CN111876388A - Bone and bone tissue targeted exosome and preparation method and application thereof - Google Patents

Bone and bone tissue targeted exosome and preparation method and application thereof Download PDF

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CN111876388A
CN111876388A CN202010722732.7A CN202010722732A CN111876388A CN 111876388 A CN111876388 A CN 111876388A CN 202010722732 A CN202010722732 A CN 202010722732A CN 111876388 A CN111876388 A CN 111876388A
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cells
pth
extracellular vesicles
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修朝阳
秦蕴豪
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Sairuicheng Suzhou Biotechnology Co ltd
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/635—Parathyroid hormone, i.e. parathormone; Parathyroid hormone-related peptides
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Abstract

The present disclosure relates to a bone and bone tissue targeted exosome and methods of making and using the same, employing a molecule comprising a PTH (parathyroid hormone) domain and an extracellular vesicle obtained or obtainable from the methods disclosed herein, the method comprising the step of introducing into a fluid which may comprise the extracellular vesicle a molecule comprising: a parathyroid hormone-related protein component (PTH component) comprising a PTH domain or an active fragment thereof.

Description

Bone and bone tissue targeted exosome and preparation method and application thereof
Technical Field
The present disclosure relates to a method of targeting extracellular vesicles employing a molecule comprising a PTH (parathyroid hormone) domain and extracellular vesicles obtained or obtainable from the methods disclosed herein.
Background
Extracellular vesicles (also known as microvesicles or microparticles) are a vehicle for loading cellular exocytosis material and are also important carriers for intercellular communication.
There is a great deal of evidence that mammalian cells and their body fluids contain a large number of extracellular vesicles, such as: healthy, stem and diseased cells, as well as plasma, saliva, urine, bile, synovial fluid, semen and breast milk.
Studies have demonstrated that extracellular vesicles can be used as a replacement for stem cells for therapy. However, the low abundance and minimal volume pose significant difficulties in isolating and using these extracellular vesicles.
A great deal of research not only explores the mechanism of the extracellular vesicles, but also provides beneficial ideas and guidance for loading effective substances by utilizing the extracellular vesicles to target and/or treat a series of diseases. Among them, targeted delivery is one of the major issues of its therapeutic potential: efficient loading of extracellular vesicles: effective to define and load the amount of active substance into the extracellular vesicles for therapeutic, diagnostic and prognostic purposes, etc., such as proteins, natural and non-natural oligonucleotides (small molecules, enzymes, probes, etc.); guidance of extracellular vesicle targeting site: defining, endowing or enhancing targeting, therapeutic proteins, markers and other extracellular vesicles with a site or cell specific recognition function, conveniently and quickly obtaining or delivering the localization and the like; elimination of harmful or pathogenic components of extracellular vesicles: the identification and elimination of pathogenic substances and signals in the extracellular vesicles are the key to reduce the toxic and side effects of the effective carrier, and the adoption of biological generation forms such as healthy cells or stem cells is an effective means
Disclosure of Invention
The present invention relates to a method of targeting extracellular vesicles using a composition comprising a PTH component and extracellular vesicles obtained or obtainable from the methods disclosed herein. The specific technical scheme is as follows:
construction, amplification and extraction of fusion plasmid: inserting PTH component gene sequence and Lamp2b gene CDS sequence into the downstream of CMV promoter of pcDNA3.1_ hygro vector, transferring the connected vector into 293T cell for culture and amplification and extracting modified plasmid;
preparation and extraction of PTH component chimeric exosomes: transfecting the modified plasmid extracted in the step 1 into 293T cells, and obtaining a stably transfected 293T cell line by adopting a puromycin resistance screening mode; adopting DMEM + 10% FBS culture medium to culture 293T stably-transformed cell line in large scale and collecting culture supernatant; extracting exosomes in supernatant by ultracentrifugation or an exosome special extraction kit;
loading of payload: mixing the extracted exosome with a target load according to the mass ratio of 1:1, adding a transfection reagent lipo2000, and stably transfecting for 4 hours;
culturing the cell line obtained by screening in the step 3 in a DMEM + 10% FBS culture medium, collecting cell culture supernatant, and collecting exosomes in the supernatant by adopting an ultracentrifugation or exosome extraction kit;
and 4, the exosomes collected in the step 4 are targeting and payload-loaded exosomes containing PTH components.
The invention also provides a method for preparing the targeted exosome for treating osteoporosis. The specific technical scheme is as follows:
preparing and collecting target exosomes of the PTH component;
synthesizing mir27a in vitro;
mixing the components in the steps 1 and 2 according to the mass ratio of 1:1, adding lipo2000 transfection reagent for transfection for 4-6 hours according to the mass ratio of 1:2-3(mir (ug): lipo2000 (ul));
collecting exosomes by ultracentrifugation;
osteoporosis rats were injected tail vein with mir27 a-loaded pth targeting exosomes obtained in step 4. Compared with the prior art, the invention has the beneficial effects that:
(1) the preparation method of the invention does not show obvious toxicity to cells, and has no obvious influence on the secretion capacity and the form of exosomes of the cells;
(2) the expression quantity of the PTH component on the surface of the prepared exosome membrane is obviously increased, the targeting capability of bones and bone tissues is obviously improved, and the specific aggregation of exosomes at the positions of the bones and the bone tissues is enhanced;
(3) the membrane modification process of the exosome cannot damage the exosome, the integrity of the exosome membrane can be kept to the maximum extent, and the content with the treatment effect is protected from loss;
(4) the preparation method is simple to operate and convenient to control.
Drawings
FIG. 1: constructing an effect graph by the fusion vector;
FIG. 2: electron microscopy analysis of targeting exosomes and 293T natural exosomes.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.
The present invention relates to a method of targeting extracellular vesicles using a composition comprising a PTH component and extracellular vesicles obtained or obtainable from the methods disclosed herein. The specific technical scheme is as follows:
construction, amplification and extraction of fusion plasmid: inserting PTH component gene sequence and Lamp2b gene CDS sequence into the downstream of CMV promoter of pcDNA3.1_ hygro vector, transferring the connected vector into 293T cell for culture and amplification and extracting modified plasmid;
preparation and extraction of PTH component chimeric exosomes: transfecting the modified plasmid extracted in the step 1 into 293T cells, and obtaining a stably transfected 293T cell line by adopting a hygromycin resistance screening mode; adopting DMEM + 10% FBS culture medium to culture 293T stably-transformed cell line in large scale and collecting culture supernatant; extracting exosomes in supernatant by ultracentrifugation or an exosome special extraction kit;
loading of payload: mixing the extracted exosome and a target load according to the mass ratio of 1:1, adding a transfection reagent lipo2000, stably transfecting for 4 hours, transferring to a fresh culture medium for culturing for 12-24 hours, adding a replacement solution, adding 5ug/ml puromycin (puro) for continuously culturing for 24-48 hours, and obtaining a cell line stably transfecting a target carrier;
culturing the cell line obtained by screening in the step 3 in a DMEM + 10% FBS culture medium, collecting cell culture supernatant, and collecting exosomes in the supernatant by adopting an ultracentrifugation or exosome extraction kit;
and 4, the exosomes collected in the step 4 are targeting and payload-loaded exosomes containing PTH components.
The invention also provides a method for preparing the targeted exosome for treating osteoporosis. The specific technical scheme is as follows:
s1, preparing and collecting target exosomes of the PTH component;
s2, synthesizing mir27a in vitro;
s3, mixing the components in the steps 1 and 2 according to the mass ratio of 1:1, adding lipo2000 transfection reagent according to the mass ratio of 1:2-3(mir (ug): lipo2000(ul)) for transfection for 4-6 hours;
s4, collecting exosomes through ultracentrifugation;
s5, injecting mir27 a-loaded pth obtained in the step 4 into the tail vein of the osteoporosis rat to target exosomes.
Compared with the prior art, the invention has the beneficial effects that:
(1) the preparation method of the invention does not show obvious toxicity to cells, and has no obvious influence on the secretion capacity and the form of exosomes of the cells;
(2) the expression quantity of the PTH component on the surface of the prepared exosome membrane is obviously increased, the targeting capability of bones and bone tissues is obviously improved, and the specific aggregation of exosomes at the positions of the bones and the bone tissues is enhanced;
(3) the membrane modification process of the exosome cannot damage the exosome, the integrity of the exosome membrane can be kept to the maximum extent, and the content with the treatment effect is protected from loss;
(4) the preparation method is simple to operate and convenient to control.
Sequence listing
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Claims (10)

1. A method for targeting extracellular vesicles with surface-exposed parathyroid hormone (PTH), the method comprising the step of introducing into a fluid that may comprise the extracellular vesicles a molecule comprising: a parathyroid hormone-related protein component (PTH component) comprising a PTH domain or an active fragment thereof.
2. The method of claim 1, wherein the extracellular vesicles are exosomes having diameters in the range of 30-150 nm.
3. The method of claims 1-2, wherein the PTH component of the extracellular vesicle-targeted is fused to one or more transmembrane proteins of: lamp-1, Lamp-2, CD13, CD86, lipocalin, synaptogusin-3, CD2, CD36, CD40, CD40L, CD41a, CD44, CD45, ICAM-1, integrin α 4, LiCAM, LFA-1, Mac-1 α and β, Vti-IA and B, CD3 and ζ, CD9, CD18, CD37, CD53, CD63, CD81, CD82, CXCR4, FcR, GluR2/3, HLA-dm (MHC II), immunoglobulins, MHC-I or MHC-II components, TCR β, tetraspanins, and combinations of two or more thereof.
4. The method of any one of claims 1-3, wherein the vesicles are released from one or more cells, such as engineered cells 293T, embryonic stem cells, bone marrow stem cells, peripheral blood stem cells, mesenchymal stem cells, perinatal stem cells, apoptotic cells, necrotic cells, cancer cells, pathogen-infected cells (such as virally-infected cells, bacterially-infected cells, or parasitically-infected cells), and combinations of two or more thereof.
5. The method of any of claims 1, 3, 4, wherein the PTH component is from the sequence shown for protein PTH or a derivative thereof, e.g., comprising the sequence shown in SEQ ID NO 1-NO 4.
6. The method of any one of claims 1, 3, 4, 5, wherein the PTH component is fused to the transmembrane protein either directly or through a linker sequence.
7. The method according to claims 1 to 4, wherein the extracellular vesicles are taggable from the group consisting of fluorescent proteins (comprising fluorescent probes (such as rhodamine dyes, FITC, FAM, CY5)), biotin, enzymes, tags (e.g. HIS-tags, FLAG-tags, myc-tags), radionuclides (in particular radioiodinates, radioisotopes, such as 99mTc), luminescent tags or compounds detectable by NMR or ESR spectroscopy, wherein detectable tags are in molecular beacons.
8. The method of any one of claims 1-4, wherein the extracellular vesicles may be loaded with a composition comprising a toxin, a polymer (e.g., a synthetic or naturally occurring polymer), a biologically active protein (e.g., an enzyme, other antibody or antibody fragment), a drug (small molecule (chemical entity)), a nucleic acid and fragments thereof (e.g., DNA, RNA, and fragments thereof), a metal chelator, a nanoparticle, or a combination of two or more thereof.
9. The method according to any one of claims 1 to 8, wherein the extracellular vesicles are useful for detecting or treating osteoporosis, osteoarthritis and rheumatoid arthritis, glioma, colorectal cancer, testicular cancer, liver cancer, biliary tract cancer, prostate cancer, pancreatic cancer, breast cancer, ovarian cancer, cervical cancer, uterine cancer, gastric cancer, esophageal cancer, thyroid cancer, renal cancer, bladder cancer, brain cancer, head and neck cancer, lung cancer, leukemia, lymphoma, myeloma, chronic myeloproliferative diseases (such as AML, CML, ALL and CLL), or alternatively other tumors or diseases.
10. A vesicle or population of vesicles obtainable from the method of any one of claims 1 to 9.
CN202010722732.7A 2020-07-24 2020-07-24 Bone and bone tissue targeted exosome and preparation method and application thereof Pending CN111876388A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114940967A (en) * 2022-05-09 2022-08-26 中山大学 Breast milk extracellular vesicle and application thereof in preparation of bone repair material
WO2024000263A1 (en) * 2022-06-29 2024-01-04 Beijing Thera Bioscience Co., Ltd. Methods for manufacturing and using extracellular vesicles

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114940967A (en) * 2022-05-09 2022-08-26 中山大学 Breast milk extracellular vesicle and application thereof in preparation of bone repair material
CN114940967B (en) * 2022-05-09 2024-05-31 中山大学 Breast milk extracellular vesicles and application thereof in preparation of bone repair materials
WO2024000263A1 (en) * 2022-06-29 2024-01-04 Beijing Thera Bioscience Co., Ltd. Methods for manufacturing and using extracellular vesicles

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