticles.
The invention relates to medical application of an artificial exosome.
The application of the artificial exosome in preparing the medicine for treating tumor and/or tumor metastasis; the tumor is preferably osteosarcoma, breast cancer or melanoma.
Advantageous effects
The invention uses the antagonist of miRNA-194-5p/miRNA-215-5p with clustered tissues wrapped by the exosome membrane to prepare the artificial exosome, fully utilizes the transfer function of the exosome membrane, and the antagonist of miRNA-194-5p/miRNA-215-5p and the exosome membrane act synergistically to obviously inhibit the growth and metastasis of tumors.
Drawings
Fig. 1 inhibits the potential of osteosarcoma transfer nanoparticles anta-mirna@exo.
Figure 2 particle size of osteosarcoma metastasis inhibiting nanoparticle anta-mirna@exo.
Fig. 3 is a transmission electron microscopy image of osteosarcoma metastasis inhibiting nanoparticle anta-mirna@exo.
Fig. 4 shows a cell delivery fluorescence profile of osteosarcoma metastasis inhibiting nanoparticles anta-mirna@exo.
The left graph shows red fluorescence labeled anta-miRNA194-5p, the left two graphs show green fluorescence labeled anta-miRNA215-5p, the left three graphs show the cytoskeleton labeled phalloidin, and the right graph shows the combination of the three fluorescence graphs.
Fig. 5 is a profile of the in vivo delivery of osteosarcoma metastasis nanoparticle anta-mirna@exo.
The upper part shows different detection time, the right ruler shows fluorescence intensity, and the fluorescence intensity gradually decreases from bottom to top on the ruler.
FIG. 6 effects of different proportions of anta-mirnas@Exo on osteosarcoma growth.
FIG. 7 inhibition of osteosarcoma metastasis nanoparticle anta-mirnas@exo inhibited osteosarcoma growth.
FIG. 8 inhibition of osteosarcoma metastasis nanoparticle anta-mirnas@exo inhibited osteosarcoma metastasis.
FIG. 9 inhibition of breast cancer metastasis nanoparticles anta-mirnas@Exo inhibited breast cancer growth.
FIG. 10 inhibition of breast cancer metastasis nanoparticles anta-mirnas@Exo inhibit breast cancer metastasis.
FIG. 11 inhibition of melanoma metastasis nanoparticles anta-mirnas@Exo inhibited melanoma growth.
FIG. 12 inhibition of melanoma metastasis nanoparticles anta-mirnas@Exo inhibited melanoma metastasis.
Detailed Description
EXAMPLE 1 preparation of nanoparticles anti-MiRNAs@Exo inhibiting osteosarcoma metastasis
The preparation method of the artificial exosome anta-miRNAs@Exo nano-particles for inhibiting the expression of osteosarcoma transfer miRNA cluster comprises the following steps:
(1) Isolation of osteosarcoma extracellular exosome (HOS-LuT 3-Exo)
The supernatant collected after 48 h of culture of osteosarcoma cells HOS-LuT3 was passaged and the exosomes were isolated by differential centrifugation. The supernatant was collected by the first centrifugation (4 ℃,2000 g,10 min) and the supernatant was collected by the second centrifugation (4 ℃,10000 g,30 min). The pellet was collected by a third centrifugation (4 ℃,110000 g,70 min), resuspended in PBS, the exosomes were collected by a fourth centrifugation (4 ℃,110000 g,70 min), and the exosomes were resuspended in an appropriate amount of PBS.
(2) Preparation of exosome membranes
The exosomes were resuspended in ice-cold Tris (0.01M) -magnesium (0.001M MgCl 2) buffer (TM), flash frozen at-80 ℃ and thawed at room temperature. The freeze-thaw cycle was repeated five more times and then mixed with 1M sucrose to a final concentration of 0.25M and centrifuged at 2000 Xg for 10 minutes. The collected supernatant was added with RNase, and the mixture was centrifuged at 3000 g for 30 minutes to collect the exosome membrane. The exosome membrane was washed with 0.25M sucrose low temperature TM buffer and centrifuged at 3000 Xg at 4℃for 30 min.
(3) Preparation of CBSA-miRNA cluster antagonist (CBSA-anta-miRNAs) nanoparticles
MiR-194-5p sequence: 5'-GCCTGTAACAGCAACTCCATGTGG-3' (SEQ ID NO. 1),
MiR-215-5p sequence: 5'-GCCGCGATGACCTATGAATTG-3' (SEQ ID NO. 2),
MiR-194-5p antagonist sequence: 5'-UCCACAUGGAGUUGCUGUUACA-3' (underlined base is subjected to thio modification), methylation modification is carried out on the whole chain of the sequence, meanwhile, 2 bases at the 5 'end are subjected to thio modification, and nucleic acid obtained by thio modification is used as miR-194-5p antagonist anta-miRNA-194-5p at the 4 bases at the 3' end.
MiR-215-5p antagonist sequence: 5'-GUCUGUCAAUUCAUAGGUCAU-3' (underlined base is thio-modified), methylation modification is carried out on the whole chain of the sequence, meanwhile, thio modification is carried out on 2 bases at the 5 'end, and nucleic acid obtained by thio modification is taken as anta-miRNA-215-5p on 4 bases at the 3' end.
Dissolving anta-miRNA-194-5p and anta-miRNA-215-5p in a mass ratio of 1:1 by using RNase-free water. The solutions of anta-miRNA-194-5p and anta-miRNA-215-5p were mixed with the CBSA solution in different ratios (anta-miRNA/cbsa=1/45, 1/40,1/35,1/30,1/25, w/w) such that the final CBSA concentration was 1 mg/mL and incubated at 4 ℃ for 30 minutes to obtain CBSA-anta-miRNAs nanoparticles containing different concentrations of miRNA cluster antagonists.
(4) Preparation of anta-miRNAs@Exo nanoparticles
The CBSA-anta-miRNAs are coated with exosome films by adopting an incubation and extrusion method to prepare the anta-miRNAs@Exo nano-particles. The CBSA-anta-miRNAs nanoparticle and exosome membrane were first mixed in a mass ratio of 1:1, incubated for 30 min at 4℃and then incubated with shaking for 1 hr at 37 ℃. The mixture was then co-extruded 20 times through a 200 nm polycarbonate film to obtain the anta-mirnas@exo nanoparticles.
Example 2 preparation of nanoparticles anti-MiRNAs@Exo inhibiting breast cancer metastasis
The preparation method of the artificial exosome anta-miRNAs@Exo nano-particles for inhibiting the expression of breast cancer metastasis miRNA clusters comprises the following steps:
(1) Isolation of breast cancer cell exosomes (MDA-MB-231-LuT 3-Exo)
The supernatant collected after 48 h of MDA-MB-231-LuT3 culture of the breast cancer cells was passaged, and exosomes were isolated by differential centrifugation. The supernatant was collected by the first centrifugation (4 ℃,2000 g,10 min) and the supernatant was collected by the second centrifugation (4 ℃,10000 g,30 min). The pellet was collected by a third centrifugation (4 ℃,110000 g,70 min), resuspended in PBS, the exosomes were collected by a fourth centrifugation (4 ℃,110000 g,70 min), and the exosomes were resuspended in an appropriate amount of PBS.
(2) Preparation of exosome membranes
The exosomes were resuspended in ice-cold Tris (0.01M) -magnesium (0.001M MgCl 2) buffer (TM), flash frozen at-80 ℃ and thawed at room temperature. The freeze-thaw cycle was repeated five more times and then mixed with 1M sucrose to a final concentration of 0.25M and centrifuged at 2000 Xg for 10 minutes. The collected supernatant was added with RNase, and the mixture was centrifuged at 3000 g for 30 minutes to collect the exosome membrane. The exosome membrane was washed with 0.25M sucrose low temperature TM buffer and centrifuged at 3000 Xg at 4℃for 30 min.
(3) Preparation of CBSA-miRNA cluster antagonist (CBSA-anta-miRNAs) nanoparticles
Dissolving anta-miRNA-194-5p and anta-miRNA-215-5p in a mass ratio of 1:1 by using RNase-free water. The anta-miRNA-194-5p and anta-miRNA-215-5p solutions were mixed with the CBSA solution in a ratio (anta-miRNA/cbsa=1/30, w/w) such that the final CBSA concentration was 1 mg/mL and incubated at 4 ℃ for 30 minutes.
(4) Preparation of anta-miRNAs@Exo nanoparticles
The CBSA-anta-miRNAs are coated with exosome films by adopting an incubation and extrusion method to prepare the anta-miRNAs@Exo nano-particles. The CBSA-anta-miRNAs nanoparticle and exosome membrane were first mixed in a mass ratio of 1:1, incubated for 30 min at 4℃and then incubated with shaking for 1 hr at 37 ℃. The mixture was then co-extruded 20 times through a 200 nm polycarbonate film to obtain the anta-mirnas@exo nanoparticles.
Example 3 preparation of nanoparticles anti-MiRNAs@Exo inhibiting melanoma metastasis
The preparation method of the artificial exosome anta-miRNAs@Exo nano-particles for inhibiting the expression of melanoma metastasis miRNA clusters comprises the following steps:
(1) Isolation of melanoma extracellular exosomes (A375-LuT 3-Exo)
The supernatant collected after 48 h of passage of melanoma cells A375-LuT3 was used for separation of exosomes by differential centrifugation. The supernatant was collected by the first centrifugation (4 ℃,2000 g,10 min) and the supernatant was collected by the second centrifugation (4 ℃,10000 g,30 min). The pellet was collected by a third centrifugation (4 ℃,110000 g,70 min), resuspended in PBS, the exosomes were collected by a fourth centrifugation (4 ℃,110000 g,70 min), and the exosomes were resuspended in an appropriate amount of PBS.
(2) Preparation of exosome membranes
The exosomes were resuspended in ice-cold Tris (0.01M) -magnesium (0.001M MgCl 2) buffer (TM), flash frozen at-80 ℃ and thawed at room temperature. The freeze-thaw cycle was repeated five more times and then mixed with 1M sucrose to a final concentration of 0.25M and centrifuged at 2000 Xg for 10 minutes. The collected supernatant was added with RNase, and the mixture was centrifuged at 3000 g for 30 minutes to collect the exosome membrane. The exosome membrane was washed with 0.25M sucrose low temperature TM buffer and centrifuged at 3000 Xg at 4℃for 30 min.
(3) Preparation of CBSA-miRNA cluster antagonist (CBSA-anta-miRNAs) nanoparticles
Dissolving anta-miRNA-194-5p and anta-miRNA-215-5p in a mass ratio of 1:1 by using RNase-free water. The anta-miRNA-194-5p and anta-miRNA-215-5p solutions were mixed with the CBSA solution in a ratio (anta-miRNA/cbsa=1/30, w/w) such that the final CBSA concentration was 1 mg/mL and incubated at 4 ℃ for 30 minutes.
(4) Preparation of anta-miRNAs@Exo nanoparticles
The CBSA-anta-miRNAs are coated with exosome films by adopting an incubation and extrusion method to prepare the anta-miRNAs@Exo nano-particles. The CBSA-anta-miRNAs nanoparticle and exosome membrane were first mixed in a mass ratio of 1:1, incubated for 30 min at 4℃and then incubated with shaking for 1 hr at 37 ℃. The mixture was then co-extruded 20 times through a 200 nm polycarbonate film to obtain the anta-mirnas@exo nanoparticles.
Example 4 preparation of fluorescent anta-mirnas@Exo nanoparticles
1. The preparation method of the bicolor fluorescent anta-miRNAs@Exo nano-particles comprises the following steps:
(1) Isolation of osteosarcoma extracellular exosome (HOS-LuT 3-Exo)
The supernatant collected after 48 h of culture of osteosarcoma cells HOS-LuT3 was passaged and the exosomes were isolated by differential centrifugation. The supernatant was collected by the first centrifugation (4 ℃,2000 g,10 min) and the supernatant was collected by the second centrifugation (4 ℃,10000 g,30 min). The pellet was collected by a third centrifugation (4 ℃,110000 g,70 min), resuspended in PBS, the exosomes were collected by a fourth centrifugation (4 ℃,110000 g,70 min), and the exosomes were resuspended in an appropriate amount of PBS.
(2) Preparation of exosome membranes
The exosomes were resuspended in ice-cold Tris (0.01M) -magnesium (0.001M MgCl 2) buffer (TM), flash frozen at-80 ℃ and thawed at room temperature. The freeze-thaw cycle was repeated five more times and then mixed with 1M sucrose to a final concentration of 0.25M and centrifuged at 2000 Xg for 10 minutes. The collected supernatant was added with RNase, and the mixture was centrifuged at 3000 g for 30 minutes to collect the exosome membrane. The exosome membrane was washed with 0.25M sucrose low temperature TM buffer and centrifuged at 3000 Xg at 4℃for 30 min.
(3) Preparation of bicolor fluorescent CBSA-miRNA cluster antagonist (CBSA-anta-miRNAs) nanoparticle
Cy5 fluorescent-labeled anta-miRNA-194-5p and FAM fluorescent-labeled anta-miRNA-215-5p were dissolved in RNase-free water at a mass ratio of 1:1. The anta-miRNA-194-5p and anta-miRNA-215-5p solutions were mixed with the CBSA solution in a ratio (fluorescent anta-miRNA/cbsa=1/30, w/w) such that the final CBSA concentration was 1 mg/mL and incubated at 4 ℃ for 30 minutes.
(4) Preparation of bicolor fluorescent anta-miRNAs@Exo nanoparticles
Fluorescent anta-miRNA@Exo nano-particles are prepared by coating fluorescent CBSA-anta-miRNA with an exosome film by adopting an incubation and extrusion method. The fluorescent CBSA-anta-miRNAs nanoparticle and exosome membrane were first mixed in a mass ratio of 1:1, incubated for 30 minutes at 4 ℃, then incubated for 1 hour with shaking at 37 ℃. The mixture was then co-extruded 20 times through a 200 nm polycarbonate film to obtain bicolor fluorescent anta-mirnas@exo nanoparticles.
2. The preparation method of the monochromatic fluorescent anta-miRNA@Exo nano particles comprises the following steps:
(1) Isolation and staining of osteosarcoma cell exosomes (HOS-LuT 3-Exo)
The supernatant collected after 48 h of culture of osteosarcoma cells HOS-LuT3 was passaged and the exosomes were isolated by differential centrifugation. The supernatant was collected by the first centrifugation (4 ℃,2000 g,10 min) and the supernatant was collected by the second centrifugation (4 ℃,10000 g,30 min). The pellet was collected by a third centrifugation (4 ℃,110000 g,70 min), resuspended in PBS, the exosomes were collected by a fourth centrifugation (4 ℃,110000 g,70 min), and the exosomes were resuspended in an appropriate amount of PBS. The exosomes were stained with the fluorescent dye PKH26 for 30 minutes and the fluorescent exosomes were collected by centrifugation (4 ℃,110000 g,70 min).
(2) Preparation of PKH26 fluorescent-labeled exosome membranes
PKH26 fluorescently labeled exosomes were resuspended in ice-cold Tris (0.01M) -magnesium (0.001M MgCl 2) buffer (TM), flash frozen at-80 ℃ and thawed at room temperature. The freeze-thaw cycle was repeated five more times and then mixed with 1M sucrose to a final concentration of 0.25M and centrifuged at 2000 Xg for 10 minutes. The collected supernatant was added with RNase, and the mixture was centrifuged at 3000 g for 30 minutes to collect the exosome membrane. The exosome membrane was washed with 0.25M sucrose low temperature TM buffer and centrifuged at 3000 Xg at 4℃for 30 min.
(3) Preparation of CBSA-miRNA antagonist (CBSA-anta-miRNA) nanoparticle
Dissolving anta-miRNA-194-5p and anta-miRNA-215-5p in a mass ratio of 1:1 by using RNase-free water. The anta-miRNA-194-5p and anta-miRNA-215-5p solutions were mixed with the CBSA solution in a ratio (anta-miRNA/cbsa=1/30, w/w) such that the final CBSA concentration was 1 mg/mL and incubated at 4 ℃ for 30 minutes.
(4) Preparation of PKH26 fluorescent-labeled anta-miRNA@Exo nano-particles
And (3) coating the CBSA-anta-miRNA with a fluorescent exosome film by adopting an incubation and extrusion method to prepare the monochromatic fluorescent anta-miRNA@Exo nano-particles. The CBSA-anta-miRNA nanoparticle and the PKH26 fluorescent-labeled exosome membrane are firstly mixed according to the mass ratio of 1:1, incubated for 30 minutes at 4 ℃, and then incubated for 1 hour at 37 ℃ with shaking. The mixture was then co-extruded 20 times through a 200 nm polycarbonate film to obtain PKH26 fluorescently labeled anta-mirna@exo nanoparticles.
Example 5 preparation of control nanoparticles
1. The preparation method of the artificial exosome anta-miRNA-194-5p@Exo nano particle for inhibiting miRNA-194-5p expression comprises the following steps:
(1) Isolation of osteosarcoma extracellular exosome (HOS-LuT 3-Exo)
The supernatant collected after 48 h of culture of osteosarcoma cells HOS-LuT3 was passaged and the exosomes were isolated by differential centrifugation. The supernatant was collected by the first centrifugation (4 ℃,2000 g,10 min) and the supernatant was collected by the second centrifugation (4 ℃,10000 g,30 min). The pellet was collected by a third centrifugation (4 ℃,110000 g,70 min), resuspended in PBS, the exosomes were collected by a fourth centrifugation (4 ℃,110000 g,70 min), and the exosomes were resuspended in an appropriate amount of PBS.
(2) Preparation of exosome membranes
The exosomes were resuspended in ice-cold Tris (0.01M) -magnesium (0.001M MgCl 2) buffer (TM), flash frozen at-80 ℃ and thawed at room temperature. The freeze-thaw cycle was repeated five more times and then mixed with 1M sucrose to a final concentration of 0.25M and centrifuged at 2000 Xg for 10 minutes. The collected supernatant was added with RNase, and the mixture was centrifuged at 3000 g for 30 minutes to collect the exosome membrane. The exosome membrane was washed with 0.25M sucrose low temperature TM buffer and centrifuged at 3000 Xg at 4℃for 30 min.
(3) Preparation of CBSA-miRNA-194-5p antagonist (CBSA-anta-miRNA) nanoparticle
The anta-miRNA-194-5p was dissolved with rnase-free water, and the anta-miRNA-194-5p solution was mixed with the CBSA solution in a ratio (anta-miRNA/cbsa=1/30, w/w) such that the final CBSA concentration was 1 mg/mL, then immediately mixed and incubated at 4 ℃ for 30 minutes.
(4) Preparation of anta-miRNA-194-5 p@Exo nanoparticles
And coating the CBSA-anta-miRNA-194-5p with an exosome film by adopting an incubation and extrusion method to prepare the anta-miRNA-194-5 p@Exo nano-particle. Firstly, mixing CBSA-anta-miRNA-194-5p nano particles and an exosome membrane according to a mass ratio of 1:1, incubating for 30 minutes at 4 ℃, and then incubating for 1 hour at 37 ℃ with shaking. The mixture was then co-extruded 20 times through a 200 nm polycarbonate film to obtain the anta-miRNA-194-5 p@exo nanoparticles.
2. The preparation method of the artificial exosome anta-miRNA-215-5p@Exo nano particle for inhibiting miRNA-215-5p expression comprises the following steps:
(1) Isolation of osteosarcoma extracellular exosome (HOS-LuT 3-Exo)
The supernatant collected after 48 h of culture of osteosarcoma cells HOS-LuT3 was passaged and the exosomes were isolated by differential centrifugation. The supernatant was collected by the first centrifugation (4 ℃,2000 g,10 min) and the supernatant was collected by the second centrifugation (4 ℃,10000 g,30 min). The pellet was collected by a third centrifugation (4 ℃,110000 g,70 min), resuspended in PBS, the exosomes were collected by a fourth centrifugation (4 ℃,110000 g,70 min), and the exosomes were resuspended in an appropriate amount of PBS.
(2) Preparation of exosome membranes
The exosomes were resuspended in ice-cold Tris (0.01M) -magnesium (0.001M MgCl 2) buffer (TM), flash frozen at-80 ℃ and thawed at room temperature. The freeze-thaw cycle was repeated five more times and then mixed with 1M sucrose to a final concentration of 0.25M and centrifuged at 2000 Xg for 10 minutes. The collected supernatant was added with RNase, and the mixture was centrifuged at 3000 g for 30 minutes to collect the exosome membrane. The exosome membrane was washed with 0.25M sucrose low temperature TM buffer and centrifuged at 3000 Xg at 4℃for 30 min.
(3) Preparation of CBSA-miRNA-215-5p antagonist (CBSA-anta-miRNA) nanoparticle
The anta-miRNA-215-5p solution was dissolved with rnase-free water, the anta-miRNA-215-5p solution was mixed with CBSA solution in a ratio (anta-miRNA/cbsa=1/30, w/w) such that the final CBSA concentration was 1 mg/mL, then immediately mixed and incubated at 4 ℃ for 30 minutes.
(4) Preparation of anta-miRNA-215-5 p@Exo nanoparticles
And coating the CBSA-anta-miRNA-215-5p with an exosome film by adopting an incubation and extrusion method to prepare the anta-miRNA-215-p@Exo nano-particle. The CBSA-anta-miRNA-215-p nano-particles and the exosome membrane are firstly mixed according to the mass ratio of 1:1, incubated for 30 minutes at 4 ℃, and then incubated for 1 hour at 37 ℃ with shaking. The mixture was then co-extruded 20 times through a 200 nm polycarbonate film to obtain the anta-miRNA-215-5 p@exo nanoparticles.
3. The preparation method of the contrast artificial exosome anta-miRNA-NC@Exo nano-particle comprises the following steps:
(1) Isolation of osteosarcoma extracellular exosome (HOS-LuT 3-Exo)
The supernatant collected after 48 h of culture of osteosarcoma cells HOS-LuT3 was passaged and the exosomes were isolated by differential centrifugation. The supernatant was collected by the first centrifugation (4 ℃,2000 g,10 min) and the supernatant was collected by the second centrifugation (4 ℃,10000 g,30 min). The pellet was collected by a third centrifugation (4 ℃,110000 g,70 min), resuspended in PBS, the exosomes were collected by a fourth centrifugation (4 ℃,110000 g,70 min), and the exosomes were resuspended in an appropriate amount of PBS.
(2) Preparation of exosome membranes
The exosomes were resuspended in ice-cold Tris (0.01M) -magnesium (0.001M MgCl 2) buffer (TM), flash frozen at-80 ℃ and thawed at room temperature. The freeze-thaw cycle was repeated five more times and then mixed with 1M sucrose to a final concentration of 0.25M and centrifuged at 2000 Xg for 10 minutes. The collected supernatant was added with RNase, and the mixture was centrifuged at 3000 g for 30 minutes to collect the exosome membrane. The exosome membrane was washed with 0.25M sucrose low temperature TM buffer and centrifuged at 3000 Xg at 4℃for 30 min.
(3) Preparation of CBSA-miRNA-NC nanoparticles
The anta-miRNA-NC solution was dissolved with rnase-free water, mixed with CBSA solution in a ratio (anta-miRNA/cbsa=1/30, w/w) such that the final CBSA concentration was 1 mg/mL, then immediately mixed and incubated at 4 ℃ for 30 minutes.
(4) Preparation of anta-miRNA-NC@Exo nanoparticles
And coating the CBSA-anta-miRNA-NC by using an exosome film by adopting an incubation and extrusion method to prepare the anta-miRNA-NC@Exo nano-particle. The CBSA-anta-miRNA-NC nanoparticle and the exosome membrane were first mixed according to a mass ratio of 1:1, incubated for 30 minutes at 4 ℃, and then incubated for 1 hour with shaking at 37 ℃. The mixture was then co-extruded 20 times through a 200 nm polycarbonate film to obtain the anta-miRNA-nc@exo nanoparticles.
Example 6 anta-biological Property analysis of miRNAs@Exo nanoparticles
In this example, CBSA-anta-miRNAs nanoparticles were derived from example 1, and nanoparticles were prepared in the ratio anta-miRNA/cbsa=1/30 (w/w), and in this example, anta-mirnas@exo nanoparticles were derived from example 1.
The zeta potential of the nanoparticles was measured using a Malvern Nano ZS90 Particle Size Potentiometer analyzer, and the measurement showed that the charge of the CBSA-anta-miRNAs nanoparticles was 21.83.+ -. 1.01 mV, whereas the charge of the anta-miRNAs@Exo nanoparticles was-24.43.+ -. 0.70 mV, similar to the charge of the exosome surface (-26.53.+ -. 1.01 mV) (FIG. 1), indicating that the surface of the anta-miRNAs@Exo nanoparticles had been successfully coated with exosome membrane. Malvern Zetasizer Nano analysis of the particle size showed that the average particle size of the anta-mirnas@exo nanoparticles was 229.13 ±7.75. 7.75 nm (fig. 2). As can be seen from fig. 3, the anta-mirnas@exo nanoparticle is in a typical vesicle structure under a transmission electron microscope, and it is confirmed that the surface of the anta-mirnas@exo nanoparticle has been successfully coated with an exosome membrane.
Example 7 anta-miRNAs@Exo nanoparticle cell delivery Capacity assessment
The dual fluorescently labeled anta-mirnas@exo nanoparticle in this example was derived from example 4.
Fluorescent anta-mirnas@exo nanoparticles were incubated with passaged osteosarcoma HOS cells for 24 hours, then the fluorescence intensity in the cells was detected, and the experimental results are shown in fig. 4, and the incubated cells show obvious FAM (green) and Cy5 (red) signals, which indicate that the fluorescent anta-mirnas@exo nanoparticles can effectively deliver miRNA-194-5 p/215-5 p cluster antagonists.
Example 8 anta-miRNAs@Exo nanoparticle in vivo delivery Capacity assessment
The fluorescent anta-mirnas@exo nanoparticles in this example were derived from example 4.
The anta-miRNA-194-5 p/215-5 p was loaded into PKH26 labeled exosome membrane vesicles, fluorescent anta-miRNAs@Exo nanoparticles were prepared, and injected into in situ osteosarcoma mice via tail vein. Observations from the mice in vivo imager showed that the mouse tibial tumor enriched BLI signal was strongest after 24 hours of injection, demonstrating that the exosome membrane vesicle system was able to deliver anta-miRNA-194-5 p/215-5 p to in situ osteosarcoma cells (fig. 5).
Example 9 effect of anta-miRNAs@Exo nanoparticles on inhibition of in situ osteosarcoma metastasis
In this example the anta-mirnas@exo nanoparticle was derived from example 1 and the exosome HOS-LuT3-Exo was derived from example 1.
To examine the effect of different proportions of anta-miRNA and CBSA on the inhibition of in situ osteosarcoma metastasis by anta-mirnas@exo nanoparticles, animals were divided into 6 groups, PBS groups respectively, with anta-miRNA and CBSA ratios of 1:45 groups, anta-miRNA and CBSA ratio of 1: group 40, anta-miRNA and CBSA ratio of 1:35 groups, anta-miRNA and CBSA ratio 1:30 groups, anta-miRNA and CBSA ratio of 1:25 groups. The osteosarcoma HOS cells are injected into the tibia of the 6-8 week old NCG-HLA-A2.1 male mice in situ. After 3d days, 1.25X10. 10 10 granules of exosome HOS-LuT3-Exo (200. Mu.L) were injected intravenously at the tail of 3 days, and the osteosarcoma metastasis model was constructed by continuous injection 5 times. Different proportions of anta-mirnas@exo of 1.25X10 10 particles were injected separately at the same time as the HOS-LuT3-Exo was injected into the tail vein. The tumor gravimetric analysis of each group showed that as the anta-miRNA/CBSA ratio increased, the mouse bone tumor burden decreased, while when the anta-miRNA/CBSA ratio exceeded 1:30, the inhibition effect is not improved, so that the anta-miRNA/CBSA ratio is 1:30 (fig. 6).
Further examining the effect of the anta-mirnas@Exo nanoparticles in inhibiting in-situ osteosarcoma metastasis, the experiment was divided into 4 groups, namely, an anta-mirnas@Exo group, an anta-miRNA-194-5p@Exo group, an anta-miRNA-215-5p@Exo group and an anta-miRNA-NC@Exo group. The osteosarcoma HOS cells are injected into the tibia of the 6-8 week old NCG-HLA-A2.1 male mice in situ. After 3 d days, 1.25X10. 10 10 granules of exosome HOS-LuT3-Exo (200. Mu.L) were injected intravenously at the tail of 3 days, and the osteosarcoma metastasis model was constructed by continuous injection 5 times. At the same time of tail vein injection of HOS-LuT3-Exo, 1.25X 10 particles of anta-miRNAs@Exo, anta-miRNA-194-5p@Exo, anta-miRNA-215-5p@Exo and anta-miRNA-NC@Exo are respectively injected. The tumor gravimetric analysis of each group showed a significant decrease in bone tumor burden in mice treated with anta-mirnas@exo (figure 7). The lung tissue H & E staining results indicated that anta-mirnas@Exo reversed osteosarcoma lung metastasis (FIG. 8).
Example 10 effect of anta-miRNAs@Exo nanoparticles on inhibition of breast cancer metastasis
In this example the anta-mirnas@exo nanoparticle was derived from example 2 and the MDA-MB-231-LuT3-Exo was derived from example 2.
To examine the effect of the anta-mirnas@exo nanoparticles on inhibition of breast cancer metastasis, animals were divided into 2 groups, namely anta-mirnas@exo group and anta-miRNA-nc@exo group. The breast cancer MDA-MB-231 cells are inoculated to the mammary gland of the NCG-HLA-A2.1 female mice with the age of 7-8 weeks. After 3 d days, 1.25X10. 10 10 exosomes MDA-MB-231-LuT3-Exo (200 μL) were injected intravenously at the tail of 3 days, and 5 times of injections were continued to construct a breast cancer metastasis model. 1.25X10 10 particles of anta-miRNAs@Exo and anta-miRNA-NC@Exo were injected separately at the same time as MDA-MB-231-LuT3-Exo was injected into the tail vein. The tumor gravimetric analysis of each group showed a significant decrease in breast tumor burden in the anta-mirnas@exo treated mice (figure 9). The lung tissue H & E staining results indicated that anta-mirnas@Exo reversed breast cancer lung metastasis (FIG. 10).
Example 11 effect of anta-miRNAs@Exo nanoparticles on inhibition of melanoma metastasis
In this example, the anta-miRNAs@Exo nanoparticles were derived from example 3 and the A375-LuT3-Exo nanoparticles were derived from example 3.
To examine the effect of the anta-mirnas@exo nanoparticles on inhibition of melanoma metastasis, animals were divided into 2 groups, the anta-mirnas@exo group and the anta-miRNA-nc@exo group, respectively. The human malignant melanoma A375 cells are passaged and injected subcutaneously into 6-8 week old NCG-HLA-A2.1 male mice. After 3d days, 1.25X10. 10 10 exosomes A375-LuT3-Exo (200. Mu.L) were injected intravenously at the tail every 3 days, and 5 injections were continued to construct osteosarcoma transfer model. 1.25X10 10 particles of anta-miRNAs@Exo and anta-miRNA-NC@Exo were injected simultaneously with the tail intravenous injection of A375-LuT 3-Exo. The tumor gravimetric analysis of each group showed a significant decrease in melanoma burden in mice treated with anta-mirnas@exo (figure 11). The lung tissue H & E staining results indicated that anta-mirnas@Exo reversed melanoma lung metastasis (FIG. 12).
The CBSA-miRNA cluster antagonist nanoparticle and the miRNA-194-5p/miRNA-215-5p cluster antagonist nanoparticle are two expressions of the same concept in the specification.
Claims (7)
1. The artificial exosome is characterized by being prepared from miRNA-194-5p/miRNA-215-5p cluster antagonist nano particles wrapped by exosome membranes; the sequence of miRNA-194-5p is shown as SEQ ID NO.1, and the sequence of miRNA-215-5p is shown as SEQ ID NO. 2; the miRNA-194-5p/miRNA-215-5p cluster antagonist nano-particles are obtained by coupling an antagonist aiming at miRNA-194-5p and an antagonist aiming at miRNA-215-5p through cationic serum albumin; the antagonist for miRNA-194-5p is nucleic acid obtained by carrying out methylation modification on 5'-UCCACAUGGAGUUGCUGUUACA-3' sequence full chain, carrying out thio modification on 2 bases at the 5 'end and carrying out thio modification on 4 bases at the 3' end; the antagonist against miRNA-215-5p is nucleic acid obtained by methylation modification of 5'-GUCUGUCAAUUCAUAGGUCAU-3' sequence full chain, thio modification of 2 bases at the 5 'end and thio modification of 4 bases at the 3' end.
2. The artificial exosome of claim 1, wherein the mass ratio of cationic bovine serum albumin to antagonists against miRNA-194-5p and miRNA-215-5p is 45-25:1.
3. The artificial exosome according to claim 2, wherein the miRNA-194-5p and miRNA-215-5p cluster nanoparticles are prepared by the following method: the antagonists aiming at miRNA-194-5p and miRNA-215-5p are dissolved by water without RNase, mixed with CBSA solution according to the proportion, and then incubated for 20-40 minutes at 0-4 ℃.
4. A method of preparing the artificial exosome of claims 1-3, comprising the steps of:
a) Preparing an exosome membrane;
b) Preparing miRNA-194-5p/miRNA-215-5p cluster antagonist nano-particles;
c) Coating miRNA-194-5p/miRNA-215-5p cluster antagonist nano particles by using an exosome membrane to form the artificial exosome.
5. The preparation method of the miRNA-194-5p/miRNA-215-5p cluster antagonist nano-particles, which is characterized in that: dissolving the antagonists aiming at miRNA-194-5p and miRNA-215-5p by using water without RNase, mixing with a cationic serum albumin solution, and then incubating for 20-40 minutes at 0-4 ℃.
6. The preparation method of claim 4, wherein the mass ratio of the cationic bovine serum albumin to the antagonist against miRNA-194-5p and miRNA-215-5p is 45-25:1; the antagonist for miRNA-194-5p is nucleic acid obtained by carrying out methylation modification on 5'-UCCACAUGGAGUUGCUGUUACA-3' sequence full chain, carrying out thio modification on 2 bases at the 5 'end and carrying out thio modification on 4 bases at the 3' end; the antagonist against miRNA-215-5p is nucleic acid obtained by methylation modification of 5'-GUCUGUCAAUUCAUAGGUCAU-3' sequence full chain, thio modification of 2 bases at the 5 'end and thio modification of 4 bases at the 3' end.
7. Use of the artificial exosome of claim 1 in the manufacture of a medicament for treating tumors and/or tumor metastases; the tumor is osteosarcoma, breast cancer or melanoma; the tumor metastasis is lung metastasis of tumor.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410020375.8A CN117530932B (en) | 2024-01-08 | 2024-01-08 | Artificial exosome for inhibiting tumor growth and lung metastasis and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410020375.8A CN117530932B (en) | 2024-01-08 | 2024-01-08 | Artificial exosome for inhibiting tumor growth and lung metastasis and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN117530932A CN117530932A (en) | 2024-02-09 |
| CN117530932B true CN117530932B (en) | 2024-04-26 |
Family
ID=89796179
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410020375.8A Active CN117530932B (en) | 2024-01-08 | 2024-01-08 | Artificial exosome for inhibiting tumor growth and lung metastasis and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117530932B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115038796A (en) * | 2019-09-09 | 2022-09-09 | 科莱鹤株式会社 | Micro RNA-containing body fluid extract |
| CN115297868A (en) * | 2020-02-21 | 2022-11-04 | 联合治疗公司 | Compositions and methods for organ protective expression and regulation of ribonucleic acids |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB201302468D0 (en) * | 2013-02-12 | 2013-03-27 | Reneuron Ltd | Stem cell product |
| US20180356373A1 (en) * | 2017-06-13 | 2018-12-13 | Velayudhan Sahadevan | Metastasis and Adaptive Resistance Inhibiting Immunotherapy Combined Online Chemotherapy with Radiotherapy's tumor Seeking Extracellular Vesicles with siRNA and Chemotherapeutics |
| EP3755344A1 (en) * | 2018-02-19 | 2020-12-30 | Combined Therapeutics, Inc. | Compositions and methods for organ-protective expression and modulation of coding ribonucleic acids |
| EP3858332A1 (en) * | 2020-01-31 | 2021-08-04 | Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. | Bottom-up assembly of synthetic extracellular vesicles |
-
2024
- 2024-01-08 CN CN202410020375.8A patent/CN117530932B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115038796A (en) * | 2019-09-09 | 2022-09-09 | 科莱鹤株式会社 | Micro RNA-containing body fluid extract |
| CN115297868A (en) * | 2020-02-21 | 2022-11-04 | 联合治疗公司 | Compositions and methods for organ protective expression and regulation of ribonucleic acids |
Non-Patent Citations (1)
| Title |
|---|
| Extracellular miRNAs for the Management of Barrett’s Esophagus and Esophageal Adenocarcinoma: A Systematic Review;Kazumi Inokuchi等;J. Clin. Med.;20201231;第10卷(第117期);第1-16页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN117530932A (en) | 2024-02-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zhou et al. | Pancreatic cancer-targeting exosomes for enhancing immunotherapy and reprogramming tumor microenvironment | |
| Liu et al. | Tumor associated macrophage-targeted microRNA delivery with dual-responsive polypeptide nanovectors for anti-cancer therapy | |
| Yan et al. | Nanosized functional miRNA liposomes and application in the treatment of TNBC by silencing Slug gene | |
| Zhao et al. | Biomimetic nanovesicles made from iPS cell-derived mesenchymal stem cells for targeted therapy of triple-negative breast cancer | |
| Ma et al. | Small extracellular vesicles deliver osteolytic effectors and mediate cancer‐induced osteolysis in bone metastatic niche | |
| CN113355290B (en) | Anti-tumor engineered exosome, preparation method and application | |
| Huang et al. | Macrophage membrane-coated nanovesicles for dual-targeted drug delivery to inhibit tumor and induce macrophage polarization | |
| You et al. | Cytosine deaminase‐producing human mesenchymal stem cells mediate an antitumor effect in a mouse xenograft model | |
| CN113846047A (en) | Kidney targeting drug-loaded exosome and application and drug for treating kidney diseases | |
| CN111068069A (en) | Immune targeting functional liposome and preparation method and application thereof | |
| Ma et al. | Tumor microenvironment targeting system for glioma treatment via fusion cell membrane coating nanotechnology | |
| KR20200136978A (en) | Use of exosomes for targeted delivery of therapeutic agents | |
| CN102784398B (en) | Composition comprising endostatin adopted as delivery system and chemically-synthesized RNA interference molecule, and application thereof | |
| Shao et al. | Phenylboronic acid-functionalized polyaminoglycoside as an effective CRISPR/Cas9 delivery system | |
| Lu et al. | Delivery of TSPAN1 siRNA by novel Th17 targeted cationic liposomes for gastric cancer intervention | |
| Ding et al. | Genetically engineered nanovesicles mobilize synergistic antitumor immunity by ADAR1 silence and PDL1 blockade | |
| CN117530932B (en) | Artificial exosome for inhibiting tumor growth and lung metastasis and application thereof | |
| Lan et al. | Cationic liposome coupled endostatin gene for treatment of peritoneal colon cancer | |
| CN110760478A (en) | Anti-tumor macrophage bioreactor system and preparation method thereof | |
| Gao et al. | Engineered exosomes loaded with miR-563 inhibit lung cancer growth | |
| CN103834035A (en) | Cationic laminarin and preparation method and application thereof | |
| Zhuang et al. | The colorectal cancer-specific microbiome regulation and immune response activation via an artificial biomimetic nanovaccine | |
| CN101148680A (en) | Method for leading plasmid carrier containing gene cure segment in cell by nano particles | |
| CN114685587B (en) | Cyclic RNAcircMDK, application thereof and liver cancer treatment preparation | |
| CN116603069B (en) | Microorganism targeting implantation system and method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |