WO2023226203A1 - 一种骨靶向细胞外囊泡及其制备方法和应用 - Google Patents

一种骨靶向细胞外囊泡及其制备方法和应用 Download PDF

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WO2023226203A1
WO2023226203A1 PCT/CN2022/112128 CN2022112128W WO2023226203A1 WO 2023226203 A1 WO2023226203 A1 WO 2023226203A1 CN 2022112128 W CN2022112128 W CN 2022112128W WO 2023226203 A1 WO2023226203 A1 WO 2023226203A1
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bone
targeting
extracellular vesicles
extracellular
extracellular vesicle
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French (fr)
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赵晓丽
郝浏智
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中国科学院深圳先进技术研究院
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    • 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
    • A61K9/5005—Wall or coating material
    • A61K9/5063—Compounds of unknown constitution, e.g. material from plants or animals
    • A61K9/5068—Cell membranes or bacterial membranes enclosing drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • 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
    • A61K47/54—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 an organic compound
    • A61K47/548—Phosphates or phosphonates, e.g. bone-seeking
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0665—Blood-borne mesenchymal stem cells, e.g. from umbilical cord blood
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0668—Mesenchymal stem cells from other natural sources
    • C—CHEMISTRY; METALLURGY
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    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes

Definitions

  • This application belongs to the technical field of biomedical materials, and specifically relates to a bone-targeting extracellular vesicle and its preparation method and application.
  • Bone regeneration is regulated by a variety of cells, especially stem cells with regenerative ability.
  • Stem cells promote bone regeneration mainly by releasing vesicles containing various proteins and nucleic acids (such as miRNA)- realized by extracellular vesicles.
  • extracellular vesicles can participate in a variety of different biochemical and cellular processes such as intercellular communication, immune regulation, energy metabolism, tissue repair, tissue regeneration and metabolism.
  • Extracellular vesicles have natural advantages as drug carriers. Their smaller size and double-layered phospholipid layer structure reduce the risk of blood vessel blockage and facilitate cellular uptake. In addition, extracellular vesicles also have excellent characteristics such as low toxicity and immunogenicity, and the surface of extracellular vesicles is rich in amino groups, which facilitates the modification of functional groups. Therefore, using extracellular vesicles as drugs to promote bone regeneration has natural advantages. However, the ability of original extracellular vesicles to target bone tissue is average, and the therapeutic effect needs to be further improved.
  • This application provides a bone-targeting extracellular vesicle and its preparation method and application.
  • the bone-targeting extracellular vesicles described in this application include extracellular vesicles modified with bone-targeting molecules on their outer surfaces and bone-promoting drugs loaded in the extracellular vesicles.
  • the bone-targeting cells described in this application The external vesicles achieve precise delivery to target organs through bone tissue targeting, and improve the bone-promoting therapeutic effect by loading bone-promoting drugs.
  • the bone-targeting molecule When preparing the bone-targeting extracellular vesicles, the bone-targeting molecule generates a triazole bond through a click chemical reaction with a bifunctional molecule and is connected and modified on the surface of the extracellular vesicles loaded with osteopromoting drugs, so
  • the bifunctional molecule is grafted on the outer surface of the extracellular vesicle loaded with bone-promoting drugs by forming a covalent bond with the amino group on the surface of the extracellular vesicle, and responds gently and conveniently to the bone-targeted modification of the extracellular vesicle without any problem. It will destroy the structure of the extracellular vesicle itself and will not affect the performance of the extracellular vesicle itself.
  • the polypeptide bone-targeting molecule used in this application has low toxic side effects and good bone-targeting effect.
  • the present application provides a bone-targeting extracellular vesicle, which includes an extracellular vesicle modified with a bone-targeting molecule on its outer surface and loaded in the extracellular vesicle. of bone-stimulating drugs.
  • This application uses extracellular vesicles of human stem cells as therapeutic tools, and modifies bone-targeting molecules on the surface of the extracellular vesicles, which can achieve precise delivery to target organs through bone tissue targeting.
  • the resulting bone-targeting extracellular vesicles can achieve bone-targeting vesicles.
  • the therapeutic effect can be further improved by loading bone-promoting drugs.
  • the bone-targeting molecule is connected and modified on the surface of the extracellular vesicle loaded with the bone-promoting drug through a click chemical reaction with a bifunctional molecule to generate a triazole bond;
  • the bifunctional molecule is connected to the extracellular vesicle by The amino groups on the surface form covalent bonds and are grafted onto the outer surface of the extracellular vesicles loaded with bone-promoting drugs;
  • the bifunctional molecule is a molecule with both diphenylcyclooctyne and N-hydroxysuccinimide .
  • the bifunctional molecule is selected from dibenzylcyclooctyl-sulfo-N-hydroxysuccinimide ester, azadibenzocyclooctyne-succinimide ester, diphenylcyclooctyne- Either PEG4-hydrogenated succinimide ester or diphenylcyclooctyne-carbon 6-succinimide ester is preferably dibenzylcyclooctyl-sulfo-N-hydroxysuccinimide ester.
  • the bone-targeting molecule is a bone-targeting molecule modified with an azide group
  • the bone-targeting molecule includes a polypeptide bone-targeting molecule and a chemical bone-targeting molecule.
  • the amino acid sequence of the polypeptide bone-targeting molecule includes (D)n, SDSSD (SEQ ID NO:6), (DSS)6 (SEQ ID NO:7) or TPLSYLKGLVTVG (SEQ ID NO:1) Any one or a combination of at least two, wherein n in (D)n is 6-10, n is a positive integer, for example, it can be 6, 7, 8, 9 or 10; the polypeptide bone targeting The molecule is linked to the azide group via lysine.
  • the chemical bone targeting molecule includes any one or a combination of at least two of bisphosphonate, alendronate, zoledronate or pamidronate.
  • the molar ratio of the bone-targeting molecule and the bifunctional molecule in the bone-targeting extracellular vesicle is 1:(5-20), for example, it can be 1:5, 1:8, 1:10, 1 :12, 1:14, 1:15, 1:18 or 1:20, etc., preferably 1:(12-18).
  • the loading amount of the bone-targeting molecule on the bone-targeting extracellular vesicle is 0.1-0.3 ⁇ mol per gram of vesicle, for example, it can be 0.1 ⁇ mol, 0.15 ⁇ mol, 0.2 ⁇ mol, 0.25 ⁇ mol or 0.3 ⁇ mol, etc.
  • the loading amount of the bone-promoting drug in the bone-targeting extracellular vesicle is 5-50%, for example, it can be 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45% % or 50% etc.
  • the structure of the bone-targeting extracellular vesicle is a double-layered phospholipid layer vesicle.
  • the particle size of the bone-targeting extracellular vesicles is 30-200 nm, for example, it can be 30 nm, 50 nm, 80 nm, 100 nm, 120 nm, 150 nm, 160 nm, 180 nm or 200 nm, etc.
  • the zeta potential of the bone-targeting extracellular vesicle is -5 to -40mV, for example, it can be -5mV, -10mV, -15mV, -20mV, -25mV, -30mV, -35mV or -40mV, etc.
  • the bone-promoting drug includes any one or a combination of at least two of nucleic acids, proteins, peptide chains or small molecule chemical drugs.
  • the nucleic acid includes any one or a combination of at least two of DNA, iRNA, micro RNA, siRNA, shRNA, mRNA, ncRNA, antisense RNA, LNA or morpholino oligonucleotide, preferably micro RNA .
  • Nucleic acids described in this application also include morpholino oligonucleotide analogs or morpholino oligonucleotide conjugates.
  • the protein includes any one or a combination of at least two of bone morphogenetic proteins, osteopontin, catenin, collagen or silk fibroin.
  • the peptide chain includes any one or a combination of at least two of teriparatide, osteogenic growth polypeptide or RGD peptide.
  • the peptide chains described in this application have bone-promoting effects, and also include parathyroid hormone-related peptides, calcitonin gene-related peptides, growth factor short peptide derivatives, extracellular matrix-derived peptides, hydroxyapatite-binding peptides or self-assembly Peptides.
  • the self-assembling peptide includes RADA16-I.
  • the small molecule chemical drugs in the bone-promoting drugs include any one or a combination of at least two of dedecenoic acid, flavonoids, quinones, Wnt pathway-related drugs or RANKL pathway-related drugs.
  • the Wnt pathway is a set of signal transduction pathways with multiple downstream channels mediated by the binding of the ligand Wnt to membrane receptors. Included drugs are sclerostin, Dickkopf-1, and secreted Frizzled-related protein.
  • the source of the extracellular vesicles is human stem cells.
  • the human stem cells include human bone marrow mesenchymal stem cells, human adipose stem cells, human umbilical cord mesenchymal stem cells, human umbilical cord blood mesenchymal stem cells, human placental mesenchymal stem cells, human dental pulp stem cells, and human periodontal ligament. Any one of stem cells, human hair follicle stem cells or human amniotic membrane mesenchymal stem cells.
  • the extracellular vesicles are obtained by the following preparation method:
  • step (b) Centrifuge the culture medium collected in step (a) with increasing centrifugal force three times to obtain the extracellular vesicles.
  • the specific steps of culturing until the confluence is 80-90% are: mixing human stem cells and complete culture medium, and culturing at 37°C for 2-3 days until the confluence is 80-90%. % (for example, it can be 80%, 85% or 90%, etc.); wherein, the amount of human stem cells added per 1 mL of complete culture medium is 5 ⁇ 10 5 -1 ⁇ 10 6 cells (for example, it can be 5 ⁇ 10 5 , 6 ⁇ 10 5 , 7 ⁇ 10 5 , 8 ⁇ 10 5 , 9 ⁇ 10 5 or 1 ⁇ 10 6 etc.).
  • the specific steps of culturing in complete culture medium are: transferring human stem cells with a confluency of 80-90% to a complete culture medium prepared with extracellular vesicle-free serum, and Cultivate at 37°C for 36-72h (for example, it can be 36h, 48h, 60h or 72h, etc.); wherein, the amount of stem cells added per 1 mL of complete culture medium is 5 ⁇ 10 5 -1 ⁇ 10 6 cells (for example, it can be 5 ⁇ 10 5 , 6 ⁇ 10 5 , 7 ⁇ 10 5 , 8 ⁇ 10 5 , 9 ⁇ 10 5 or 1 ⁇ 10 6 etc.).
  • the complete culture medium includes ⁇ -MEM culture medium or DMEM culture medium, and 8-12% fetal bovine serum (for example, 8%, 10% fetal bovine serum) needs to be added to the complete culture medium. % or 12%, etc.) and 0.5-2% double antibody (for example, it can be 0.5%, 1%, 1.5% or 2%, etc.).
  • step (b) the specific steps of the centrifugal treatment with three increasing centrifugal forces are:
  • the culture collected in step (a) is based on 0-10°C (for example, it can be 0°C, 2°C, 4°C, 6°C, 8°C or 10°C, etc.), 800-1200g (for example, it can be 800g, 900g, 1000g) , 1100g or 1200g, etc.), centrifuge for 10-30min (for example, it can be 10min, 15min, 20min, 25min or 30min, etc.), and collect the supernatant;
  • the supernatant liquid is heated at 0-10°C (for example, it can be 0°C, 2°C, 4°C, 6°C, 8°C or 10°C, etc.), 8000-12000g (for example, it can be 8000g, 9000g, 10000g, 11000g or 12000g Centrifuge for 30-60min (for example, 30min, 35min, 40min, 45min, 50min, 55min or 60min, etc.) under centrifugal force, and collect the supernatant;
  • the supernatant is heated at 0-10°C (for example, it can be 0°C, 2°C, 4°C, 6°C, 8°C or 10°C, etc.), 80000-120000g (for example, it can be 80000g, 90000g, 100000g, 110000g or 120000g Centrifuge for 90-120 min (for example, 90 min, 95 min, 100 min, 105 min, 110 min, 115 min or 120 min, etc.) under centrifugal force, collect the precipitate, and obtain the extracellular vesicles.
  • 90-120 min for example, 90 min, 95 min, 100 min, 105 min, 110 min, 115 min or 120 min, etc.
  • step (b) the extracellular vesicles need to be resuspended and stored in sterile PBS buffer.
  • the mass ratio of the extracellular vesicles and sterile PBS buffer is 1:(1-5), for example, it can be 1:1, 1:2, 1:3, 1:4 or 1:5, etc. .
  • the extracellular vesicles are prepared by the following preparation method:
  • Human stem cells with a confluence of 80-90% are transferred to a complete medium prepared with extracellular vesicle-free serum and cultured at 37°C for 36-72 hours.
  • the complete medium contains 8-12% fetal bovine serum and 0.5 -2% double antibody complete culture medium; the amount of human stem cells added per 1 mL of complete culture medium is 5 ⁇ 10 5 -1 ⁇ 10 6 cells;
  • Collect the culture medium and perform three centrifugal treatments with increasing centrifugal force The specific steps of the three centrifugal treatments with increasing centrifugal force are: centrifuge the collected culture for 10-30 minutes at a centrifugal force of 0-10°C and 800-1200g, and collect the supernatant. liquid; then centrifuge the supernatant at 0-10°C and 8000-12000g for 30-60min to collect the supernatant; finally, centrifuge the supernatant at 0-10°C and 80000-120000g for 90-120min , collect the precipitate to obtain the extracellular vesicles; resuspend and store the obtained extracellular vesicles and sterile PBS buffer at a mass ratio of 1: (1-5).
  • this application provides a method for preparing bone-targeted extracellular vesicles as described in the first aspect.
  • the method for preparing bone-targeted extracellular vesicles includes the following steps:
  • Modification of bone-targeting molecules Modify bone-targeting molecules on the outer surface of extracellular vesicles through click chemical reactions to obtain extracellular vesicles with bone-targeting molecules modified on the outer surface;
  • This application obtains extracellular vesicles secreted by human stem cells through step-by-step centrifugation, uses click chemistry to modify the surface of the extracellular vesicles with polypeptide bone-targeting molecules, and uses an electroporation method to load bone-promoting related drugs to obtain the bone Targeting extracellular vesicles.
  • the bone-targeted modification of extracellular vesicles in this application is gentle and convenient, does not destroy the structure of the extracellular vesicles themselves, and does not affect the performance of the extracellular vesicles themselves, and the polypeptide bone used in this application Targeted molecules have low toxic side effects and good targeting effects.
  • step (1) the specific steps of modification are:
  • the bifunctional molecule is selected from dibenzylcyclooctyl-sulfo-N-hydroxysuccinimide ester, azadibenzocyclooctyne-succinimide ester, diphenylcyclooctyne- Any one of PEG4-hydrogenated succinimide ester or diphenylcyclooctyne-carbon 6-succinimide ester; preferably dibenzylcyclooctyl-sulfo-N-hydroxysuccinimide ester.
  • the concentration of the bifunctional molecule in the mixed liquid obtained after the mixing is 10-50 ⁇ M, for example, it can be 10 ⁇ M, 15 ⁇ M, 20 ⁇ M, 25 ⁇ M, 30 ⁇ M, 35 ⁇ M, 40 ⁇ M, 45 ⁇ M or 50 ⁇ M, etc.;
  • the concentration of the extracellular vesicles is 0.5-1 mg/mL, for example, it can be 0.5 mg/mL, 0.6 mg/mL, 0.7 mg/mL, 0.8 mg/mL, 0.9 mg/mL or 1 mg/mL, etc.
  • the temperature of the grafting reaction is 30-40°C, for example, it can be 30°C, 33°C, 35°C, 37°C or 40°C, etc.; and the time of the grafting reaction is 3-6h, for example, it can be 3h, 3.5h, 4h, 4.5h, 5h, 5.5h or 6h, etc.
  • step (A) after the grafting reaction is completed, the ungrafted bifunctional molecules need to be removed by ultrafiltration and centrifugation.
  • the concentration of extracellular vesicles with bifunctional molecules grafted on their surfaces in the solution obtained after resuspension is 0.5-5 mg/mL, for example, it can be 0.5 mg/mL or 1 mg/mL. , 1.5mg/mL, 2mg/mL, 2.5mg/mL, 3mg/mL, 3.5mg/mL, 4mg/mL, 4.5mg/mL or 5mg/mL, etc.
  • the concentration of the bone-targeting molecule in the mixed solution obtained after mixing is 10-50 ⁇ M, for example, it can be 10 ⁇ M, 15 ⁇ M, 20 ⁇ M, 25 ⁇ M, 30 ⁇ M, 35 ⁇ M, 40 ⁇ M, 45 ⁇ M or 50 ⁇ M, etc. ;
  • the concentration of the extracellular vesicles with bifunctional molecules grafted on the surface is 0.5-5mg/mL, for example, it can be 0.5mg/mL, 1mg/mL, 2mg/mL, 3mg/mL, 4mg/mL or 5mg /mL etc.
  • the temperature of the coupling reaction is 0-10°C, for example, it can be 0°C, 2°C, 4°C, 6°C, 8°C or 10°C; the temperature of the coupling reaction
  • the time is 8-12h, for example, it can be 8h, 9h, 10h, 11h or 12h, etc.
  • step (B) after the coupling reaction is completed, the unmodified bone targeting molecules need to be removed by ultrafiltration centrifugation.
  • step (B) the extracellular vesicles modified with bone-targeting molecules on their outer surfaces need to be resuspended and stored in sterile PBS buffer.
  • the mass ratio of the extracellular vesicles modified with bone-targeting molecules on their outer surfaces and the sterile PBS buffer is 1:(1-5), for example, it can be 1:1, 1:1.5, or 1:2 , 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5 or 1:5, etc.
  • the specific steps of loading are: mixing the extracellular vesicles with bone-targeting molecules modified on their outer surfaces, bone-promoting drugs and electroporation buffer, performing electroporation, and completing the poration. Then, it is allowed to stand to obtain the bone-targeting extracellular vesicles.
  • the concentration of extracellular vesicles with bone-targeting molecules modified on their outer surfaces in the mixed solution obtained after mixing is 0.5-5 mg/mL, for example, it can be 0.5 mg/mL, 1 mg/mL, 2 mg/mL, or 3 mg.
  • the concentration of the bone-promoting drug is 0.01-0.5 ⁇ g/ ⁇ L, for example, it can be 0.01 ⁇ g/ ⁇ L, 0.02 ⁇ g/ ⁇ L, 0.03 ⁇ g/ ⁇ L, 0.05 ⁇ g/ ⁇ L , 0.08 ⁇ g/ ⁇ L, 0.1 ⁇ g/ ⁇ L, 0.2 ⁇ g/ ⁇ L, 0.3 ⁇ g/ ⁇ L, 0.4 ⁇ g/ ⁇ L or 0.5 ⁇ g/ ⁇ L, etc.
  • the electroporation buffer includes a mixture of iodixanol and potassium dihydrogen phosphate-potassium chloride solution with a volume ratio of (18-23):(77-82), and the volume ratio can be, for example, 18 :82, 19:81, 20:80, 21:79, 22:78 or 23:77, etc.;
  • the mass concentration of potassium dihydrogen phosphate in the potassium dihydrogen phosphate-potassium chloride solution is 1-1.2mM (for example It can be 1mM, 1.1mM or 1.2mM, etc.), the mass concentration of potassium chloride is 23-27mM (for example, it can be 23mM, 24mM, 25mM, 26mM or 27mM, etc.), and the solvent is water.
  • the electroporation voltage is 100-400V, for example, it can be 100V, 150V, 250V, 300V, 350V or 400V, etc.
  • the electroporation time is 1-20ms, for example, it can be 1ms, 2ms, 5ms. , 8ms, 12ms, 16ms, 18ms or 20ms, etc.
  • the standing temperature is 30-40°C, for example, it can be 30°C, 33°C, 35°C, 37°C or 40°C, etc.; and the standing time is 25-35min, for example, it can be 25min. , 28min, 30min, 31min, 32min or 35min, etc.
  • unloaded bone-promoting drugs need to be removed by ultrafiltration centrifugation after said standing.
  • the preparation method of the bone-targeting extracellular vesicles includes the following steps:
  • (B) Resuspend the obtained extracellular vesicles with bifunctional molecules grafted on the surface in PBS buffer, and the concentration of the extracellular vesicles with bifunctional molecules grafted on the surface in the solution obtained after resuspension is 0.5- 5 mg/mL, and then mix the resuspended solution with the bone-targeting molecule.
  • the concentration of the bone-targeting molecule in the mixed solution obtained after mixing is 10-50 ⁇ M, and the surface is grafted with extracellular bifunctional molecules.
  • the concentration of vesicles is 0.5-5mg/mL; the coupling reaction is carried out at 0-10°C for 8-12 hours.
  • the unmodified bone-targeting molecules need to be removed by ultrafiltration centrifugation to obtain the bone-targeting molecule modified on the outer surface.
  • Molecule extracellular vesicles resuspend and preserve the extracellular vesicles with bone-targeting molecules modified on their outer surfaces and sterile PBS buffer at a mass ratio of 1: (1-5);
  • the extracellular vesicles with bone-targeting molecules modified on their outer surfaces, bone-promoting drugs and electroporation buffer are mixed.
  • the extracellular vesicles with bone-targeting molecules modified on their outer surfaces are The concentration is 0.5-5mg/mL, and the concentration of the bone-promoting drug is 0.01-0.5 ⁇ g/ ⁇ L; electroporation is performed, the voltage of the electroporation is 100-400V, and the time is 1-20ms; after the completion of the perforation, the electroporation time is 30-30ms.
  • the present application provides a pharmaceutical composition comprising the bone-targeting extracellular vesicle described in the first aspect.
  • the pharmaceutical composition includes pharmaceutically acceptable excipients.
  • this application provides any one of the bone-targeting extracellular vesicles described in the first aspect, the preparation method of the bone-targeting extracellular vesicles described in the second aspect, or the pharmaceutical composition described in the third aspect.
  • the bone-targeting molecule described in this application is connected and modified on the surface of the extracellular vesicle loaded with the bone-promoting drug through a click chemical reaction with a bifunctional molecule to generate a triazole bond, and the bifunctional molecule interacts with the extracellular
  • the amino groups on the surface of the vesicles form covalent bonds and are grafted onto the outer surface of the extracellular vesicles loaded with bone-promoting drugs.
  • the preparation method of the bone-targeting extracellular vesicles reacts to the bone-targeting modification of the extracellular vesicles. Gentle and convenient, it will not damage the structure of the extracellular vesicles themselves, nor will it affect the performance of the extracellular vesicles themselves.
  • the bone-targeting extracellular vesicles described in this application can improve the therapeutic effect after being loaded with bone-promoting drugs, overcoming the shortcomings of poor therapeutic effect and weak targeting of original stem cell extracellular vesicles.
  • the polypeptide bone-targeting molecule described in this application has good targeting effect and is conducive to delivering extracellular vesicles to the surface of bone tissue to improve the therapeutic effect.
  • Figure 1 is the transmission electron microscope detection result of extracellular vesicles in Example 1 (scale bar 100 nm).
  • Figure 2 is the transmission electron microscope detection result of bone-targeting extracellular vesicles in Example 1 (scale bar 200 nm).
  • Figure 3 is the transmission electron microscope detection result of bone-targeting extracellular vesicles in Example 1 (scale bar 200 nm).
  • Figure 4 is the fluorescence intensity curve of bone-targeted extracellular vesicles in Test Example 2.
  • Figure 5 is a graph showing the statistical results of particle size distribution of the bone-targeting extracellular vesicles (Example 1) described in Test Example 2.
  • Figures 6A and 6B show the distribution of bone-targeted extracellular vesicles in various mouse tissues in Test Example 3.
  • Figures 7A and 7B are the staining results of the in vitro bone-promoting effect test in Test Example 4.
  • This embodiment provides a bone-targeting extracellular vesicle, the extracellular vesicle is produced by human bone marrow mesenchymal stem cells, the bone-targeting molecule is a polypeptide bone-targeting molecule, and the polypeptide bone-targeting molecule is The amino acid sequence of the molecule is (D) 8 (SEQ ID NO: 8).
  • the polypeptide bone-targeting molecule is connected to the azide group through lysine.
  • the bone-promoting drug includes micro RNA.
  • the micro RNA The nucleotide sequence is UUCAAGUAAUCCAGGAUAGGCU (SEQ ID NO: 2).
  • the preparation method of the extracellular vesicles is as follows:
  • Human bone marrow mesenchymal stem cells with a confluency of 80-90% were transferred to complete medium prepared with extracellular vesicle-free serum, and cultured at 37°C and 5% CO2 for 48 hours. Human bone marrow in every 1 mL of complete medium The amount of mesenchymal stem cells added is 5 ⁇ 10 5 cells; the complete medium is ⁇ -MEM medium containing 10% fetal bovine serum and 1% double antibody;
  • centrifuge 1000g for 10 minutes at 4°C to obtain supernatant one, which is then centrifuged at 4°C. Centrifuge at 10,000g for 30 minutes to obtain supernatant two, and then centrifuge said supernatant two at 100,000g for 90 minutes at 4°C; and
  • the extracellular vesicles need to be resuspended and stored in sterile PBS buffer.
  • the mass ratio of the extracellular vesicles and sterile PBS buffer is 1:1.
  • the preparation method of the bone-targeting extracellular vesicles is as follows:
  • concentration of sulfo-N-hydroxysuccinimide ester is 30 ⁇ M, and the concentration of the extracellular vesicles is 0.8 mg/mL; after mixing, the grafting reaction is carried out at 37°C for 5 hours; after the grafting reaction is completed, ultrafiltration is required Centrifuge to remove ungrafted dibenzylcyclooctyl-sulfo-N-hydroxysuccinimide ester.
  • the molecular weight cutoff of the ultrafiltration centrifuge tube used in the ultrafiltration centrifugation is 100kDa, and the surface grafted dibenzylcyclooctyl-N-hydroxysuccinimide ester is obtained. Extracellular vesicles of hydroxy-sulfo-N-hydroxysuccinimide ester;
  • the mixture obtained after mixing The concentration of the bone-targeting molecule in the solution is 30 ⁇ M, and the concentration of the extracellular vesicles grafted with dibenzylcyclooctyl-sulfo-N-hydroxysuccinimide ester on the surface is 3 mg/mL; at 4 °C coupling reaction for 10 hours.
  • the unmodified bone-targeting molecules need to be removed by ultrafiltration centrifugation.
  • the molecular weight cutoff of the ultrafiltration centrifuge tube used in the ultrafiltration centrifugation is 100kDa, and the outer surface is modified with bone-targeting molecules.
  • the extracellular vesicles with bone-targeting molecules modified on their outer surfaces, bone-promoting drugs and electroporation buffer are mixed.
  • the extracellular vesicles with bone-targeting molecules modified on their outer surfaces are The concentration is 3 mg/mL, and the concentration of the bone-promoting drug is 0.01 ⁇ g/ ⁇ L; electroporation is performed, and the voltage of the electroporation is 350V and the time is 10 ms; after the perforation is completed, it is allowed to stand at 37°C for 30 minutes.
  • the unloaded bone-promoting drugs are removed by ultrafiltration centrifugation using an ultrafiltration centrifuge tube with a molecular weight cutoff of 100 kDa to obtain the bone-targeting extracellular vesicles.
  • the electroporation buffer is a mixture of iodixanol (density gradient separation liquid iodixanol, optiprep, Axis-Shield) and potassium dihydrogen phosphate-potassium chloride solution with a volume ratio of 21:79.
  • the transmission electron microscope detection results of the bone-targeting extracellular vesicles are shown in Figure 3. It can be seen from Figure 3 that the morphology of the bone-targeting extracellular vesicles is spherical and the size is about 120 nm.
  • This embodiment provides a bone-targeting extracellular vesicle, the extracellular vesicle is produced by human adipose stem cells, the bone-targeting molecule is a polypeptide bone-targeting molecule, and the amino acid of the polypeptide bone-targeting molecule
  • the sequence is SDSSD (SEQ ID NO: 6)
  • the polypeptide bone-targeting molecule is connected to the azide group through lysine
  • the bone-promoting drug includes micro RNA
  • the nucleotide sequence of the micro RNA is UAGCACCAUUUGAAAUCAGUGUU(SEQ ID NO:3).
  • the method for preparing the extracellular vesicles is as follows:
  • Human adipose stem cells with a confluency of 80-90% were transferred to complete medium prepared with extracellular vesicle-free serum and cultured at 37°C and 5% CO2 for 36 hours.
  • the amount of human adipose stem cells added per 1 mL of complete culture medium was The amount is 8 ⁇ 10 5 ; the complete culture medium is ⁇ -MEM culture medium containing 8% fetal calf serum and 0.5% double antibody;
  • the extracellular vesicles need to be resuspended and stored in sterile PBS buffer.
  • the mass ratio of the extracellular vesicles and sterile PBS buffer is 1:1.
  • the preparation method of the bone-targeting extracellular vesicles is as follows:
  • concentration of sulfo-N-hydroxysuccinimide ester is 10 ⁇ M, and the concentration of the extracellular vesicles is 0.5 mg/mL; after mixing, the grafting reaction is carried out at 30°C for 6 hours; after the grafting reaction is completed, ultrafiltration is required Centrifuge to remove ungrafted dibenzylcyclooctyl-sulfo-N-hydroxysuccinimide ester.
  • the molecular weight cutoff of the ultrafiltration centrifuge tube used in the ultrafiltration centrifugation is 100kDa, and the surface grafted dibenzylcyclooctyl-N-hydroxysuccinimide ester is obtained. Extracellular vesicles of hydroxy-sulfo-N-hydroxysuccinimide ester;
  • the mixture obtained after mixing The concentration of the bone-targeting molecule in the liquid is 10 ⁇ M, and the concentration of the extracellular vesicles grafted with dibenzylcyclooctyl-sulfo-N-hydroxysuccinimide ester on the surface is 0.5 mg/mL; in The coupling reaction is carried out at 2°C for 12 hours. After the coupling reaction is completed, the unmodified bone targeting molecules need to be removed by ultrafiltration centrifugation. The molecular weight cutoff of the ultrafiltration centrifuge tube used in the ultrafiltration centrifugation is 100kDa, and the outer surface is modified with bone targets. To prepare extracellular vesicles of molecules, resuspend and store the extracellular vesicles with bone-targeting molecules modified on their outer surfaces and sterile PBS buffer at a mass ratio of 1:1.5.
  • the extracellular vesicles with bone-targeting molecules modified on their outer surfaces, bone-promoting drugs and electroporation buffer are mixed.
  • the extracellular vesicles with bone-targeting molecules modified on their outer surfaces are The concentration is 0.5 mg/mL, and the concentration of the bone-promoting drug is 0.02 ⁇ g/ ⁇ L; electroporation is performed, and the voltage of the electroporation is 400V and the time is 10 ms; after the perforation is completed, let it stand at 30°C for 35 minutes.
  • the unloaded bone-promoting drugs need to be removed by ultrafiltration centrifugation using an ultrafiltration centrifuge tube with a molecular weight cutoff of 100 kDa to obtain the bone-targeting extracellular vesicles.
  • the electroporation buffer is a mixture of iodixanol (density gradient separation liquid iodixanol, optiprep, Axis-Shield) and potassium dihydrogen phosphate-potassium chloride solution with a volume ratio of 21:79.
  • This embodiment provides a bone-targeting extracellular vesicle, the extracellular vesicle is produced by human umbilical cord mesenchymal stem cells, the bone-targeting molecule is a polypeptide bone-targeting molecule, and the polypeptide bone-targeting molecule is The amino acid sequence of the molecule is (DSS) 6 (SEQ ID NO: 7).
  • the polypeptide bone-targeting molecule is connected to the azide group through lysine.
  • the bone-promoting drug includes micro RNA.
  • the micro RNA The nucleotide sequence is UUCACAGUGGCUAAGUUCCGC (SEQ ID NO: 4).
  • the method for preparing the extracellular vesicles is as follows:
  • Human umbilical cord mesenchymal stem cells with a confluency of 80-90% were transferred to complete medium prepared with extracellular vesicle-free serum, and cultured at 37°C and 5% CO2 for 72 hours.
  • Human umbilical cord mesenchymal stem cells per 1 mL of complete medium The added amount of mesenchymal stem cells is 1 ⁇ 10 6 cells; the complete medium is DMEM medium containing 12% fetal bovine serum and 2% double antibodies;
  • the extracellular vesicles need to be resuspended and stored in sterile PBS buffer.
  • the mass ratio of the extracellular vesicles and sterile PBS buffer is 1:1.
  • the preparation method of the bone-targeting extracellular vesicles is as follows:
  • the molecular weight cutoff of the ultrafiltration centrifuge tube used in the ultrafiltration centrifugation is 100kDa, and obtain the surface grafted dibenzylcyclooctyl group. -Extracellular vesicles of sulfo-N-hydroxysuccinimide ester;
  • the mixed solution obtained after mixing The concentration of the bone-targeting molecule described in is 50 ⁇ M, and the concentration of the extracellular vesicles grafted with dibenzylcyclooctyl-sulfo-N-hydroxysuccinimide ester on the surface is 5 mg/mL; at 6°C
  • the coupling reaction is carried out for 8 hours.
  • the unmodified bone-targeting molecules need to be removed by ultrafiltration centrifugation.
  • the molecular weight cutoff of the ultrafiltration centrifuge tube used in the ultrafiltration centrifugation is 100kDa, and the outer surface is modified with bone-targeting molecules.
  • Extracellular vesicles, the extracellular vesicles modified with bone-targeting molecules on their outer surfaces and sterile PBS buffer are resuspended and stored at a mass ratio of 1:2.
  • the extracellular vesicles with bone-targeting molecules modified on their outer surfaces, bone-promoting drugs and electroporation buffer are mixed.
  • the extracellular vesicles with bone-targeting molecules modified on their outer surfaces are The concentration is 5 mg/mL, and the concentration of the bone-promoting drug is 0.5 ⁇ g/ ⁇ L; electroporation is performed, and the voltage of the electroporation is 200V and the time is 15 ms; after the perforation is completed, it is allowed to stand at 40°C for 25 minutes.
  • the unloaded bone-promoting drugs are removed by ultrafiltration centrifugation using an ultrafiltration centrifuge tube with a molecular weight cutoff of 100 kDa to obtain the bone-targeting extracellular vesicles.
  • the electroporation buffer is a mixture of iodixanol (density gradient separation liquid iodixanol, optiprep, Axis-Shield) and potassium dihydrogen phosphate-potassium chloride solution with a volume ratio of 21:79.
  • This embodiment provides a bone-targeting extracellular vesicle.
  • the bone-promoting drug is siRNA
  • the nucleotide sequence of the siRNA is GAAGUGGUUCAGAAGAUGACGCCCAAACCCAAACCCAAACCCAAACCCAAA (SEQ IDNO:5).
  • the preparation method of the bone-targeting extracellular vesicles is as described in Example 1.
  • This embodiment provides a bone-targeting extracellular vesicle.
  • the only difference between the bone-targeting extracellular vesicle and Example 1 is that the bone-promoting drug is a small molecule chemical drug, and the small molecule chemical drug is Bone morphogenetic protein.
  • the preparation method of the bone-targeting extracellular vesicles is as described in Example 1.
  • This embodiment provides a bone-targeting extracellular vesicle.
  • the only difference between the preparation method of the bone-targeting extracellular vesicle and Example 1 is that the grafting reaction time in step (A) is 12 hours, The temperature of the grafting reaction is 25°C, and the remaining steps are as described in Example 1.
  • This embodiment provides a bone-targeting extracellular vesicle.
  • the only difference between the preparation method of the bone-targeting extracellular vesicle and Example 1 is that the grafting reaction time in step (A) is 2 hours, The temperature of the grafting reaction is 60°C, and the remaining steps are as described in Example 1.
  • This embodiment provides a bone-targeting extracellular vesicle.
  • the only difference between the preparation method of the bone-targeting extracellular vesicle and Example 1 is that the dibenzyl ring in the mixed solution in step (A)
  • the concentration of octyl-sulfo-N-hydroxysuccinimide ester is 5 ⁇ M; refer to Example 1 for the remaining steps.
  • This embodiment provides a bone-targeting extracellular vesicle.
  • the only difference between the preparation method of the bone-targeting extracellular vesicle and Example 1 is that the bone-targeting extracellular vesicle in the mixed solution in step (B)
  • the concentration of the molecule is 5 ⁇ M, and the remaining steps are as described in Example 1.
  • This embodiment provides a bone-targeting extracellular vesicle.
  • the only difference between the preparation method of the bone-targeting extracellular vesicle and Example 1 is that the electroporation voltage in step (2) is 700V, so The electroporation time is 2 ms, and the remaining steps are as described in Example 1.
  • This embodiment provides a bone-targeting extracellular vesicle.
  • the only difference between the preparation method of the bone-targeting extracellular vesicle and Example 1 is that the electroporation voltage in step (2) is 50V, so The electroporation time is 25 ms, and the remaining steps are as described in Example 1.
  • This comparative example provides a non-targeted extracellular vesicle, the extracellular vesicle is produced by human bone marrow mesenchymal stem cells, the bone-promoting drug includes microRNA, and the nucleotide sequence of the microRNA is UUCAAGUAAUCCAGGAUAGGCU ( SEQ ID NO:2).
  • the extracellular vesicles do not contain bone-targeting molecules.
  • the method for preparing the extracellular vesicles is as follows:
  • Human bone marrow mesenchymal stem cells with a confluency of 80-90% were transferred to complete medium prepared with extracellular vesicle-free serum, and cultured at 37°C and 5% CO2 for 48 hours. Human bone marrow in every 1 mL of complete medium The amount of mesenchymal stem cells added is 5 ⁇ 10 5 cells; the complete medium is ⁇ -MEM medium containing 10% fetal bovine serum and 1% double antibody;
  • centrifuge 1000g for 10 minutes at 4°C to obtain supernatant one, which is then centrifuged at 4°C. Centrifuge at 10,000g for 30 minutes to obtain supernatant two, and then centrifuge said supernatant two at 100,000g for 90 minutes at 4°C; and
  • the precipitate was collected to obtain extracellular vesicles.
  • the method for preparing the non-targeted extracellular vesicles is as follows:
  • the extracellular vesicles, bone-promoting drugs and electroporation buffer are mixed.
  • the concentration of the extracellular vesicles in the mixed solution obtained after mixing is 3 mg/mL, and the concentration of the bone-promoting drugs is 0.03 ⁇ g/ ⁇ L.
  • Carry out electroporation the electroporation voltage is 350V and the time is 10ms; after the perforation is completed, let it stand at 37°C for 30 minutes. After standing, the unloaded bone-promoting drugs need to be removed by ultrafiltration centrifugation.
  • the ultrafiltration centrifugation is used The ultrafiltration centrifuge tube has a molecular weight cutoff of 100 kDa to obtain the non-targeted extracellular vesicles.
  • This comparative example provides a non-bone-targeted extracellular vesicle.
  • the only difference between the non-bone-targeted extracellular vesicle and Example 1 is that (G) 6 (SEQ ID NO: 9) is used to replace Example 1.
  • the preparation method of the non-bone-targeted extracellular vesicles is as described in Example 1.
  • This comparative example provides a bone-targeting vesicle.
  • the only difference between the bone-targeting vesicle and Example 1 is that the vesicle is a liposome vesicle.
  • the preparation method of the liposome vesicles includes the following steps:
  • the formed multicolloidal vesicles were then extruded through a 0.2 ⁇ m polycarbonate membrane (Whatman) 10 times using an Avanti mini extruder (Avanti Polar Lipids), and then through a 0.1 ⁇ m membrane 10 times to gradually form the final Liposomes.
  • the preparation method of the bone-targeting vesicles refers to step (1) and step (2) of Example 1.
  • This test example is to test the adsorption rate of the bone-targeting extracellular vesicles in Examples 1-11 and the vesicles in Comparative Examples 1-3. Taking the extracellular vesicle mimic as an example, the adsorption rate test is The method includes the following steps:
  • Rhodamine B fluorescently labeled polypeptide targeting molecule with the amino acid sequence (D) 8 (SEQ ID NO: 8) was modified on the extracellular vesicle mimic, and prepared into a 1 mg/mL suspension in PBS , use a fluorescence spectrophotometer to measure the fluorescence intensity of the suspension, set the excitation wavelength to 550nm, and the emission wavelength to 580nm; after measurement, mix the extracellular vesicle mimic suspension with 10 mg/mL hydroxyapatite particles at 25°C Incubate for 5 hours in an air bath shaker.
  • D amino acid sequence
  • the suspension is centrifuged at 4000 rpm for 10 minutes to precipitate the hydroxyapatite and extracellular vesicle mimics adsorbed on it.
  • a fluorescence spectrophotometer to measure the content of the supernatant. Fluorescence intensity, excitation wavelength 550nm, emission wavelength 580nm, calculate adsorption rate through the following formula:
  • I1 Fluorescence intensity before incubation with hydroxyapatite
  • I2 Fluorescence intensity after incubation with hydroxyapatite.
  • adsorption rates of the bone-targeting extracellular vesicles in Examples 1-11 and the vesicles in Comparative Examples 1-3 were measured with reference to the adsorption rate test method of extracellular vesicle mimics. Appropriate modification conditions can be selected by calculating the adsorption rate. The test results of adsorption rate are shown in Table 1.
  • This test example is to conduct particle size distribution test, drug loading test, vesicle concentration test, and bone-targeting molecule modification on the bone-targeting extracellular vesicles in Examples 1-11 and the vesicles in Comparative Examples 1-3. Efficiency testing and zeta potential measurements of bone-targeting extracellular vesicles.
  • the extracellular vesicles and PBS were mixed at a ratio of 1:1 and the particle size distribution was measured using a Zetasizer nanoparticle size analyzer.
  • the detection method of drug loading test includes the following steps:
  • Vesicle concentration was tested using a BCA kit according to the instructions.
  • the detection method for bone-targeting molecule modification efficiency testing includes the following steps:
  • the extracellular vesicles were mixed with PBS at a ratio of 1:1 and the zeta potential was measured using a Zetasizer nanoparticle size analyzer.
  • Example 1 It can be seen from the comparison between Example 1 and Examples 6-7 that prolonging the reaction time or raising the reaction temperature will cause the vesicles to break and thereby reduce the yield of the vesicles.
  • Example 1 It can be seen from the comparison between Example 1 and Examples 8-9 that lowering the reagent concentration will lead to insufficient modification efficiency. Similarly, too high a reagent concentration will also cause vesicles to be broken and the modification efficiency will decrease.
  • Example 1 It can be seen from the comparison between Example 1 and Examples 10-11 that a lower voltage cannot penetrate the vesicles, resulting in the inability to load more drugs, and a higher voltage will crush the vesicles, resulting in the inability to load the drug.
  • the detection method includes the following steps:
  • the polypeptide bone-targeting molecules in the bone-targeting extracellular vesicles provided in this application have good targeting effects and are conducive to delivering the extracellular vesicles to the surface of bone tissue to improve the therapeutic effect.
  • the bone Loading bone-promoting drugs into targeted extracellular vesicles can further enhance the therapeutic effect and overcome the shortcomings of poor therapeutic effect and weak targeting of original stem cell extracellular vesicles.
  • the bone-targeted extracellular vesicles are used in the preparation of orthopedic treatments. It has important application value in products related to diseases.

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Abstract

本申请提供了一种骨靶向细胞外囊泡及其制备方法和应用，所述骨靶向细胞外囊泡包括外表面修饰有骨靶向分子的细胞外囊泡和装载于所述细胞外囊泡内的促成骨药物。本申请中所述骨靶向细胞外囊泡通过骨组织靶向实现靶器官精准递送，并且通过装载促成骨药物提高促成骨治疗效果。在制备所述骨靶向细胞外囊泡时，对细胞外囊泡的骨靶向修饰反应温和便捷，不会破坏细胞外囊泡本身的结构，也不会影响细胞外囊泡本身的性能，且本申请中所使用的多肽类骨靶向分子的毒副作用较低，骨靶向效果好。

Description

一种骨靶向细胞外囊泡及其制备方法和应用 技术领域
本申请属于生物医用材料技术领域，具体涉及一种骨靶向细胞外囊泡及其制备方法和应用。
背景技术
创伤、感染和肿瘤切除等原因均会导致骨组织缺损，尽管骨组织能够自我再生，但再生能力有限。此外，人的老龄化会进一步延长骨再生的时间，且肥胖等原因也会导致骨骼再生能力的减弱。因此，临床上促进骨再生是治疗骨缺损疾病的重点。
目前临床使用的促进骨再生的药物价格昂贵或存在毒副作用，因而限制了其广泛使用。近年来的研究发现，骨的再生受到多种细胞的调控，尤其是具有再生能力的干细胞，而干细胞促进骨再生主要是通过释放包含有各类蛋白和核酸类(如miRNA)物质的囊泡-细胞外囊泡实现的。研究发现细胞外囊泡能参与细胞间通讯、免疫调节、能量代谢、组织修复、组织再生和新陈代谢等多种不同的生化和细胞过程。
细胞外囊泡作为药物载体具有天然的优势，较小的尺寸和双层磷脂层结构降低了血管堵塞的风险，有利于细胞摄取。此外细胞外囊泡还具有较低毒性和免疫原性等优良特点，且细胞外囊泡的表面富含氨基，便于功能基团的修饰。因此，使用细胞外囊泡作为促进骨再生的药物具有天然的优势，但原始的细胞外囊泡靶向骨组织的能力一般，治疗效果有待进一步提升。
因此，提供一种具有骨靶向能力装载小分子药物的细胞外囊泡及其制备方法具有重要意义。
发明内容
本申请提供了一种骨靶向细胞外囊泡及其制备方法和应用。本申请中所述骨靶向细胞外囊泡包括外表面修饰有骨靶向分子的细胞外囊泡和装载于所述细胞外囊泡内的促成骨药物，本申请中所述骨靶向细胞外囊泡通过骨组织靶向实现靶器官精准递送，并且通过装载促成骨药物提高促成骨治疗效果。在制备所述骨靶向细胞外囊泡时，所述骨靶向分子通过与双功能分子的点击化学反应生成三氮唑键连接修饰在所述装载促成骨药物的细胞外囊泡表面，所述双功能分子通过与细胞外囊泡表面的氨基形成共价键接枝在所述装载促成骨药物的细胞外囊泡的外表面，对细胞外囊泡的骨靶向修饰反应温和便捷，不会破坏细胞外囊泡本身的结构，也不会影响细胞外囊泡本身的性能，且本申请中所使用的多肽类骨靶向分子的毒副作用较低，骨靶向效果好。
第一方面，本申请提供一种骨靶向细胞外囊泡，所述骨靶向细胞外囊泡包括外表面修饰有骨靶向分子的细胞外囊泡和装载于所述细胞外囊泡内的促成骨药物。
本申请采用人干细胞的细胞外囊泡为治疗工具，在细胞外囊泡的表面修饰骨靶向分子，可以通过骨组织靶向实现靶器官精准递送，所得骨靶向细胞外囊泡能够实现骨组织靶向并提高治疗效果。此外，通过装载促成骨药物可进一步提高治疗效果。
优选地，所述骨靶向分子通过与双功能分子的点击化学反应生成三氮唑键连接修饰在所述装载促成骨药物的细胞外囊泡表面；所述双功能分子通过与细胞外囊泡表面的氨基形成共价键接枝在所述装载促成骨药物的细胞外囊泡的外表面；所述双功能分子为同时带有二苯基环辛炔和N-羟基琥珀酰亚胺的分子。
优选地，所述双功能分子选自二苄环辛基-磺基-N-羟基琥珀酰亚胺酯、氮杂二苯并环辛炔-琥珀酰亚胺酯、二苯基环辛炔-PEG4-氢化琥珀酰亚胺酯或二苯基环辛炔-碳6-琥珀酰亚胺酯中任意一种，优选为二苄环辛基-磺基-N-羟基琥珀酰亚胺酯。
优选地，所述骨靶向分子为修饰有叠氮基团的骨靶向分子，所述骨靶向分子包括多肽类骨靶向分子和化学骨靶向分子。
优选地，所述多肽类骨靶向分子的氨基酸序列包括(D)n、SDSSD(SEQ ID NO:6)、(DSS)6(SEQ ID NO:7)或TPLSYLKGLVTVG(SEQ ID NO:1)中任意一种或至少两种的组合，其中，所述(D)n中n为6-10，n为正整数，例如可以是6、7、8、9或10；所述多肽类骨靶向分子通过赖氨酸与叠氮基团相连接。
优选地，所述化学骨靶向分子包括双膦酸盐、阿仑膦酸盐、唑来膦酸盐或帕米膦酸盐中任意一种或至少两种的组合。
优选地，所述骨靶向细胞外囊泡中的骨靶向分子和双功能分子的摩尔比为1:(5-20)，例如可以是1:5、1:8、1:10、1:12、1:14、1:15、1:18或1:20等，优选为1:(12-18)。
优选地，所述骨靶向细胞外囊泡上的骨靶向分子的负载量为每克囊泡0.1-0.3μmol，例如可以是0.1μmol、0.15μmol、0.2μmol、0.25μmol或0.3μmol等。
优选地，所述骨靶向细胞外囊泡中促成骨药物的装载量为5-50％，例如可以是5％、10％、15％、20％、25％、30％、40％、45％或50％等。
优选地，所述骨靶向细胞外囊泡的结构为双层磷脂层囊泡。
优选地，所述骨靶向细胞外囊泡的粒径为30-200nm，例如可以是30nm、50nm、80nm、100nm、120nm、150nm、160nm、180nm或200nm等。
优选地，所述骨靶向细胞外囊泡的zeta电势为-5～-40mV，例如可以是-5mV、-10mV、-15mV、-20mV、-25mV、-30mV、-35mV或-40mV等。
优选地，所述促成骨药物包括核酸、蛋白质、肽链或小分子化学药物中任意一种或至少两种的组合。
优选地，所述核酸包括DNA、iRNA、micro RNA、siRNA、shRNA、mRNA、ncRNA、反义RNA、LNA或吗啉代寡核苷酸中任意一种或至少两种的组合，优选为micro RNA。
本申请中所述核酸还包括吗啉代寡核苷酸类似物或吗啉代寡核苷酸缀合物。
优选地，所述蛋白质包括骨形态发生蛋白、骨桥蛋白、连环蛋白、胶原蛋白或丝素蛋白 中任意一种或至少两种的组合。
优选地，所述肽链包括特立帕肽、成骨生长多肽或RGD肽中任意一种或至少两种的组合。
本申请中所述肽链具有促成骨作用，还包括甲状旁腺激素相关肽、降钙素基因相关肽、生长因子短肽衍生物、细胞外基质衍生肽、羟基磷灰石结合肽或自组装肽。所述自组装肽包括RADA16-Ⅰ。
本申请中，所述促成骨药物中小分子化学药物包括十烯酸类、黄酮类、醌类、Wnt通路相关药物或RANKL通路相关药物中任意一种或至少两种的组合。Wnt通路由配体Wnt和膜受体结合介导的一组多下游通道的信号转导途径。所包含药物如硬骨素、Dickkopf-1、分泌型卷曲相关蛋白。
优选地，所述细胞外囊泡的来源为人干细胞。
优选地，所述人干细胞包括人骨髓间充质干细胞、人脂肪干细胞、人脐带间充质干细胞、人脐带血间充质干细胞、人胎盘间充质干细胞、人牙髓干细胞、人牙周膜干细胞、人毛囊干细胞或人羊膜间充质干细胞中任意一种。
优选地，所述细胞外囊泡由以下制备方法得到：
(a)将人源干细胞培养至汇合度为80-90％后，再于完全培养基中进行培养，收集培养基；
(b)将步骤(a)收集得到的培养基通过三次递增离心力的离心处理，得到所述细胞外囊泡。
优选地，步骤(a)中，所述培养至汇合度为80-90％的具体步骤为：将人源干细胞和完全培养基混合，于37℃培养2-3天至汇合度为80-90％(例如可以是80％、85％或90％等)；其中，每1mL完全培养基中人源干细胞的添加量为5×10 5-1×10 6个细胞(例如可以是5×10 5、6×10 5、7×10 5、8×10 5、9×10 5或1×10 6等)。
优选地，步骤(a)中，所述于完全培养基中进行培养的具体步骤为：将汇合度为80-90％的人源干细胞转移至无细胞外囊泡血清配制的完全培养基，于37℃培养36-72h(例如可以是36h、48h、60h或72h等)；其中，每1mL完全培养基中干细胞的添加量为5×10 5-1×10 6个细胞(例如可以是5×10 5、6×10 5、7×10 5、8×10 5、9×10 5或1×10 6等)。
优选地，步骤(a)中，所述完全培养基包括α-MEM培养基或DMEM培养基，且所述完全培养基中还需添加8-12％胎牛血清(例如可以是8％、10％或12％等)和0.5-2％双抗(例如可以是0.5％、1％、1.5％或2％等)。
优选地，步骤(b)中，所述三次递增离心力的离心处理的具体步骤为：
将步骤(a)收集得到的培养基于0-10℃(例如可以是0℃、2℃、4℃、6℃、8℃或10℃等)、800-1200g(例如可以是800g、900g、1000g、1100g或1200g等)的离心力下离心10-30min(例如可以是10min、15min、20min、25min或30min等)，收集上清液；
再将上清液于0-10℃(例如可以是0℃、2℃、4℃、6℃、8℃或10℃等)、8000-12000g(例如可以是8000g、9000g、10000g、11000g或12000g等)的离心力下离心30-60min (例如可以是30min、35min、40min、45min、50min、55min或60min等)，收集上清液；
最后将上清液于0-10℃(例如可以是0℃、2℃、4℃、6℃、8℃或10℃等)、80000-120000g(例如可以是80000g、90000g、100000g、110000g或120000g等)的离心力下离心90-120min(例如可以是90min、95min、100min、105min、110min、115min或120min等)，收集沉淀，得到所述细胞外囊泡。
优选地，步骤(b)中，所述细胞外囊泡需在无菌PBS缓冲液中重悬保存。
优选地，所述细胞外囊泡和无菌PBS缓冲液的质量比为1:(1-5)，例如可以是1:1、1:2、1:3、1:4或1:5等。
作为本申请的优选技术方案，所述细胞外囊泡由以下制备方法制备得到：
将人源干细胞和完全培养基混合，于37℃培养2-3天至汇合度为80-90％；其中，每1mL完全培养基中人源干细胞的添加量为5×10 5-1×10 6个细胞；
将汇合度为80-90％的人源干细胞转移至无细胞外囊泡血清配制的完全培养基，于37℃培养36-72h，所述完全培养基为含有8-12％胎牛血清和0.5-2％双抗的完全培养基；其中，每1mL完全培养基中人源干细胞的添加量为5×10 5-1×10 6个细胞；
收集培养基，进行三次递增离心力的离心处理，所述三次递增离心力的离心处理的具体步骤为：将收集得到的培养基于0-10℃、800-1200g的离心力下离心10-30min，收集上清液；再将上清液于0-10℃、8000-12000g的离心力下离心30-60min，收集上清液；最后将上清液于0-10℃、80000-120000g的离心力下离心90-120min，收集沉淀，得到所述细胞外囊泡；将所得细胞外囊泡与无菌PBS缓冲液按质量比为1:(1-5)重悬保存。
第二方面，本申请提供一种第一方面所述的骨靶向细胞外囊泡的制备方法，所述骨靶向细胞外囊泡的制备方法包括如下步骤：
(1)骨靶向分子的修饰：通过点击化学反应将骨靶向分子修饰在细胞外囊泡的外表面，得到外表面修饰有骨靶向分子的细胞外囊泡；
(2)促成骨药物的装载：通过电穿孔处理将促成骨药物装载于所述外表面修饰有骨靶向分子的细胞外囊泡内，得到所述骨靶向细胞外囊泡。
本申请通过分步离心获得人源干细胞分泌的细胞外囊泡，使用点击化学的方式在细胞外囊泡的表面修饰多肽类骨靶向分子，使用电穿孔方法装载促成骨相关药物得到所述骨靶向细胞外囊泡。本申请中对细胞外囊泡的骨靶向修饰反应温和便捷，不会破坏细胞外囊泡本身的结构，也不会影响细胞外囊泡本身的性能，且本申请中所使用的多肽类骨靶向分子的毒副作用较低，靶向效果好。
优选地，步骤(1)中，修饰的具体步骤为：
(A)将含有细胞外囊泡的PBS缓冲液和双功能分子混合后，进行接枝反应，得到表面接枝有双功能分子的细胞外囊泡；
(B)将得到的表面接枝有双功能分子的细胞外囊泡重悬于PBS缓冲液后，再与骨靶向分子混合，进行偶联反应，得到外表面修饰有骨靶向分子的细胞外囊泡。
优选地，所述双功能分子选自二苄环辛基-磺基-N-羟基琥珀酰亚胺酯、氮杂二苯并环辛炔-琥珀酰亚胺酯、二苯基环辛炔-PEG4-氢化琥珀酰亚胺酯或二苯基环辛炔-碳6-琥珀酰亚胺酯中任意一种；优选为二苄环辛基-磺基-N-羟基琥珀酰亚胺酯。
优选地，步骤(A)中，所述混合后得到的混合液中双功能分子的浓度为10-50μM，例如可以是10μM、15μM、20μM、25μM、30μM、35μM、40μM、45μM或50μM等；以及所述细胞外囊泡的浓度为0.5-1mg/mL，例如可以是0.5mg/mL、0.6mg/mL、0.7mg/mL、0.8mg/mL、0.9mg/mL或1mg/mL等。
优选地，步骤(A)中，所述接枝反应的温度为30-40℃，例如可以是30℃、33℃、35℃、37℃或40℃等；以及所述接枝反应的时间为3-6h，例如可以是3h、3.5h、4h、4.5h、5h、5.5h或6h等。
优选地，步骤(A)中，所述接枝反应结束后需通过超滤离心去除未接枝的双功能分子。
优选地，步骤(B)中，所述重悬后得到的溶液中表面接枝有双功能分子的细胞外囊泡的浓度为0.5-5mg/mL，例如可以是0.5mg/mL、1mg/mL、1.5mg/mL、2mg/mL、2.5mg/mL、3mg/mL、3.5mg/mL、4mg/mL、4.5mg/mL或5mg/mL等。
优选地，步骤(B)中，所述混合后得到的混合液中骨靶向分子的浓度为10-50μM，例如可以是10μM、15μM、20μM、25μM、30μM、35μM、40μM、45μM或50μM等；以及所述表面接枝有双功能分子的细胞外囊泡的浓度为0.5-5mg/mL，例如可以是0.5mg/mL、1mg/mL、2mg/mL、3mg/mL、4mg/mL或5mg/mL等。
优选地，步骤(B)中，所述偶联反应的温度为0-10℃，例如可以是0℃、2℃、4℃、6℃、8℃或10℃等；所述偶联反应的时间为8-12h，例如可以是8h、9h、10h、11h或12h等。
优选地，步骤(B)中，所述偶联反应结束后需通过超滤离心去除未修饰的骨靶向分子。
优选地，步骤(B)中，所述外表面修饰有骨靶向分子的细胞外囊泡需在无菌PBS缓冲液中重悬保存。
优选地，所述外表面修饰有骨靶向分子的细胞外囊泡和无菌PBS缓冲液的质量比为1:(1-5)，例如可以是1:1、1:1.5、1:2、1:2.5、1:3、1:3.5、1:4、1:4.5或1:5等。
优选地，步骤(2)中，所述装载的具体步骤为：将所述外表面修饰有骨靶向分子的细胞外囊泡、促成骨药物和电穿孔缓冲液混合，进行电穿孔，穿孔完成后再进行静置，得到所述骨靶向细胞外囊泡。
优选地，所述混合后得到的混合液中外表面修饰有骨靶向分子的细胞外囊泡的浓度为0.5-5mg/mL，例如可以是0.5mg/mL、1mg/mL、2mg/mL、3mg/mL、4mg/mL或5mg/mL等；以及所述促成骨药物的浓度为0.01-0.5μg/μL，例如可以是0.01μg/μL、0.02μg/μL、0.03μg/μL、0.05μg/μL、0.08μg/μL、0.1μg/μL、0.2μg/μL、0.3μg/μL、0.4μg/μL或0.5μg/μL等。
优选地，所述电穿孔缓冲液包括体积比为(18-23):(77-82)的碘克沙醇和磷酸二氢钾-氯化钾溶液的混合液，所述体积比例如可以是18:82、19:81、20:80、21:79、22:78或23:77等；所述磷酸二氢钾-氯化钾溶液中磷酸二氢钾的质量浓度为1-1.2mM(例如可以是1mM、1.1mM 或1.2mM等)，氯化钾的质量浓度为23-27mM(例如可以是23mM、24mM、25mM、26mM或27mM等)，溶剂为水。
优选地，所述电穿孔的电压为100-400V，例如可以是100V、150V、250V、300V、350V或400V等；以及所述电穿孔的时间为1-20ms，例如可以是1ms、2ms、5ms、8ms、12ms、16ms、18ms或20ms等。
优选地，所述静置的温度为30-40℃，例如可以是30℃、33℃、35℃、37℃或40℃等；以及所述静置的时间为25-35min，例如可以是25min、28min、30min、31min、32min或35min等。
优选地，所述静置后需通过超滤离心去除未装载的促成骨药物。
作为本申请的优选技术方案，所述骨靶向细胞外囊泡的制备方法包括如下步骤：
(1)骨靶向分子的修饰：
(A)将含有细胞外囊泡的PBS缓冲液和双功能分子混合，得到的混合液，所述混合液中所述双功能分子的浓度为10-50μM，所述细胞外囊泡的浓度为0.5-1mg/mL；混合后在30-40℃接枝反应3-6h；接枝反应结束后需通过超滤离心去除未接枝的双功能分子，得到表面接枝有双功能分子的细胞外囊泡；
(B)将得到的表面接枝有双功能分子的细胞外囊泡重悬于PBS缓冲液，重悬后得到的溶液中所述表面接枝有双功能分子的细胞外囊泡浓度为0.5-5mg/mL，再将重悬得到的溶液与骨靶向分子混合，混合后得到的混合液中所述骨靶向分子的浓度为10-50μM，所述表面接枝有双功能分子的细胞外囊泡的浓度为0.5-5mg/mL；在0-10℃偶联反应8-12h，偶联反应结束后需通过超滤离心去除未修饰的骨靶向分子，得到外表面修饰有骨靶向分子的细胞外囊泡，将所述外表面修饰有骨靶向分子的细胞外囊泡和无菌PBS缓冲液按质量比为1:(1-5)重悬保存；
(2)促成骨药物的装载：
将所述外表面修饰有骨靶向分子的细胞外囊泡、促成骨药物和电穿孔缓冲液混合，混合后得到的混合液中所述外表面修饰有骨靶向分子的细胞外囊泡的浓度为0.5-5mg/mL，所述促成骨药物的浓度为0.01-0.5μg/μL；进行电穿孔，所述电穿孔的电压为100-400V、时间为1-20ms；穿孔完成后在30-40℃静置25-35min，静置后需通过超滤离心去除未装载的促成骨药物，得到所述骨靶向细胞外囊泡。
第三方面，本申请提供一种药物组合物，所述药物组合物中包含第一方面所述的骨靶向细胞外囊泡。
优选地，所述药物组合物包括药学上可接受的辅料。
第四方面，本申请提供第一方面所述的骨靶向细胞外囊泡、第二方面所述的骨靶向细胞外囊泡的制备方法或第三方面所述的药物组合物中任意一种或至少两种的组合在制备治疗骨科疾病的产品中的应用。
本申请所述的数值范围不仅包括上述例举的点值，还包括没有例举出的上述数值范围之 间的任意的点值，限于篇幅及出于简明的考虑，本申请不再穷尽列举所述范围包括的具体点值。
相对于现有技术，本申请具有以下有益效果：
(1)本申请所述骨靶向分子通过与双功能分子的点击化学反应生成三氮唑键连接修饰在所述装载促成骨药物的细胞外囊泡表面，所述双功能分子通过与细胞外囊泡表面的氨基形成共价键接枝在所述装载促成骨药物的细胞外囊泡的外表面，所述骨靶向细胞外囊泡的制备方法对细胞外囊泡的骨靶向修饰反应温和便捷，不会破坏细胞外囊泡本身的结构，也不会影响细胞外囊泡本身的性能。
(2)本申请所述骨靶向细胞外囊泡装载促成骨药物后可以提高治疗效果，克服了原始干细胞细胞外囊泡的治疗效果差和靶向性弱的缺点。
(3)本申请所述多肽类骨靶向分子的靶向效果好，有利于将细胞外囊泡递送至骨组织表面从而提高治疗效果。
(4)相比于脂质体制备中会使用大量有机溶剂而导致的潜在毒副作用，细胞外囊泡良好的生物安全性有利于其进一步临床应用，且骨靶向修饰过程条件温和、无有机溶剂参与。
附图说明
图1是实施例1中细胞外囊泡的透射电镜检测结果，(比例尺100nm)。
图2是实施例1中骨靶向细胞外囊泡的透射电镜检测结果，(比例尺200nm)。
图3是实施例1中骨靶向细胞外囊泡的透射电镜检测结果，(比例尺200nm)。
图4是测试例2中骨靶向细胞外囊泡的荧光强度曲线。
图5是测试例2中所述骨靶向细胞外囊泡(实施例1)的粒径分布统计结果图。
图6A和图6B是测试例3中骨靶向细胞外囊泡在小鼠各组织的分布情况。
图7A和图7B是测试例4中的体外促成骨效果检测染色结果。
具体实施方式
下面通过具体实施方式来进一步说明本申请的技术方案。本领域技术人员应该明了，所述实施例仅仅是帮助理解本申请，不应视为对本申请的具体限制。
实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购获得的常规产品。
以下实施例和对比例中所用各组分来源如下所示：
组分 厂家 牌号
二苄环辛基-磺基-N-羟基琥珀酰亚胺酯 Sigma-Aldrich 762040
α-MEM培养基 Gibco C12571500BT
DMEM培养基 Gibco C11995500BT
碘克沙醇 Serumwerk Bernburg No.1893
PBS缓冲液 BI 02-024-1-ACS
超滤离心管 Millipore RT-UFC510096
实施例1
本实施例提供一种骨靶向细胞外囊泡，所述细胞外囊泡由人骨髓间充质干细胞产生，所述骨靶向分子为多肽类骨靶向分子，所述多肽类骨靶向分子的氨基酸序列为(D)8(SEQ ID NO:8)，所述多肽类骨靶向分子通过赖氨酸与叠氮基团相连接，所述促成骨药物包括micro RNA，所述micro RNA的核苷酸序列为UUCAAGUAAUCCAGGAUAGGCU(SEQ ID NO:2)。
所述细胞外囊泡的制备方法如下所示：
将人骨髓间充质干细胞和完全培养基混合，于37℃、5％CO 2培养2-3天至汇合度为80-90％；其中，每1mL完全培养基中人骨髓间充质干细胞的添加量为5×10 5个细胞；
将汇合度为80-90％的人骨髓间充质干细胞转移至无细胞外囊泡血清配制的完全培养基，于37℃、5％CO 2培养48h，其中，每1mL完全培养基中人骨髓间充质干细胞的添加量为5×10 5个细胞；所述完全培养基为含有10％胎牛血清和1％双抗的α-MEM培养基；
收集培养基，进行三次递增离心力的离心处理，所述三次递增离心力的离心处理的具体步骤为：在4℃以1000g离心10min，得到上清液一，将所述上清液一在4℃以10000g离心30min，得到上清液二，再将所述上清液二在4℃以100000g离心90min；以及
收集沉淀，得到细胞外囊泡，所述细胞外囊泡需在无菌PBS缓冲液中重悬保存，所述细胞外囊泡和无菌PBS缓冲液的质量比为1:1。
所得细胞外囊泡的透射电镜检测结果如图1所示，从图1可以看出，通过离心方法收集到的细胞外囊泡形貌为球形，尺寸在100nm左右。
所述骨靶向细胞外囊泡的制备方法如下所示：
(1)骨靶向分子的修饰：
(A)将含有细胞外囊泡的PBS缓冲液和二苄环辛基-磺基-N-羟基琥珀酰亚胺酯混合，得到的混合液，所述混合液中所述二苄环辛基-磺基-N-羟基琥珀酰亚胺酯的浓度为30μM，所述细胞外囊泡的浓度为0.8mg/mL；混合后在37℃接枝反应5h；接枝反应结束后需通过超滤离心去除未接枝的二苄环辛基-磺基-N-羟基琥珀酰亚胺酯，所述超滤离心使用的超滤离心管的截留分子量为100kDa，得到表面接枝有二苄环辛基-磺基-N-羟基琥珀酰亚胺酯的细胞外囊泡；
(B)将得到的表面接枝有二苄环辛基-磺基-N-羟基琥珀酰亚胺酯的细胞外囊泡重悬于PBS缓冲液，重悬后得到的溶液中所述表面接枝有二苄环辛基-磺基-N-羟基琥珀酰亚胺酯的细胞外囊泡浓度为0.5mg/mL，再将重悬得到的溶液与骨靶向分子混合，混合后得到的混合液中所述骨靶向分子的浓度为30μM，所述表面接枝有二苄环辛基-磺基-N-羟基琥珀酰亚胺酯的细胞外囊泡的浓度为3mg/mL；在4℃偶联反应10h，偶联反应结束后需通过超滤离心去除未修饰的骨靶向分子，所述超滤离心使用的超滤离心管的截留分子量为100kDa，得到外 表面修饰有骨靶向分子的细胞外囊泡，将所述外表面修饰有骨靶向分子的细胞外囊泡和无菌PBS缓冲液按质量比为1:1重悬保存；所述外表面修饰有骨靶向分子的细胞外囊泡的透射电镜检测结果如图2所示，从图2可以看出，所述外表面修饰有骨靶向分子的细胞外囊泡的形貌为球形，尺寸仍在100nm左右。
(2)促成骨药物的装载：
将所述外表面修饰有骨靶向分子的细胞外囊泡、促成骨药物和电穿孔缓冲液混合，混合后得到的混合液中所述外表面修饰有骨靶向分子的细胞外囊泡的浓度为3mg/mL，所述促成骨药物的浓度为0.01μg/μL；进行电穿孔，所述电穿孔的电压为350V、时间为10ms；穿孔完成后在37℃静置30min，静置后需通过超滤离心去除未装载的促成骨药物，所述超滤离心使用的超滤离心管的截留分子量为100kDa，得到所述骨靶向细胞外囊泡。
所述电穿孔缓冲液为体积比为21:79的碘克沙醇(密度梯度分离液碘克沙醇，optiprep，Axis-Shield)和磷酸二氢钾-氯化钾溶液的混合液，所述磷酸二氢钾-氯化钾溶液中磷酸二氢钾的质量浓度为1.15mM、pH＝7.2，氯化钾的质量浓度为25mM，溶剂为水。
所述骨靶向细胞外囊泡的透射电镜检测结果如图3所示，从图3可以看出，所述骨靶向细胞外囊泡的形貌为球形，尺寸在120nm左右。
实施例2
本实施例提供一种骨靶向细胞外囊泡，所述细胞外囊泡由人脂肪干细胞产生，所述骨靶向分子为多肽类骨靶向分子，所述多肽类骨靶向分子的氨基酸序列为SDSSD(SEQ ID NO:6)，所述多肽类骨靶向分子通过赖氨酸与叠氮基团相连接，所述促成骨药物包括micro RNA，所述micro RNA的核苷酸序列为UAGCACCAUUUGAAAUCAGUGUU(SEQ ID NO:3)。
所述细胞外囊泡的制备的方法如下所示：
将人脂肪干细胞和完全培养基混合，于37℃、5％CO 2培养2-3天至汇合度为80-90％；其中，每1mL完全培养基中人脂肪干细胞的添加量为8×10 5个；
将汇合度为80-90％的人脂肪干细胞转移至无细胞外囊泡血清配制的完全培养基，于37℃、5％CO 2培养36h，其中，每1mL完全培养基中人脂肪干细胞的添加量为8×10 5个；所述完全培养基为含有8％胎牛血清和0.5％双抗的α-MEM培养基；
收集培养基，进行三次递增离心力的离心处理，所述三次递增离心力的离心处理的具体步骤为：在4℃以800g离心30min，得到上清液一，将所述上清液一在4℃以8000g离心60min，得到上清液二，再将所述上清液二在4℃以80000g离心120min；以及
收集沉淀，得到细胞外囊泡，所述细胞外囊泡需在无菌PBS缓冲液中重悬保存，所述细胞外囊泡和无菌PBS缓冲液的质量比为1:1。
所述骨靶向细胞外囊泡的制备方法如下所示：
(1)骨靶向分子的修饰：
(A)将含有细胞外囊泡的PBS缓冲液和二苄环辛基-磺基-N-羟基琥珀酰亚胺酯混合，得到的混合液，所述混合液中所述二苄环辛基-磺基-N-羟基琥珀酰亚胺酯的浓度为10μM，所 述细胞外囊泡的浓度为0.5mg/mL；混合后在30℃接枝反应6h；接枝反应结束后需通过超滤离心去除未接枝的二苄环辛基-磺基-N-羟基琥珀酰亚胺酯，所述超滤离心使用的超滤离心管的截留分子量为100kDa，得到表面接枝有二苄环辛基-磺基-N-羟基琥珀酰亚胺酯的细胞外囊泡；
(B)将得到的表面接枝有二苄环辛基-磺基-N-羟基琥珀酰亚胺酯的细胞外囊泡重悬于PBS缓冲液，重悬后得到的溶液中所述表面接枝有二苄环辛基-磺基-N-羟基琥珀酰亚胺酯的细胞外囊泡浓度为0.75mg/mL，再将重悬得到的溶液与骨靶向分子混合，混合后得到的混合液中所述骨靶向分子的浓度为10μM，所述表面接枝有二苄环辛基-磺基-N-羟基琥珀酰亚胺酯的细胞外囊泡的浓度为0.5mg/mL；在2℃偶联反应12h，偶联反应结束后需通过超滤离心去除未修饰的骨靶向分子，所述超滤离心使用的超滤离心管的截留分子量为100kDa，得到外表面修饰有骨靶向分子的细胞外囊泡，将所述外表面修饰有骨靶向分子的细胞外囊泡和无菌PBS缓冲液按质量比为1:1.5重悬保存。
(2)促成骨药物的装载：
将所述外表面修饰有骨靶向分子的细胞外囊泡、促成骨药物和电穿孔缓冲液混合，混合后得到的混合液中所述外表面修饰有骨靶向分子的细胞外囊泡的浓度为0.5mg/mL，所述促成骨药物的浓度为0.02μg/μL；进行电穿孔，所述电穿孔的电压为400V、时间为10ms；穿孔完成后在30℃静置35min，静置后需通过超滤离心去除未装载的促成骨药物，所述超滤离心使用的超滤离心管的截留分子量为100kDa，得到所述骨靶向细胞外囊泡。
所述电穿孔缓冲液为体积比为21:79的碘克沙醇(密度梯度分离液碘克沙醇，optiprep，Axis-Shield)和磷酸二氢钾-氯化钾溶液的混合液，所述磷酸二氢钾-氯化钾溶液中磷酸二氢钾的质量浓度为1.15mM、pH＝7.2，氯化钾的质量浓度为25mM，溶剂为水。
实施例3
本实施例提供一种骨靶向细胞外囊泡，所述细胞外囊泡由人脐带间充质干细胞产生，所述骨靶向分子为多肽类骨靶向分子，所述多肽类骨靶向分子的氨基酸序列为(DSS)6(SEQ ID NO:7)，所述多肽类骨靶向分子通过赖氨酸与叠氮基团相连接，所述促成骨药物包括micro RNA，所述micro RNA的核苷酸序列为UUCACAGUGGCUAAGUUCCGC(SEQ ID NO:4)。
所述细胞外囊泡的制备的方法如下所示：
将人脐带间充质干细胞和完全培养基混合，于37℃、5％CO 2培养2-3天至汇合度为80-90％；其中，每1mL完全培养基中人脐带间充质干细胞的添加量为1×10 6个；
将汇合度为80-90％的人脐带间充质干细胞转移至无细胞外囊泡血清配制的完全培养基，于37℃、5％CO 2培养72h，其中，每1mL完全培养基中人脐带间充质干细胞的添加量为1×10 6个；所述完全培养基为含有12％胎牛血清和2％双抗的DMEM培养基；
收集培养基，进行三次递增离心力的离心处理，所述三次递增离心力的离心处理的具体步骤为：在4℃以1200g离心10min，得到上清液一，将所述上清液一在4℃以12000g离心30min，得到上清液二，再将所述上清液二在4℃以120000g离心90min；以及
收集沉淀，得到细胞外囊泡，所述细胞外囊泡需在无菌PBS缓冲液中重悬保存，所述细胞外囊泡和无菌PBS缓冲液的质量比为1:1。
所述骨靶向细胞外囊泡的制备方法如下所示：
(1)骨靶向分子的修饰：
(A)将含有细胞外囊泡的PBS缓冲液和二苄环辛基-磺基-N-羟基琥珀酰亚胺酯混合，得到的混合液，所述混合液中所述二苄环辛基-磺基-N-羟基琥珀酰亚胺酯的浓度为50μM，所述细胞外囊泡的浓度为1mg/mL；混合后在40℃接枝反应3h；接枝反应结束后需通过超滤离心去除未接枝的二苄环辛基-磺基-N-羟基琥珀酰亚胺酯，所述超滤离心使用的超滤离心管的截留分子量为100kDa，得到表面接枝有二苄环辛基-磺基-N-羟基琥珀酰亚胺酯的细胞外囊泡；
(B)将得到的表面接枝有二苄环辛基-磺基-N-羟基琥珀酰亚胺酯的细胞外囊泡重悬于PBS缓冲液，重悬后得到的溶液中所述表面接枝有二苄环辛基-磺基-N-羟基琥珀酰亚胺酯的细胞外囊泡浓度为1mg/mL，再将重悬得到的溶液与骨靶向分子混合，混合后得到的混合液中所述骨靶向分子的浓度为50μM，所述表面接枝有二苄环辛基-磺基-N-羟基琥珀酰亚胺酯的细胞外囊泡的浓度为5mg/mL；在6℃偶联反应8h，偶联反应结束后需通过超滤离心去除未修饰的骨靶向分子，所述超滤离心使用的超滤离心管的截留分子量为100kDa，得到外表面修饰有骨靶向分子的细胞外囊泡，将所述外表面修饰有骨靶向分子的细胞外囊泡和无菌PBS缓冲液按质量比为1:2重悬保存。
(2)促成骨药物的装载：
将所述外表面修饰有骨靶向分子的细胞外囊泡、促成骨药物和电穿孔缓冲液混合，混合后得到的混合液中所述外表面修饰有骨靶向分子的细胞外囊泡的浓度为5mg/mL，所述促成骨药物的浓度为0.5μg/μL；进行电穿孔，所述电穿孔的电压为200V、时间为15ms；穿孔完成后在40℃静置25min，静置后需通过超滤离心去除未装载的促成骨药物，所述超滤离心使用的超滤离心管的截留分子量为100kDa，得到所述骨靶向细胞外囊泡。
所述电穿孔缓冲液为体积比为21:79的碘克沙醇(密度梯度分离液碘克沙醇，optiprep，Axis-Shield)和磷酸二氢钾-氯化钾溶液的混合液，所述磷酸二氢钾-氯化钾溶液中磷酸二氢钾的质量浓度为1.15mM、pH＝7.2，氯化钾的质量浓度为25mM，溶剂为水。
实施例4
本实施例提供一种骨靶向细胞外囊泡，所述骨靶向细胞外囊泡与实施例1的区别仅在于，所述促成骨药物为siRNA，所述siRNA的核苷酸序列为GAAGUGGUUCAGAAGAUGACGCCCAAACCCAAACCCAAACCCAAACCCAAA(SEQ IDNO:5)。所述骨靶向细胞外囊泡的制备方法参照实施例1。
实施例5
本实施例提供一种骨靶向细胞外囊泡，所述骨靶向细胞外囊泡与实施例1的区别仅在于，所述促成骨药物为小分子化学药物，所述小分子化学药物为骨形态发生蛋白。所述骨靶向细 胞外囊泡的制备方法参照实施例1。
实施例6
本实施例提供一种骨靶向细胞外囊泡，所述骨靶向细胞外囊泡的制备方法与实施例1的区别仅在于，步骤(A)中所述接枝反应的时间为12h，所述接枝反应的温度为25℃，其余步骤参照实施例1。
实施例7
本实施例提供一种骨靶向细胞外囊泡，所述骨靶向细胞外囊泡的制备方法与实施例1的区别仅在于，步骤(A)中所述接枝反应的时间为2h，所述接枝反应的温度为60℃，其余步骤参照实施例1。
实施例8
本实施例提供一种骨靶向细胞外囊泡，所述骨靶向细胞外囊泡的制备方法与实施例1的区别仅在于，步骤(A)中所述混合液中所述二苄环辛基-磺基-N-羟基琥珀酰亚胺酯的浓度为5μM；其余步骤参照实施例1。
实施例9
本实施例提供一种骨靶向细胞外囊泡，所述骨靶向细胞外囊泡的制备方法与实施例1的区别仅在于，步骤(B)中所述混合液中所述骨靶向分子的浓度为5μM，其余步骤参照实施例1。
实施例10
本实施例提供一种骨靶向细胞外囊泡，所述骨靶向细胞外囊泡的制备方法与实施例1的区别仅在于，步骤(2)中所述电穿孔的电压为700V，所述电穿孔的时间为2ms，其余步骤参照实施例1。
实施例11
本实施例提供一种骨靶向细胞外囊泡，所述骨靶向细胞外囊泡的制备方法与实施例1的区别仅在于，步骤(2)中所述电穿孔的电压为50V，所述电穿孔的时间为25ms，其余步骤参照实施例1。
对比例1
本对比例提供一种非靶向细胞外囊泡，所述细胞外囊泡由人骨髓间充质干细胞产生，所述促成骨药物包括micro RNA，所述micro RNA的核苷酸序列为UUCAAGUAAUCCAGGAUAGGCU(SEQ ID NO:2)。本对比例与实施例1的区别在于所述细胞外囊泡中不含有骨靶向分子。
所述细胞外囊泡的制备的方法如下所示：
将人骨髓间充质干细胞和完全培养基混合，于37℃、5％CO 2培养2-3天至汇合度为80-90％；其中，每1mL完全培养基中人骨髓间充质干细胞的添加量为5×10 5个细胞；
将汇合度为80-90％的人骨髓间充质干细胞转移至无细胞外囊泡血清配制的完全培养基，于37℃、5％CO 2培养48h，其中，每1mL完全培养基中人骨髓间充质干细胞的添加量为5×10 5 个细胞；所述完全培养基为含有10％胎牛血清和1％双抗的α-MEM培养基；
收集培养基，进行三次递增离心力的离心处理，所述三次递增离心力的离心处理的具体步骤为：在4℃以1000g离心10min，得到上清液一，将所述上清液一在4℃以10000g离心30min，得到上清液二，再将所述上清液二在4℃以100000g离心90min；以及
收集沉淀，得到细胞外囊泡。
所述非靶向细胞外囊泡的制备的方法如下所示：
将所述细胞外囊泡、促成骨药物和电穿孔缓冲液混合，混合后得到的混合液中所述细胞外囊泡的浓度为3mg/mL，所述促成骨药物的浓度为0.03μg/μL；进行电穿孔，所述电穿孔的电压为350V、时间为10ms；穿孔完成后在37℃静置30min，静置后需通过超滤离心去除未装载的促成骨药物，所述超滤离心使用的超滤离心管的截留分子量为100kDa，得到所述非靶向细胞外囊泡。
对比例2
本对比例提供一种非骨靶向细胞外囊泡，所述非骨靶向细胞外囊泡与实施例1的区别仅在于，用(G)6(SEQ ID NO:9)替代实施例1中的骨靶向分子。所述非骨靶向细胞外囊泡的制备方法参照实施例1。
对比例3
本对比例提供一种骨靶向囊泡，所述骨靶向囊泡与实施例1的区别仅在于所述囊泡为脂质体囊泡。
所述脂质体囊泡的制备方法包括如下步骤：
将溴化三甲基-2,3-二油酰氧基丙基铵、二油酰磷脂酰乙醇胺、胆固醇、二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000以63:16:16:5的摩尔比溶解在氯仿中，并在60℃下用旋转蒸发器在负压下蒸发成薄膜，用10mM磷酸盐缓冲盐水(PBS，pH＝7.4)在60℃的水浴中预孵化得到的脂质薄膜，形成多胶体囊泡。然后用Avanti迷你挤出机(Avanti Polar Lipids)将形成的多胶体囊泡通过0.2μm的聚碳酸酯膜(Whatman)挤出10次，再通过0.1μm的膜挤出10次，逐步形成最后的脂质体。
所述骨靶向囊泡的制备方法参照实施例1的步骤(1)和步骤(2)。
测试例1
本测试例是对实施例1-11中的骨靶向细胞外囊泡和对比例1-3中的囊泡进行吸附率测试，以细胞外囊泡模拟物为例，所述吸附率测试的方法包括如下步骤：
将罗丹明B荧光标记的氨基酸序列为(D)8(SEQ ID NO:8)的多肽类靶向分子修饰于细胞外囊泡模拟物，将其在PBS中制备成1mg/mL的混悬液，使用荧光分光光度计测定所述混悬液的荧光强度，设置激发波长550nm，发射波长580nm；测定后将细胞外囊泡模拟物混悬液与10mg/mL的羟基磷灰石颗粒在25℃气浴摇床中共孵育5h，孵育完成后将混悬液以4000rpm离心10min，使羟基磷灰石及吸附在其上的细胞外囊泡模拟物沉淀，使用荧光分光光度计测定上清液中的荧光强度，激发波长550nm，发射波长580nm，通过下列公式计算吸附率：
吸附率＝(I1-I2)/I1×100％
I1：与羟基磷灰石孵育前荧光强度；
I2：与羟基磷灰石孵育后荧光强度。
实施例1中骨靶向细胞外囊泡孵育前和孵育后的荧光强度曲线图如图4所示，从图中可以看出，孵育后的上清液的荧光强度明显下降，说明所述骨靶向细胞外囊泡的靶向效果好，吸附率高。
参照细胞外囊泡模拟物的吸附率测试测试方法，检测实施例1-11中的骨靶向细胞外囊泡和对比例1-3中的囊泡的吸附率。通过计算吸附率可以选择出合适的修饰条件。吸附率的检测结果如表1所示。
表1
样品 吸附率
实施例1 75％
实施例2 75％
实施例3 75％
实施例4 75％
实施例5 75％
实施例6 75％
实施例7 75％
实施例8 30％
实施例9 40％
实施例10 75％
实施例11 75％
对比例1 22％
对比例2 22％
对比例3 60％
从表1的结果可知，与修饰非多肽类骨靶向分子相比，修饰多肽类骨靶向分子后可以显著提高细胞外囊泡的骨靶向性，降低双功能分子浓度会降低骨靶向性，降低骨靶向分子浓度会降低骨靶向性，使用脂质体制备的骨靶向性也比使用细胞外囊泡低。
测试例2
本测试例是对实施例1-11中的骨靶向细胞外囊泡和对比例1-3中的囊泡进行粒径分布测试、载药量测试、囊泡浓度测试、骨靶向分子修饰效率测试和骨靶向细胞外囊泡的zeta电势测量。
将细胞外囊泡与PBS按照1:1混合后使用Zetasizer纳米粒度仪测定粒径分布。
载药量测试的检测方法包括如下步骤：
将药物使用荧光标记后测定荧光强度F1，装载进入细胞外囊泡，按照1:1的比例稀释于 PBS中使用荧光分光光度计测定荧光强度F2，加入TritonX后测定荧光强度F3，装载效率＝(F3-F2)/F1×100％。
囊泡浓度测试采用BCA试剂盒按说明书测试。
骨靶向分子修饰效率测试的检测方法包括如下步骤：
使用荧光分光光度计测定荧光标记的多肽类骨靶向分子的标准曲线，将质量为M1的荧光多肽类骨靶向分子用于修饰细胞外囊泡，将收集到的细胞外囊泡按照1:1分散到PBS中测定荧光强度，按照标准曲线测定出相应含量M2，修饰效率＝M2/M1×100％。
将细胞外囊泡与PBS按照1:1混合后使用Zetasizer纳米粒度仪测定zeta电势。
实施例1中所述骨靶向细胞外囊泡的粒径分布统计结果图如图5所示，通过对修饰后细胞外囊泡的粒径统计可以发现粒径成正态分布，粒径较为均一，平均粒径为124nm。
粒径分布、载药量、骨靶向分子修饰效率(骨靶向分子、二苄环辛基-磺基-N-羟基琥珀酰亚胺酯和细胞外囊泡的摩尔比)和zeta电势检测结果统计如表2所示。
表2
样品 平均粒径 载药量 囊泡浓度 修饰效率 zeta电势
实施例1 125 15％ 1 20％ -20mV
实施例2 125 20％ 1 25％ -20mV
实施例3 125 25％ 1 30％ -20mV
实施例4 125 15％ 1 20％ -20mV
实施例5 125 15％ 1 20％ -20mV
实施例6 125 15％ 0.5 20％ -20mV
实施例7 125 15％ 0.5 20％ -20mV
实施例8 125 15％ 1 10％ -20mV
实施例9 125 15％ 1 10％ -20mV
实施例10 125 0％ 0.5 20％ -20mV
实施例11 125 5％ 1 20％ -20mV
对比例1 125 15％ 1 20％ -20mV
对比例2 125 15％ 1 20％ -20mV
对比例3 125 5％ 无 10％ -20mV
从表2的结果可知，随着获得细胞外囊泡的总量上升，载药量和修饰效率也随之上升，zeta电势则没有明显变化。
通过实施例1与实施例6-7的对比可知，延长反应时间或提高反应温度会导致囊泡破碎从而使囊泡的产量减少。
通过实施例1与实施例8-9的对比可知，降低试剂浓度会导致修饰效率不足，同样试剂浓度过高也会导致囊泡破碎导致修饰效率下降。
通过实施例1与实施例10-11的对比可知，较低的电压无法穿透囊泡导致无法装载更多 药物，较高的电压会击碎囊泡导致无法载药。
通过实施例1与对比例3的对比可知，使用脂质体由于自带氨基很少导致修饰效率很低且无法使用电穿孔的方法载药导致载药效率较低。
测试例3
检测实施例1所述骨靶向细胞外囊泡和对比例2中细胞外囊泡的体内分布效果。
取100μL所述骨靶向细胞外囊泡用Cy5.5进行染色30min(参照说明书)，染色完成后，使用干净的100kDa超滤离心管按说明书操作去除游离染料，通过尾静脉注射入小鼠体内，6h后将心肝脾肺肾和腿骨取出，在活体成像设备下拍摄荧光染料在各器官的分布情况，所述骨靶向细胞外囊泡在小鼠各组织的分布情况如图6A和图6B所示，结果表明修饰多肽类骨靶向分子后的细胞外囊泡在骨组织的靶向性能得到有效提高，荧光强度值是对照组(对比例2中的细胞外囊泡)的4倍。
测试例4
检测实施例1所述骨靶向细胞外囊泡的体外促成骨效果，所述检测的方法包括如下步骤：
将人胎儿骨髓间充质干细胞按照每孔1×10 5的数量接种于六孔板，使用促成骨诱导培养基进行21天培养，每三天更换一次培养基，随着培养基更换加入每孔100μg的骨靶向细胞外囊泡，21天后进行成骨分化的茜素红染色，染色结果如图7A和图7B所示。茜素红染色结果越红表明成骨分化效果越好，从图7A和图7B看出加入囊泡后可以显著促进体外干细胞成骨分化。
综上，本申请所提供的骨靶向细胞外囊泡中的多肽类骨靶向分子的靶向效果好，有利于将细胞外囊泡递送至骨组织表面从而提高治疗效果，在所述骨靶向细胞外囊泡中装载促成骨药物能进一步增强治疗效果，克服了原始干细胞细胞外囊泡的治疗效果差和靶向性弱的缺点，所述骨靶向细胞外囊泡在制备治疗骨科疾病的产品中具有重要应用价值。
申请人声明，以上所述仅为本申请的具体实施方式，但本申请的保护范围并不局限于此，所属技术领域的技术人员应该明了，任何属于本技术领域的技术人员在本申请揭露的技术范围内，可轻易想到的变化或替换，均落在本申请的保护范围和公开范围之内。

Claims (14)

  1. 一种骨靶向细胞外囊泡，其包括外表面修饰有骨靶向分子的细胞外囊泡和装载于所述细胞外囊泡内的促成骨药物。
  2. 根据权利要求1所述的骨靶向细胞外囊泡，其中，所述骨靶向分子通过与双功能分子的点击化学反应生成三氮唑键连接修饰在所述装载促成骨药物的细胞外囊泡表面；所述双功能分子通过与细胞外囊泡表面的氨基形成共价键接枝在所述装载促成骨药物的细胞外囊泡的外表面；其中，所述双功能分子为同时带有二苯基环辛炔和N-羟基琥珀酰亚胺的分子。
  3. 根据权利要求1或2所述的骨靶向细胞外囊泡，其中，所述双功能分子选自二苄环辛基-磺基-N-羟基琥珀酰亚胺酯、氮杂二苯并环辛炔-琥珀酰亚胺酯、二苯基环辛炔-PEG4-氢化琥珀酰亚胺酯或二苯基环辛炔-碳6-琥珀酰亚胺酯中任意一种，优选为二苄环辛基-磺基-N-羟基琥珀酰亚胺酯；
    优选地，所述骨靶向分子为修饰有叠氮基团的骨靶向分子，其中，所述骨靶向分子包括多肽类骨靶向分子和化学骨靶向分子；
    优选地，所述多肽类骨靶向分子的氨基酸序列包括(D)n、SDSSD(SEQ ID NO:6)、(DSS)6(SEQ ID NO:7)或TPLSYLKGLVTVG(SEQ ID NO:1)中任意一种或至少两种的组合，其中，所述(D)n中n为6-10，n为正整数；所述多肽类骨靶向分子通过赖氨酸与叠氮基团相连接；
    优选地，所述化学骨靶向分子包括双膦酸盐、阿仑膦酸盐、唑来膦酸盐或帕米膦酸盐中任意一种或至少两种的组合。
  4. 根据权利要求1-3中任一项所述的骨靶向细胞外囊泡，其中，所述骨靶向细胞外囊泡中骨靶向分子和双功能分子的摩尔比为1:(5-20)，优选为1:(12-18)；
    优选地，所述骨靶向细胞外囊泡上的骨靶向分子的负载量为每克囊泡0.1-0.3μmol；
    优选地，所述骨靶向细胞外囊泡中促成骨药物的装载量为5-50％；
    优选地，所述骨靶向细胞外囊泡的结构为双层磷脂层囊泡；
    优选地，所述骨靶向细胞外囊泡的粒径为30-200nm；
    优选地，所述骨靶向细胞外囊泡的zeta电势为-5～-40mV。
  5. 根据权利要求1-4中任一项所述的骨靶向细胞外囊泡，其中，所述促成骨药物包括核酸、蛋白质、肽链或小分子化学药物中任意一种或至少两种的组合；
    优选地，所述核酸包括DNA、iRNA、micro RNA、siRNA、shRNA、mRNA、ncRNA、反义RNA、LNA或吗啉代寡核苷酸中任意一种或至少两种的组合，优选为micro RNA；
    优选地，所述蛋白质包括骨形态发生蛋白、骨桥蛋白、连环蛋白、胶原蛋白或丝素蛋白中任意一种或至少两种的组合；
    优选地，所述肽链包括特立帕肽、成骨生长多肽或RGD肽中任意一种或至少两种的组合。
  6. 根据权利要求1-5中任一项所述的骨靶向细胞外囊泡，其中，所述细胞外囊泡的来源为人源干细胞；
    优选地，所述人干细胞包括人骨髓间充质干细胞、人脂肪干细胞、人脐带间充质干细胞、 人脐带血间充质干细胞、人胎盘间充质干细胞、人牙髓干细胞、人牙周膜干细胞、人毛囊干细胞或人羊膜间充质干细胞中任意一种。
  7. 根据权利要求1-6中任一项所述的骨靶向细胞外囊泡，其中，所述细胞外囊泡由以下制备方法制备得到：
    (a)将人源干细胞培养至汇合度为80-90％后，再于完全培养基中进行培养，收集培养基；和
    (b)将步骤(a)收集得到的培养基通过三次递增离心力的离心处理，得到所述细胞外囊泡。
  8. 根据权利要求7所述的骨靶向细胞外囊泡，其中，步骤(a)中，所述培养至汇合度为80-90％的具体步骤为：将人源干细胞和完全培养基混合，于37℃培养2-3天至汇合度为80-90％；其中，每1mL完全培养基中人源干细胞的添加量为5×10 5-1×10 6个细胞；
    优选地，步骤(a)中，所述于完全培养基中进行培养的具体步骤为：将汇合度为80-90％的人源干细胞转移至无细胞外囊泡血清配制的完全培养基，于37℃培养36-72h；其中，每1mL完全培养基中人源干细胞的添加量为5×10 5-1×10 6个细胞；
    优选地，步骤(a)中，所述完全培养基包括α-MEM培养基或DMEM培养基，且所述完全培养基中还需添加8-12％胎牛血清和0.5-2％双抗；
    优选地，步骤(b)中，所述三次递增离心力的离心处理的具体步骤为：将步骤(a)收集得到的培养基于0-10℃、800-1200g的离心力下离心10-30min，收集上清液；再将上清液于0-10℃、8000-12000g的离心力下离心30-60min，收集上清液；最后将上清液于0-10℃、80000-120000g的离心力下离心90-120min，收集沉淀，得到所述细胞外囊泡；
    优选地，步骤(b)中，所述细胞外囊泡需在无菌PBS缓冲液中重悬保存；
    优选地，所述细胞外囊泡和无菌PBS缓冲液的质量比为1:(1-5)。
  9. 一种权利要求1-8中任一项所述骨靶向细胞外囊泡的制备方法，其包括如下步骤：
    (1)骨靶向分子的修饰：通过点击化学反应将骨靶向分子修饰在细胞外囊泡的外表面，得到外表面修饰有骨靶向分子的细胞外囊泡；和
    (2)促成骨药物的装载：通过电穿孔处理将促成骨药物装载于所述外表面修饰有骨靶向分子的细胞外囊泡内，得到所述骨靶向细胞外囊泡。
  10. 根据权利要求9所述的骨靶向细胞外囊泡的制备方法，其中，步骤(1)中，所述修饰的具体步骤为：
    (A)将含有细胞外囊泡的PBS缓冲液和双功能分子混合后，进行接枝反应，得到表面接枝有双功能分子的细胞外囊泡；和
    (B)将得到的表面接枝有双功能分子的细胞外囊泡重悬于PBS缓冲液后，再与骨靶向分子混合，进行偶联反应，得到外表面修饰有骨靶向分子的细胞外囊泡；
    优选地，所述双功能分子选自二苄环辛基-磺基-N-羟基琥珀酰亚胺酯、氮杂二苯并环辛炔-琥珀酰亚胺酯、二苯基环辛炔-PEG4-氢化琥珀酰亚胺酯或二苯基环辛炔-碳6-琥珀酰亚胺 酯中任意一种，优选为二苄环辛基-磺基-N-羟基琥珀酰亚胺酯。
  11. 根据权利要求10所述的骨靶向细胞外囊泡的制备方法，其中，步骤(A)中，所述混合后得到的混合液中双功能分子的浓度为10-50μM，所述细胞外囊泡的浓度为0.5-1mg/mL；
    优选地，步骤(A)中，所述接枝反应的温度为30-40℃，所述接枝反应的时间为3-6h；
    优选地，步骤(A)中，所述接枝反应结束后需通过超滤离心去除未接枝的双功能分子；
    优选地，步骤(B)中，所述重悬后得到的溶液中表面接枝有双功能分子的细胞外囊泡的浓度为0.5-5mg/mL；
    优选地，步骤(B)中，所述混合后得到的混合液中骨靶向分子的浓度为10-50μM，所述表面接枝有双功能分子的细胞外囊泡的浓度为0.5-5mg/mL；
    优选地，步骤(B)中，所述偶联反应的温度为0-10℃，所述偶联反应的时间为8-12h；
    优选地，步骤(B)中，所述偶联反应结束后需通过超滤离心去除未修饰的骨靶向分子；
    优选地，步骤(B)中，所述外表面修饰有骨靶向分子的细胞外囊泡需在无菌PBS缓冲液中重悬保存；
    优选地，所述外表面修饰有骨靶向分子的细胞外囊泡和无菌PBS缓冲液的质量比为1:(1-5)。
  12. 根据权利要求9-11中任一项所述的骨靶向细胞外囊泡的制备方法，其中，步骤(2)中，所述装载的具体步骤为：将所述外表面修饰有骨靶向分子的细胞外囊泡、促成骨药物和电穿孔缓冲液混合，进行电穿孔，穿孔完成后再进行静置，得到所述骨靶向细胞外囊泡；
    优选地，所述混合后得到的混合液中外表面修饰有骨靶向分子的细胞外囊泡的浓度为0.5-5mg/mL，所述促成骨药物的浓度为0.01-0.5μg/μL；
    优选地，所述电穿孔缓冲液包括体积比为(18-23):(77-82)的碘克沙醇和磷酸二氢钾-氯化钾溶液的混合液；所述磷酸二氢钾-氯化钾溶液中磷酸二氢钾的质量浓度为1-1.2mM，氯化钾的质量浓度为23-27mM，溶剂为水；
    优选地，所述电穿孔的电压为100-400V，所述电穿孔的时间为1-20ms；
    优选地，所述静置的温度为30-40℃，所述静置的时间为25-35min；
    优选地，所述静置后需通过超滤离心去除未装载的促成骨药物。
  13. 一种药物组合物，其包含权利要求1-8中任一项所述的骨靶向细胞外囊泡；
    优选地，所述药物组合物包括药学上可接受的辅料。
  14. 权利要求1-8中任一项所述的骨靶向细胞外囊泡、权利要求9-12中任一项所述的骨靶向细胞外囊泡的制备方法或权利要求13所述的药物组合物中任意一种或至少两种的组合在制备治疗骨科疾病的产品中的应用。
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