CN114917202A

CN114917202A - Bone-targeted extracellular vesicle and preparation method and application thereof

Info

Publication number: CN114917202A
Application number: CN202210586023.XA
Authority: CN
Inventors: 赵晓丽; 郝浏智
Original assignee: Shenzhen Institute of Advanced Technology of CAS
Current assignee: Shenzhen Institute of Advanced Technology of CAS
Priority date: 2022-05-26
Filing date: 2022-05-26
Publication date: 2022-08-19
Also published as: WO2023226203A1

Abstract

The invention provides a bone-targeting extracellular vesicle and a preparation method and application thereof. The bone targeting extracellular vesicles of the invention achieve precise delivery of target organs through bone tissue targeting, and enhance bone treatment effects through loading bone-promoting drugs. When the bone targeting extracellular vesicles are prepared, the bone targeting modification reaction on the extracellular vesicles is mild and convenient, the structure of the extracellular vesicles cannot be damaged, the performance of the extracellular vesicles cannot be influenced, and the bone targeting polypeptide used in the invention has low toxic and side effects and a good bone targeting effect.

Description

Bone-targeted extracellular vesicle and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biomedical materials, and particularly relates to a bone targeting extracellular vesicle and a preparation method and application thereof.

Background

Bone tissue defects are caused by trauma, infection, and tumor resection, and although bone tissue is capable of self-regeneration, it has limited ability to regenerate. In addition, aging of a human body further prolongs the bone regeneration time, and obesity and the like cause deterioration of the bone regeneration ability. Therefore, promoting bone regeneration is the focus of treating bone defect diseases clinically.

The existing medicines for promoting bone regeneration in clinical use are expensive or have toxic and side effects, so that the wide use of the medicines is limited. In recent years, researches show that the regeneration of bones is regulated by various cells, particularly stem cells with regeneration capacity, and the promotion of the bone regeneration by the stem cells is mainly realized by releasing vesicle-extracellular vesicle containing various proteins and nucleic acid (such as miRNA) substances. Researches show that the extracellular vesicles can participate in various biochemical and cellular processes such as intercellular communication, immunoregulation, energy metabolism, tissue repair, tissue regeneration and metabolism.

The extracellular vesicle has natural advantages as a drug carrier, and the small size and the double-layer phospholipid layer structure reduce the risk of vascular blockage and are beneficial to cell uptake. In addition, the extracellular vesicles have the excellent characteristics of low toxicity, immunogenicity and the like, and the surfaces of the extracellular vesicles are rich in amino groups, so that modification of functional groups is facilitated. Therefore, the use of extracellular vesicles as a drug for promoting bone regeneration has natural advantages, but the ability of the original extracellular vesicles to target bone tissue is general and the therapeutic effect needs to be further improved.

Therefore, the provision of the extracellular vesicle with bone targeting capability and loaded with the small molecule drug and the preparation method thereof have important significance.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a bone targeting extracellular vesicle and a preparation method and application thereof. The bone-targeting extracellular vesicle comprises an extracellular vesicle with the outer surface modified with a bone-targeting molecule and a bone-promoting drug loaded in the extracellular vesicle, wherein the bone-targeting extracellular vesicle realizes the accurate delivery of a target organ through the targeting of bone tissues, and improves the bone-promoting treatment effect through the loading of the bone-promoting drug. When the bone targeting extracellular vesicles are prepared, the bone targeting molecules are connected and modified on the surfaces of the extracellular vesicles loaded with the bone-promoting drugs by generating triazole bonds through click chemical reaction with bifunctional molecules, the bifunctional molecules are grafted on the outer surfaces of the extracellular vesicles loaded with the bone-promoting drugs by forming covalent bonds with amino groups on the surfaces of the extracellular vesicles, the bone targeting modification reaction on the extracellular vesicles is mild and convenient, the structures of the extracellular vesicles cannot be damaged, and the performances of the extracellular vesicles cannot be influenced.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a bone-targeting extracellular vesicle, which comprises an extracellular vesicle modified with a bone-targeting molecule on the outer surface, and a bone-promoting drug loaded in the extracellular vesicle.

The invention adopts the extracellular vesicles of human stem cells as a therapeutic tool, modifies the surface of the extracellular vesicles with bone targeting molecules, can realize accurate delivery of target organs through bone tissue targeting, and can realize bone tissue targeting and improve the therapeutic effect. In addition, the therapeutic effect can be further improved by loading bone-promoting drugs.

Preferably, the bone targeting molecule is connected and modified on the extracellular vesicle surface loaded with the bone-promoting drug by generating triazole bond through click chemical reaction with the bifunctional molecule; the bifunctional molecule is grafted on the outer surface of the extracellular vesicle loaded with the bone-promoting drug through forming a covalent bond with an amino group on the surface of the extracellular vesicle; the bifunctional molecule is a molecule simultaneously carrying diphenyl cyclooctyne and N-hydroxysuccinimide.

Preferably, the bifunctional molecule is selected from any one of dibenzylcyclooctyl-sulfo-N-hydroxysuccinimide ester, aza-dibenzocyclooctyne-succinimide ester, diphenyl cyclooctyne-PEG 4-hydrogenated succinimide ester or diphenyl cyclooctyne-C6-succinimide ester, preferably dibenzylcyclooctyl-sulfo-N-hydroxysuccinimide ester.

Preferably, the bone targeting molecule is modified with azide groups, and the bone targeting molecule comprises polypeptide bone targeting molecules and chemical bone targeting molecules.

Preferably, the amino acid sequence of the polypeptide bone targeting molecule comprises any one of (D) n, SDSSD, (DSS)6 or TPLSYLKGLVTVG (SEQ ID NO:1), or a combination of at least two thereof, wherein n in (D) n is 6 to 10, n is a positive integer, e.g., 6, 7, 8, 9 or 10; the polypeptide bone targeting molecule is connected with the azide group through lysine.

Preferably, the chemical bone targeting molecule comprises any one of or a combination of at least two of bisphosphonates, alendronate, zoledronate or pamidronate.

Preferably, the molar ratio of the bone targeting molecule and the bifunctional molecule in the bone targeting extracellular vesicle is 1 (5-20), and may be, for example, 1:5, 1:8, 1:10, 1:12, 1:14, 1:15, 1:18 or 1:20, and preferably 1 (12-18).

Preferably, the loading of the bone targeting molecule on the bone targeting extracellular vesicles is 0.1-0.3. mu. mol per gram of vesicles, for example, 0.1. mu. mol, 0.15. mu. mol, 0.2. mu. mol, 0.25. mu. mol, or 0.3. mu. mol, etc.

Preferably, the loading of bone-targeting extracellular vesicles in the bone-targeting extracellular vesicles contributes to 5-50%, e.g. 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45% or 50% etc.

Preferably, the structure of the bone-targeting extracellular vesicle is a bilayer phospholipid layer vesicle.

Preferably, the bone-targeting extracellular vesicles have a particle size of 30-200nm, e.g. 30nm, 50nm, 80nm, 100nm, 120nm, 150nm, 160nm, 180nm or 200nm, etc.

Preferably, the zeta potential of the bone-targeting extracellular vesicle is between-5 and-40 mV, which may be, for example, -5mV, -10mV, -15mV, -20mV, -25mV, -30mV, -35mV, or-40 mV.

Preferably, the bone-promoting drug comprises any one of, or a combination of at least two of, a nucleic acid, a protein, a peptide chain, or a small molecule chemical drug.

Preferably, the nucleic acid comprises any one of 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.

The nucleic acids of the invention also include morpholino oligonucleotide analogs or morpholino oligonucleotide conjugates.

Preferably, the protein comprises any one of bone morphogenic protein, osteopontin, catenin, collagen or silk fibroin or a combination of at least two thereof.

Preferably, the peptide chain comprises any one of teriparatide, osteogenic growth polypeptide or RGD peptide or a combination of at least two thereof.

The peptide chain has the effect of promoting bone, and also comprises parathyroid hormone related peptide, calcitonin gene related peptide, growth factor short peptide derivative, extracellular matrix derived peptide, hydroxyapatite combined peptide or self-assembly peptide. The self-assembling peptides include RADA 16-I.

In the invention, the micromolecular chemical drugs in the osteogenesis promoting drug comprise any one or the combination of at least two of decaenoic acids, flavonoids, quinones, Wnt pathway related drugs or RANKL pathway related drugs. The Wnt pathway is a signal transduction pathway mediated by ligand Wnt and membrane receptor binding in a group of multiple downstream channels. The drug includes sclerostin, Dickkopf-1, and secreted frizzled-related protein.

Preferably, the source of the extracellular vesicles is human stem cells.

Preferably, the human stem cell comprises any one of human bone marrow mesenchymal stem cell, human adipose stem cell, human umbilical cord mesenchymal stem cell, human umbilical cord blood mesenchymal stem cell, human placenta mesenchymal stem cell, human dental pulp stem cell, human periodontal ligament stem cell, human hair follicle stem cell or human amniotic mesenchymal stem cell.

Preferably, the extracellular vesicles are obtained by the following preparation method:

(a) culturing the human stem cells until the confluence degree is 80-90%, culturing in a complete culture medium, and collecting the culture medium;

(b) subjecting the culture medium collected in step (a) to centrifugation with three increasing centrifugal forces to obtain the extracellular vesicles.

Preferably, in step (a), the specific steps of culturing to reach 80-90% of confluence are: mixing human stem cells and complete culture medium, and culturing at 37 deg.C for 2-3 days until the confluency is 80-90% (such as 80%, 85% or 90%); wherein the amount of human stem cells added per 1mL of complete medium is 5X 10 ⁵ -1×10 ⁶ A cell (for example, 5X 10) ⁵ 、6×10 ⁵ 、7×10 ⁵ 、8×10 ⁵ 、9×10 ⁵ Or 1X 10 ⁶ Etc.).

Preferably, in step (a), the cultivation in complete medium comprises the following specific steps: transferring human stem cells with 80-90% confluency to complete culture medium prepared from extracellular vesicle-free serum, and culturing at 37 deg.C for 36-72h (such as 36h, 48h, 60h or 72 h); wherein the amount of stem cells added per 1mL of complete medium is 5X 10 ⁵ -1×10 ⁶ A cell (for example, 5X 10) ⁵ 、6×10 ⁵ 、7×10 ⁵ 、8×10 ⁵ 、9×10 ⁵ Or 1X 10 ⁶ Etc.).

Preferably, in step (a), the complete culture medium comprises α -MEM or DMEM, and 8-12% fetal bovine serum (e.g., 8%, 10%, 12%, etc.) and 0.5-2% diabody (e.g., 0.5%, 1%, 1.5%, 2%, etc.) are added to the complete culture medium.

Preferably, in the step (b), the centrifugation treatment with three times of increasing centrifugal force comprises the following specific steps:

centrifuging the culture collected in step (a) for 10-30min (for example, 10min, 15min, 20min, 25min or 30 min) under a centrifugal force of 0-10 deg.C (for example, 0 deg.C, 2 deg.C, 4 deg.C, 6 deg.C, 8 deg.C or 10 deg.C), 800-1200g (for example, 800g, 900g, 1000g, 1100g or 1200 g), and collecting the supernatant;

centrifuging the supernatant at 0-10 deg.C (such as 0 deg.C, 2 deg.C, 4 deg.C, 6 deg.C, 8 deg.C or 10 deg.C), 8000-;

finally, the supernatant is centrifuged for 90-120min (for example, 90min, 95min, 100min, 105min, 110min, 115min or 120 min) at a centrifugal force of 0-10 ℃ (for example, 0 ℃, 2 ℃, 4 ℃, 6 ℃, 8 ℃ or 10 ℃, etc.), 80000- & ltSUB & gt 120000g (for example, 80000g, 90000g, 100000g, 110000g or 120000g, etc.), and the precipitate is collected to obtain the extracellular vesicles.

Preferably, in step (b), the extracellular vesicles are preserved without resuspension in sterile PBS buffer.

Preferably, the mass ratio of the extracellular vesicles to the sterile PBS buffer is 1 (1-5), and may be, for example, 1:1, 1:2, 1:3, 1:4, or 1: 5.

As a preferred embodiment of the present invention, the extracellular vesicles are prepared by the following preparation method:

mixing human stem cells and complete culture medium, and culturing at 37 deg.C for 2-3 days until the confluency is 80-90%; wherein the amount of human stem cells added per 1mL of complete medium is 5X 10 ⁵ -1×10 ⁶ A cell;

transferring the human stem cells with 80-90% of confluency to a complete culture medium prepared by extracellular vesicle serum, and culturing for 36-72h at 37 ℃, wherein the complete culture medium is a complete culture medium containing 8-12% of fetal bovine serum and 0.5-2% of double antibody; wherein the amount of human stem cells added per 1mL of complete medium is 5X 10 ⁵ -1×10 ⁶ (ii) individual cells;

collecting the culture medium, and carrying out centrifugation treatment with three increasing centrifugal forces, wherein the centrifugation treatment with the three increasing centrifugal forces comprises the following specific steps: centrifuging the collected culture medium for 10-30min under the centrifugal force of 800-1200g at 0-10 ℃, and collecting the supernatant; centrifuging the supernatant at 0-10 deg.C and 8000-; finally, centrifuging the supernatant for 90-120min at the centrifugal force of 120000g at the temperature of 0-10 ℃ and 80000-; and (3) resuspending and storing the obtained extracellular vesicles and sterile PBS buffer solution according to the mass ratio of 1 (1-5).

In a second aspect, the present invention provides a method for preparing the bone-targeting extracellular vesicles of the first aspect, wherein the method for preparing the bone-targeting extracellular vesicles comprises the following steps:

(1) modification of bone targeting molecules: modifying the bone targeting molecules on the outer surface of the extracellular vesicles through a click chemical reaction to obtain the extracellular vesicles with the outer surfaces modified with the bone targeting molecules;

(2) loading of bone-promoting drugs: loading a bone-promoting drug into the extracellular vesicles of which the outer surfaces are modified with bone-targeting molecules through electroporation treatment to obtain the bone-targeting extracellular vesicles.

The invention obtains the extracellular vesicles secreted by the human stem cells through fractional centrifugation, modifies bone targeting polypeptides on the surfaces of the extracellular vesicles in a click chemistry mode, and loads bone-promoting related drugs by an electroporation method to obtain the bone targeting extracellular vesicles. The bone targeting modification reaction on the extracellular vesicles is mild and convenient, the structure of the extracellular vesicles cannot be damaged, the performance of the extracellular vesicles cannot be influenced, and the bone targeting polypeptide used in the invention has low toxic and side effects and a good targeting effect.

Preferably, in step (1), the specific steps of modification are:

(A) mixing the PBS buffer solution containing the extracellular vesicles with the bifunctional molecules, and then carrying out grafting reaction to obtain the extracellular vesicles with the bifunctional molecules grafted on the surfaces;

(B) and (3) resuspending the obtained extracellular vesicles with the surface grafted with the bifunctional molecules in a PBS buffer solution, mixing the extracellular vesicles with the bone targeting molecules, and performing coupling reaction to obtain the extracellular vesicles with the external surfaces modified with the bone targeting molecules.

Preferably, in step (A), the concentration of bifunctional molecules in the mixture obtained after mixing is 10 to 50. mu.M, for example, 10. mu.M, 15. mu.M, 20. mu.M, 25. mu.M, 30. mu.M, 35. mu.M, 40. mu.M, 45. mu.M, or 50. mu.M, and the concentration of extracellular vesicles is 0.5 to 1mg/mL, for example, 0.5mg/mL, 0.6mg/mL, 0.7mg/mL, 0.8mg/mL, 0.9mg/mL, or 1 mg/mL.

Preferably, in step (A), the temperature of the grafting reaction is 30-40 ℃, for example, 30 ℃, 33 ℃, 35 ℃, 37 ℃ or 40 ℃ and the like, and the time of the grafting reaction is 3-6h, for example, 3h, 3.5h, 4h, 4.5h, 5h, 5.5h or 6h and the like.

Preferably, in step (A), the non-grafted bifunctional molecule is removed by centrifugation by ultrafiltration after the grafting reaction has ended.

Preferably, in step (B), the concentration of the extracellular vesicles with the bifunctional molecules grafted on the surface in the solution obtained after resuspension is 0.5-5mg/mL, and may be, for example, 0.5mg/mL, 1mg/mL, 1.5mg/mL, 2mg/mL, 2.5mg/mL, 3mg/mL, 3.5mg/mL, 4mg/mL, 4.5mg/mL, or 5 mg/mL.

Preferably, in step (B), the concentration of the bone targeting molecule in the mixed solution obtained after mixing is 10-50 μ M, for example, 10 μ M, 15 μ M, 20 μ M, 25 μ M, 30 μ M, 35 μ M, 40 μ M, 45 μ M, or 50 μ M, and the concentration of the extracellular vesicle with the bifunctional molecule grafted on the surface is 0.5-5mg/mL, for example, 0.5mg/mL, 1mg/mL, 2mg/mL, 3mg/mL, 4mg/mL, or 5 mg/mL.

Preferably, in step (B), the temperature of the coupling reaction is 0-10 ℃, for example, 0 ℃, 2 ℃, 4 ℃, 6 ℃, 8 ℃ or 10 ℃ and the like, and the time of the coupling reaction is 8-12h, for example, 8h, 9h, 10h, 11h or 12h and the like.

Preferably, in step (B), the unmodified bone targeting molecule is removed by ultrafiltration and centrifugation after the coupling reaction is completed.

Preferably, in step (B), the extracellular vesicles modified with the bone targeting molecule on the outer surface are stored in sterile PBS buffer in a resuspension manner.

Preferably, the mass ratio of the extracellular vesicle modified with the bone targeting molecule on the outer surface to the sterile PBS buffer solution is 1 (1-5), and may be, for example, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5 or 1: 5.

Preferably, in the step (2), the loading specifically comprises the following steps: and mixing the extracellular vesicles the outer surfaces of which are modified with bone targeting molecules, bone promoting drugs and electroporation buffer solution, performing electroporation, and standing after the electroporation is finished to obtain the bone targeting extracellular vesicles.

Preferably, the concentration of the extracellular vesicles with the bone targeting molecules modified on the outer surface in the mixed solution obtained by mixing is 0.5-5mg/mL, for example, 0.5mg/mL, 1mg/mL, 2mg/mL, 3mg/mL, 4mg/mL, or 5mg/mL, and the concentration of the bone-promoting drug is 0.01-0.5. mu.g/μ L, for example, 0.01. mu.g/μ L, 0.02. mu.g/μ L, 0.03. mu.g/μ L, 0.05. mu.g/μ L, 0.8. mu.g/μ L, 0.1. mu.g/μ L, 0.2. mu.g/μ L, 0.3. mu.g/μ L, 0.4. mu.g/μ L, or 0.5. mu.g/μ L.

Preferably, the electroporation buffer comprises a mixed solution of iodixanol and potassium dihydrogen phosphate-potassium chloride solution in a volume ratio of (18-23) to (77-82), such as 18:82, 19:81, 20:80, 21:79, 22:78 or 23: 77; the potassium dihydrogen phosphate-potassium chloride solution contains potassium dihydrogen phosphate in a mass concentration of 1-1.2mM (for example, 1mM, 1.1mM, or 1.2 mM), potassium chloride in a mass concentration of 23-27mM (for example, 23mM, 24mM, 25mM, 26mM, or 27 mM), and water as a solvent.

Preferably, the voltage of the electroporation is 100-400V, such as 100V, 150V, 250V, 300V, 350V or 400V, and the like, and the time of the electroporation is 1-20ms, such as 1ms, 2ms, 5ms, 8ms, 12ms, 16ms, 18ms or 20ms, and the like.

Preferably, the temperature of the standing is 30-40 ℃, for example, 30 ℃, 33 ℃, 35 ℃, 37 ℃ or 40 ℃ and the like, and the time of the standing is 25-35min, for example, 25min, 28min, 30min, 32min or 35min and the like.

Preferably, the resting is followed by removal of the unloaded bone-contributing drug by ultrafiltration centrifugation.

As a preferred technical scheme of the invention, the preparation method of the bone targeting extracellular vesicle comprises the following steps:

(1) modification of bone targeting molecules:

(A) mixing a PBS buffer solution containing extracellular vesicles with a bifunctional molecule to obtain a mixed solution, wherein the concentration of the bifunctional molecule in the mixed solution is 10-50 mu M, and the concentration of the extracellular vesicles is 0.5-1 mg/mL; mixing, and performing grafting reaction at 30-40 ℃ for 3-6 h; after the grafting reaction is finished, removing the non-grafted bifunctional molecules through ultrafiltration and centrifugation to obtain extracellular vesicles with the surface grafted with the bifunctional molecules;

(B) resuspending the obtained extracellular vesicles with the surface grafted with the bifunctional molecules in a PBS buffer solution, wherein the concentration of the extracellular vesicles with the surface grafted with the bifunctional molecules in the resuspended solution is 0.5-5mg/mL, mixing the resuspended solution with bone targeting molecules, wherein the concentration of the bone targeting molecules in the mixed solution obtained after mixing is 10-50 mu M, and the concentration of the extracellular vesicles with the surface grafted with the bifunctional molecules is 0.5-5 mg/mL; performing coupling reaction for 8-12h at 0-10 ℃, removing unmodified bone targeting molecules through ultrafiltration and centrifugation after the coupling reaction is finished to obtain extracellular vesicles with the external surfaces modified with the bone targeting molecules, and resuspending and storing the extracellular vesicles with the external surfaces modified with the bone targeting molecules and a sterile PBS buffer solution according to a mass ratio of 1 (1-5);

(2) loading of bone-promoting drugs:

mixing the extracellular vesicles the outer surfaces of which are modified with the bone targeting molecules, bone-promoting drugs and an electroporation buffer solution, wherein the concentration of the extracellular vesicles the outer surfaces of which are modified with the bone targeting molecules in the mixed solution is 0.5-5mg/mL, and the concentration of the bone-promoting drugs is 0.01-0.5 mu g/mu L; performing electroporation, wherein the voltage of the electroporation is 100-400V, and the time is 1-20 ms; and standing at 30-40 ℃ for 25-35min after perforation is finished, and removing unloaded bone-promoting drugs through ultrafiltration and centrifugation after standing to obtain the bone-targeting extracellular vesicles.

In a third aspect, the present invention provides a pharmaceutical composition comprising the bone-targeting extracellular vesicle of the first aspect.

Preferably, the pharmaceutical composition comprises pharmaceutically acceptable excipients.

In a fourth aspect, the present invention provides the use of any one or a combination of at least two of the bone-targeting extracellular vesicles of the first aspect, the preparation method of the bone-targeting extracellular vesicles of the second aspect, or the pharmaceutical composition of the third aspect in the preparation of a product for treating orthopedic disorders.

The recitation of numerical ranges herein includes not only the above-recited numerical values, but also any numerical values between non-recited numerical ranges, and is not intended to be exhaustive or to limit the invention to the precise numerical values encompassed within the range for brevity and clarity.

Compared with the prior art, the invention has the following beneficial effects:

(1) the bone targeting molecule is connected and modified on the surface of the extracellular vesicle loaded with the bone promoting drug by generating triazole bond through click chemical reaction with a bifunctional molecule, the bifunctional molecule is grafted on the outer surface of the extracellular vesicle loaded with the bone promoting drug through covalent bond formation with amino on the surface of the extracellular vesicle, and the preparation method of the bone targeting extracellular vesicle is mild and convenient in bone targeting modification reaction of the extracellular vesicle, does not damage the structure of the extracellular vesicle and does not influence the performance of the extracellular vesicle.

(2) After the bone targeting extracellular vesicles are loaded with bone promoting drugs, the treatment effect can be improved, and the defects of poor treatment effect and weak targeting of the original stem cell extracellular vesicles are overcome.

(3) The bone targeting polypeptide has good targeting effect, and is beneficial to delivering extracellular vesicles to the surface of bone tissue so as to improve the treatment effect.

(4) Compared with potential toxic and side effects caused by using a large amount of organic solvents in liposome preparation, the good biological safety of the extracellular vesicles is beneficial to further clinical application of the extracellular vesicles, and the conditions in the bone targeting modification process are mild without the participation of organic solvents.

Drawings

FIG. 1 shows the results of transmission electron microscopy of the extracellular vesicles of example 1 (scale bar 100 nm).

FIG. 2 shows the result of transmission electron microscopy of the bone-targeting extracellular vesicles of example 1 (scale bar 200 nm).

FIG. 3 shows the result of transmission electron microscopy of the bone-targeting extracellular vesicles in example 1 (scale bar 200 nm).

Fig. 4 is a fluorescence intensity curve of the bone-targeting extracellular vesicles in test example 2.

Fig. 5 is a graph of the particle size distribution statistics of the bone-targeting extracellular vesicles (example 1) described in test example 2.

Fig. 6A and 6B are graphs showing the distribution of bone-targeting extracellular vesicles in various tissues of mice in test example 3.

Fig. 7A and 7B are staining results of in vitro osteogenesis promoting effect test in test example 4.

Detailed Description

The technical solution of the present invention is further described below by way of specific embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitation of the present invention.

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.

The sources of the components used in the following examples and comparative examples are as follows:

example 1

The embodiment provides a bone targeting extracellular vesicle, which is generated by a human mesenchymal stem cell, wherein the bone targeting molecule is a polypeptide bone targeting molecule, the amino acid sequence of the polypeptide bone targeting molecule is (D)8, the polypeptide bone targeting molecule is connected with an azide group through lysine, the bone-promoting drug comprises micro RNA, and the nucleotide sequence of the micro RNA is UUCAAGUAAUCCAGGAUAGGCU (SEQ ID NO: 2).

The method for preparing the extracellular vesicles is as follows:

mixing human bone marrow mesenchymal stem cells with complete culture medium at 37 deg.C under 5% CO ₂ Culturing for 2-3 days until the confluency is 80-90%; wherein the addition amount of human mesenchymal stem cells in each 1mL of complete culture medium is 5 × 10 ⁵ (ii) individual cells;

transferring 80-90% confluent human mesenchymal stem cells to complete culture medium prepared from extracellular vesicle-free serum at 37 deg.C under 5% CO ₂ Culturing for 48h, wherein the addition amount of human bone marrow mesenchymal stem cells in each 1mL complete culture medium is 5 × 10 ⁵ A cell; the complete medium is alpha-MEM medium containing 10% fetal bovine serum and 1% double antibody.

Collecting the culture medium, and carrying out centrifugation treatment with three increasing centrifugal forces, wherein the centrifugation treatment with the three increasing centrifugal forces comprises the following specific steps: centrifuging 1000g at 4 ℃ for 10min to obtain a first supernatant, centrifuging 10000g of the first supernatant at 4 ℃ for 30min to obtain a second supernatant, centrifuging 100000g of the second supernatant at 4 ℃ for 90min, collecting the precipitate to obtain extracellular vesicles, wherein the extracellular vesicles are required to be resuspended in a sterile PBS buffer solution, and the mass ratio of the extracellular vesicles to the sterile PBS buffer solution is 1: 1; the transmission electron microscope detection result of the obtained extracellular vesicles is shown in fig. 1, and it can be seen from fig. 1 that the extracellular vesicles collected by the centrifugation method are spherical in shape and about 100nm in size.

The preparation method of the bone targeting extracellular vesicle is as follows:

(1) modification of bone targeting molecules:

(A) mixing PBS buffer solution containing extracellular vesicles and dibenzylcyclooctyl-sulfo-N-hydroxysuccinimide ester to obtain a mixed solution, wherein the concentration of the dibenzylcyclooctyl-sulfo-N-hydroxysuccinimide ester in the mixed solution is 30 mu M, and the concentration of the extracellular vesicles is 0.8 mg/mL; mixing, and grafting at 37 deg.C for 5 h; after the grafting reaction is finished, removing ungrafted dibenzylcyclooctyl-sulfo-N-hydroxysuccinimide ester through ultrafiltration centrifugation, wherein the molecular weight cut-off of an ultrafiltration centrifugal tube used in the ultrafiltration centrifugation is 100kDa, and obtaining extracellular vesicles with dibenzylcyclooctyl-sulfo-N-hydroxysuccinimide ester grafted on the surface;

(B) resuspending the obtained extracellular vesicles with the surface grafted with dibenzylcyclooctyl-sulfo-N-hydroxysuccinimide ester in a PBS buffer solution, wherein the concentration of the extracellular vesicles with the surface grafted with dibenzylcyclooctyl-sulfo-N-hydroxysuccinimide ester in the resuspended solution is 0.5mg/mL, then mixing the resuspended solution with bone targeting molecules, wherein the concentration of the bone targeting molecules in the mixed solution obtained after mixing is 30 mu M, and the concentration of the extracellular vesicles with the surface grafted with dibenzylcyclooctyl-sulfo-N-hydroxysuccinimide ester is 3 mg/mL; performing coupling reaction for 10 hours at 4 ℃, removing unmodified bone targeting molecules through ultrafiltration centrifugation after the coupling reaction is finished, wherein the molecular weight cut-off of an ultrafiltration centrifugal tube used in the ultrafiltration centrifugation is 100kDa, obtaining extracellular vesicles with the external surfaces modified with the bone targeting molecules, and suspending and storing the extracellular vesicles with the external surfaces modified with the bone targeting molecules and a sterile PBS buffer solution according to the mass ratio of 1: 1; the transmission electron microscope detection result of the extracellular vesicles modified with the bone targeting molecules on the outer surface is shown in fig. 2, and as can be seen from fig. 2, the extracellular vesicles modified with the bone targeting molecules on the outer surface are spherical in shape and are still about 100nm in size.

(2) Loading of bone drug is facilitated:

mixing the extracellular vesicles the outer surfaces of which are modified with the bone targeting molecules, bone-promoting drugs and an electroporation buffer solution, wherein the concentration of the extracellular vesicles the outer surfaces of which are modified with the bone targeting molecules in the mixed solution is 3mg/mL, and the concentration of the bone-promoting drugs is 0.01 [ mu ] g/[ mu ] L; performing electroporation, wherein the voltage of the electroporation is 350V, and the time is 10 ms; and standing at 37 ℃ for 30min after perforation is finished, removing unloaded bone-promoting drugs by ultrafiltration centrifugation after standing, wherein the molecular weight cut-off of an ultrafiltration centrifugal tube used by the ultrafiltration centrifugation is 100kDa, and thus obtaining the bone-targeting extracellular vesicles.

The electroporation buffer solution is a mixed solution of iodixanol (density gradient separation solution iodixanol, optiprep, Axis-Shield) and a potassium dihydrogen phosphate-potassium chloride solution in a volume ratio of 21:79, wherein the mass concentration of potassium dihydrogen phosphate in the potassium dihydrogen phosphate-potassium chloride solution is 1.15mM, the pH value is 7.2, the mass concentration of potassium chloride is 25mM, and the solvent is water.

The transmission electron microscope detection result of the bone-targeting extracellular vesicle is shown in fig. 3, and as can be seen from fig. 3, the morphology of the bone-targeting extracellular vesicle is spherical, and the size of the bone-targeting extracellular vesicle is about 120 nm.

Example 2

The embodiment provides a bone targeting extracellular vesicle, which is generated by a human adipose-derived stem cell, wherein the bone targeting molecule is a polypeptide bone targeting molecule, the amino acid sequence of the polypeptide bone targeting molecule is SDSSD, the polypeptide bone targeting molecule is connected with an azide group through lysine, the bone promoting drug comprises micro RNA, and the nucleotide sequence of the micro RNA is UAGCACCAUUUGAAAUCAGUGUU (SEQ ID NO: 3).

The method for preparing the extracellular vesicles is as follows:

mixing human adipose stem cells with complete medium, and culturing at 37 deg.C under 5% CO ₂ Culturing for 2-3 days until the confluency is 80-90%; wherein the addition amount of human adipose-derived stem cells per 1mL of complete medium is 8X 10 ⁵ A plurality of;

transferring human adipose-derived stem cells with confluency of 80-90% to complete culture medium prepared from extracellular vesicle serum at 37 deg.C under 5% CO ₂ Culturing for 36h, wherein the addition amount of human adipose-derived stem cells per 1mL of complete culture medium is 8 × 10 ⁵ A plurality of; the complete medium is alpha-MEM medium containing 8% fetal bovine serum and 0.5% double antibody.

Collecting the culture medium, and carrying out centrifugation treatment with three increasing centrifugal forces, wherein the centrifugation treatment with the three increasing centrifugal forces comprises the following specific steps: centrifuging the supernatant I at 800g for 30min at 4 ℃ to obtain a supernatant I, centrifuging the supernatant I at 8000g for 60min at 4 ℃ to obtain a supernatant II, centrifuging the supernatant II at 80000g for 120min at 4 ℃, collecting the precipitate to obtain extracellular vesicles, wherein the extracellular vesicles do not need to be resuspended in sterile PBS buffer, and the mass ratio of the extracellular vesicles to the sterile PBS buffer is 1:1.

(1) modification of bone targeting molecules:

(A) mixing PBS buffer solution containing extracellular vesicles and dibenzylcyclooctyl-sulfo-N-hydroxysuccinimide ester to obtain mixed solution, wherein the concentration of the dibenzylcyclooctyl-sulfo-N-hydroxysuccinimide ester in the mixed solution is 10 mu M, and the concentration of the extracellular vesicles is 0.5 mg/mL; mixing and then carrying out grafting reaction for 6h at the temperature of 30 ℃; after the grafting reaction is finished, removing ungrafted dibenzylcyclooctyl-sulfo-N-hydroxysuccinimide ester through ultrafiltration centrifugation, wherein the molecular weight cut-off of an ultrafiltration centrifugal tube used in the ultrafiltration centrifugation is 100kDa, and obtaining extracellular vesicles with dibenzylcyclooctyl-sulfo-N-hydroxysuccinimide ester grafted on the surface;

(B) resuspending the obtained extracellular vesicles with the surface grafted with dibenzylcyclooctyl-sulfo-N-hydroxysuccinimide ester in a PBS buffer solution, wherein the concentration of the extracellular vesicles with the surface grafted with dibenzylcyclooctyl-sulfo-N-hydroxysuccinimide ester in the resuspended solution is 0.75mg/mL, then mixing the resuspended solution with bone targeting molecules, wherein the concentration of the bone targeting molecules in the mixed solution obtained after mixing is 10 mu M, and the concentration of the extracellular vesicles with the surface grafted with dibenzylcyclooctyl-sulfo-N-hydroxysuccinimide ester is 0.5 mg/mL; performing coupling reaction for 12 hours at the temperature of 2 ℃, removing unmodified bone targeting molecules through ultrafiltration centrifugation after the coupling reaction is finished, wherein the molecular weight cut-off of an ultrafiltration centrifugal tube used in the ultrafiltration centrifugation is 100kDa, obtaining extracellular vesicles with the external surfaces modified with the bone targeting molecules, and suspending and storing the extracellular vesicles with the external surfaces modified with the bone targeting molecules and a sterile PBS buffer solution according to the mass ratio of 1: 1.5.

(2) Loading of bone-promoting drugs:

mixing the extracellular vesicles the outer surfaces of which are modified with the bone targeting molecules, bone-promoting drugs and an electroporation buffer solution, wherein the concentration of the extracellular vesicles the outer surfaces of which are modified with the bone targeting molecules in the mixed solution is 0.5mg/mL, and the concentration of the bone-promoting drugs is 0.02 mu g/mu L; performing electroporation, wherein the voltage of the electroporation is 400V, and the time is 10 ms; and standing for 35min at 30 ℃ after perforation is finished, removing unloaded bone-promoting drugs through ultrafiltration centrifugation after standing, wherein the molecular weight cut-off of an ultrafiltration centrifugal tube used in the ultrafiltration centrifugation is 100kDa, and obtaining the bone-targeting extracellular vesicles.

Example 3

The embodiment provides a bone-targeting extracellular vesicle, which is generated by human umbilical cord mesenchymal stem cells, wherein the bone-targeting molecule is a polypeptide bone-targeting molecule, the amino acid sequence of the polypeptide bone-targeting molecule is (DSS)6, the polypeptide bone-targeting molecule is connected with an azide group through lysine, the bone-promoting drug comprises micro RNA, and the nucleotide sequence of the micro RNA is UUCACAGUGGCUAAGUUCCGC (SEQ ID NO: 4).

The method for preparing the extracellular vesicles is as follows:

mixing human umbilical cord mesenchymal stem cells with complete medium at 37 deg.C and 5% CO ₂ Culturing for 2-3 days until the confluency is 80-90%; wherein the addition amount of human umbilical cord mesenchymal stem cells in each 1mL of complete culture medium is 1 × 10 ⁶ A plurality of;

transferring human umbilical cord mesenchymal stem cells with confluence degree of 80-90% to complete culture medium prepared by cell-free outer vesicle serum, and culturing at 37 deg.C with 5% CO ₂ Culturing for 72h, wherein the human umbilical cord is cultured in every 1mL of complete mediumThe addition amount of the mesenchymal stem cells is 1 × 10 ⁶ A plurality of; the complete culture medium is a DMEM culture medium containing 12% fetal calf serum and 2% double antibody.

Collecting the culture medium, and carrying out three times of centrifugal treatment with increasing centrifugal force, wherein the three times of centrifugal treatment with increasing centrifugal force comprises the following specific steps: centrifuging 1200g of the supernatant I at 4 ℃ for 10min to obtain a supernatant I, centrifuging 12000g of the supernatant I at 4 ℃ for 30min to obtain a supernatant II, centrifuging 120000g of the supernatant II at 4 ℃ for 90min, collecting the precipitate to obtain extracellular vesicles, wherein the extracellular vesicles do not need to be resuspended in sterile PBS (phosphate buffer solution) and the mass ratio of the extracellular vesicles to the sterile PBS is 1:1.

(1) modification of bone targeting molecules:

(A) mixing PBS buffer solution containing extracellular vesicles with dibenzylcyclooctyl-sulfo-N-hydroxysuccinimide ester to obtain mixed solution, wherein the concentration of the dibenzylcyclooctyl-sulfo-N-hydroxysuccinimide ester in the mixed solution is 50 mu M, and the concentration of the extracellular vesicles is 1 mg/mL; mixing and then carrying out grafting reaction for 3h at the temperature of 40 ℃; after the grafting reaction is finished, removing ungrafted dibenzylcyclooctyl-sulfo-N-hydroxysuccinimide ester through ultrafiltration centrifugation, wherein the molecular weight cut-off of an ultrafiltration centrifugal tube used in the ultrafiltration centrifugation is 100kDa, and obtaining extracellular vesicles with dibenzylcyclooctyl-sulfo-N-hydroxysuccinimide ester grafted on the surface;

(B) resuspending the obtained extracellular vesicles with the surface grafted with dibenzylcyclooctyl-sulfo-N-hydroxysuccinimide ester in a PBS (phosphate buffer solution), wherein the concentration of the extracellular vesicles with the surface grafted with dibenzylcyclooctyl-sulfo-N-hydroxysuccinimide ester in the resuspended solution is 1mg/mL, then mixing the resuspended solution with bone targeting molecules, wherein the concentration of the bone targeting molecules in the mixed solution obtained after mixing is 50 μ M, and the concentration of the extracellular vesicles with dibenzylcyclooctyl-sulfo-N-hydroxysuccinimide ester grafted on the surface is 5 mg/mL; coupling reaction is carried out for 8 hours at 6 ℃, unmodified bone targeting molecules are removed through ultrafiltration centrifugation after the coupling reaction is finished, the molecular weight cut-off of an ultrafiltration centrifugal tube used in the ultrafiltration centrifugation is 100kDa, extracellular vesicles with the outer surfaces modified with the bone targeting molecules are obtained, and the extracellular vesicles with the outer surfaces modified with the bone targeting molecules and sterile PBS buffer solution are subjected to resuspension and storage according to the mass ratio of 1:2.

(2) Loading of bone drug is facilitated:

mixing the extracellular vesicles the outer surfaces of which are modified with the bone targeting molecules, bone-promoting drugs and an electroporation buffer solution, wherein the concentration of the extracellular vesicles the outer surfaces of which are modified with the bone targeting molecules in the mixed solution is 5mg/mL, and the concentration of the bone-promoting drugs is 0.5 [ mu ] g/[ mu ] L; performing electroporation, wherein the voltage of the electroporation is 200V, and the time is 15 ms; and standing for 25min at 40 ℃ after perforation is finished, removing unloaded bone-promoting drugs by ultrafiltration centrifugation after standing, wherein the molecular weight cut-off of an ultrafiltration centrifugal tube used by the ultrafiltration centrifugation is 100kDa, and obtaining the bone-targeting extracellular vesicles.

Example 4

This example provides a bone-targeting extracellular vesicle that differs from example 1 only in that the bone-contributing drug is an siRNA, the nucleotide sequence of which is GAAGUGGUUCAGAAGAUGACGCCCAAACCCAAACCCAAACCCAAACCCAAA (SEQ ID NO: 5). The preparation method of the bone targeting extracellular vesicles refers to example 1.

Example 5

This example provides a bone-targeting extracellular vesicle that differs from example 1 only in that the bone-contributing drug is a small molecule chemical drug that is a bone morphogenic protein. The preparation method of the bone targeting extracellular vesicles is described in example 1.

Example 6

This example provides a bone-targeting extracellular vesicle, which is prepared by a method different from that of example 1 only in that the time for the grafting reaction in step (a) is 12 hours, the temperature for the grafting reaction is 25 ℃, and the rest of the steps are referred to example 1.

Example 7

This example provides a bone-targeting extracellular vesicle prepared by a method different from that of example 1 only in that the grafting reaction time in step (a) was 2 hours, the temperature of the grafting reaction was 60 ℃, and the rest of the steps were referred to example 1.

Example 8

This example provides a bone-targeting extracellular vesicle prepared by a method different from that of example 1 only in that the concentration of the azadibenzocyclooctyne-succinimide ester in the mixed solution of step (a) is 5 μ M; the rest of the procedure was referred to example 1.

Example 9

This example provides a bone-targeting extracellular vesicle prepared by a method different from that of example 1 only in that the concentration of the bone-targeting polypeptide in the mixed solution in step (B) was 5 μ M, and the rest of the steps were performed in example 1.

Example 10

This example provides a bone-targeting extracellular vesicle, which is prepared by a method different from that of example 1 only in that the voltage for electroporation in step (2) is 700V and the time for electroporation is 2ms, and the rest of the steps refer to example 1.

Example 11

This example provides a bone-targeting extracellular vesicle, which is prepared by a method different from that of example 1 only in that the voltage for electroporation in step (2) is 50V and the time for electroporation is 25ms, and the rest of the steps refer to example 1.

Comparative example 1

This comparative example provides a non-targeted extracellular vesicle produced by a human mesenchymal stem cell, the osteogenesis-promoting drug comprises a micro RNA having a nucleotide sequence of UUCAAGUAAUCCAGGAUAGGCU (SEQ ID NO: 2). This example differs from example 1 in that the extracellular vesicles do not contain a bone targeting polypeptide.

The method for preparing the extracellular vesicles is as follows:

transferring 80-90% confluent human mesenchymal stem cells to complete culture medium prepared from extracellular vesicle-free serum at 37 deg.C under 5% CO ₂ Culturing for 48h, wherein the addition amount of human bone marrow mesenchymal stem cells in each 1mL complete culture medium is 5 × 10 ⁵ (ii) individual cells; the complete medium is alpha-MEM medium containing 10% fetal bovine serum and 1% double antibody.

Collecting the culture medium, and carrying out centrifugation treatment with three increasing centrifugal forces, wherein the centrifugation treatment with the three increasing centrifugal forces comprises the following specific steps: centrifuging at 4 deg.C at 1000g for 10min to obtain supernatant I, centrifuging at 4 deg.C at 10000g for 30min to obtain supernatant II, centrifuging at 4 deg.C at 100000g for 90min, and collecting precipitate to obtain extracellular vesicle.

The preparation method of the non-targeting extracellular vesicle is as follows:

mixing the extracellular vesicles, the bone-promoting drug and the electroporation buffer solution, wherein the concentration of the extracellular vesicles in the mixed solution is 3mg/mL, and the concentration of the bone-promoting drug is 0.03 mu g/mu L; performing electroporation, wherein the voltage of the electroporation is 350V, and the time is 10 ms; and standing at 37 ℃ for 30min after perforation is finished, removing unloaded bone-promoting drugs by ultrafiltration centrifugation after standing, wherein the molecular weight cut-off of an ultrafiltration centrifugal tube used by the ultrafiltration centrifugation is 100kDa, and thus obtaining the non-target extracellular vesicles.

Comparative example 2

This comparative example provides a non-bone targeting extracellular vesicle that differs from example 1 only in that the bone targeting polypeptide of example 1 was replaced with (G) 6. The preparation method of the non-bone targeting extracellular vesicles refers to example 1.

Comparative example 3

This comparative example provides a bone-targeting vesicle that differs from example 1 only in that the vesicle is a liposome vesicle.

The preparation method of the liposome vesicle comprises the following steps:

trimethyl-2, 3-dioleoyloxypropylammonium bromide, dioleoylphosphatidylethanolamine, cholesterol, distearoylphosphatidylethanolamine-polyethylene glycol 2000 were dissolved in chloroform at a molar ratio of 63:16:16:5, and evaporated to a thin film at 60 ℃ under negative pressure with a rotary evaporator, and the resulting lipid thin film was pre-incubated with 10mM phosphate buffered saline (PBS, pH7.4) in a water bath at 60 ℃ to form a multi-colloidal vesicle. The formed multi-colloidal vesicles were then extruded 10 times through a 0.2 μm polycarbonate membrane (Whatman) with an Avanti Mini extruder (Avanti Polar Lipids) and 10 times through a 0.1 μm membrane to gradually form the final liposomes.

The preparation method of the bone targeting vesicle is referred to step (2) and step (3) of example 1.

Test example 1

This test performed the adsorption rate test of the bone-targeting extracellular vesicles of examples 1 to 11 and the vesicles of comparative examples 1 to 3, taking extracellular vesicle mimics as an example, the method of the adsorption rate test comprising the following steps:

modifying polypeptide targeting molecules with rhodamine B fluorescence-labeled amino acid sequence (D)8 on extracellular vesicle mimics, preparing the extracellular vesicle mimics into 1mg/mL suspension in PBS, measuring the fluorescence intensity of the suspension by using a fluorescence spectrophotometer, and setting an excitation wavelength of 550nm and an emission wavelength of 580 nm; after the determination, incubating the extracellular vesicle simulant suspension and 10mg/mL hydroxyapatite particles in a gas bath shaking table at 25 ℃ for 5h, centrifuging the suspension at 4000rpm for 10min after the incubation is finished, precipitating the hydroxyapatite and the extracellular vesicle simulant adsorbed on the hydroxyapatite, determining the fluorescence intensity in the supernatant by using a fluorescence spectrophotometer, wherein the excitation wavelength is 550nm, the emission wavelength is 580nm, and the adsorption rate is calculated by the following formula:

adsorption rate (I1-I2)/I1X 100%

I1: fluorescence intensity before incubation with hydroxyapatite;

i2: fluorescence intensity after incubation with hydroxyapatite.

The fluorescence intensity curve graph before and after incubation of the bone targeting extracellular vesicles in example 1 is shown in fig. 4, and it can be seen from the graph that the fluorescence intensity of the supernatant after incubation is significantly reduced, which indicates that the bone targeting extracellular vesicles have good targeting effect and high adsorption rate.

The adsorption rates of the bone-targeting extracellular vesicles in examples 1 to 11 and the vesicles in comparative examples 1 to 3 were measured with reference to the test method for the adsorption rate test of extracellular vesicle mimics. Appropriate modification conditions can be selected by calculating the adsorption rate. The results of the adsorption rate measurement are shown in table 1.

TABLE 1

Sample(s)	Adsorption rate
		Example 1	75％
Example 2	75％
		Example 3	75％
Example 4	75％
		Example 5	75％
Example 6	75％
		Example 7	75％
Example 8	30％
		Example 9	40％
Example 10	75％
		Example 11	75％
Comparative example 1	22％
		Comparative example 2	22％
Comparative example 3	60％

From the results in table 1, it is known that, compared with the modified non-bone targeting polypeptide, the modified bone targeting polypeptide can significantly improve the bone targeting property of the extracellular vesicles, the reduced concentration of the bifunctional molecule can reduce the bone targeting property, the reduced concentration of the bone targeting molecule can reduce the bone targeting property, and the bone targeting property prepared by using the liposome is lower than that of the extracellular vesicles.

Test example 2

This test performed a particle size distribution test, a drug loading test, a vesicle concentration test, a bone targeting molecule modification efficiency test, and a zeta potential measurement of the bone targeting extracellular vesicles on the bone targeting extracellular vesicles in examples 1 to 11 and the vesicles in comparative examples 1 to 3.

The extracellular vesicles were mixed with PBS at a ratio of 1:1 and the particle size distribution was determined using a Zetasizer nano particle sizer.

The detection method for the drug loading rate test comprises the following steps:

the fluorescence intensity of the drug is measured after the drug is labeled by fluorescence, F1 is loaded into extracellular vesicles, the drug is diluted in PBS according to the ratio of 1:1, the fluorescence intensity is measured by a fluorescence spectrophotometer, F2 is used, TritonX is added, the fluorescence intensity is measured, F3 is obtained, and the loading efficiency is (F3-F2)/F1 multiplied by 100 percent.

Vesicle concentration assay the BCA kit was used to test as per instructions.

The detection method for testing the modification efficiency of the bone targeting molecule comprises the following steps:

measuring a standard curve of the fluorescently-labeled bone targeting polypeptide by using a fluorescence spectrophotometer, using the fluorescent bone targeting polypeptide with the mass of M1 to modify extracellular vesicles, dispersing the collected extracellular vesicles into PBS according to the ratio of 1:1 to measure the fluorescence intensity, and measuring the corresponding content M2 according to the standard curve, wherein the modification efficiency is M2/M1 multiplied by 100%.

Extracellular vesicles were mixed with PBS at 1:1 and zeta potential was measured using a Zetasizer nano-particle sizer.

The statistical result of the particle size distribution of the bone-targeting extracellular vesicles described in example 1 is shown in fig. 5, and it can be found that the particle size of the modified extracellular vesicles is normally distributed, the particle size is uniform, and the average particle size is 124 nm.

Statistics of particle size distribution, drug loading, bone targeting molecule modification efficiency (molar ratio of bone targeting molecule, dibenzylcyclooctyl-sulfo-N-hydroxysuccinimide ester and extracellular vesicles) and zeta-potential measurements are shown in table 2.

TABLE 2

Sample (I)	Average particle diameter	Drug loading	Vesicle concentration	Efficiency of decoration	Zeta potential
						Example 1	125	15％	1	20％	-20mV
Example 2	125	20％	1	25％	-20mV
						Example 3	125	25％	1	30％	-20mV
Example 4	125	15％	1	20％	-20mV
						Example 5	125	15％	1	20％	-20mV
Example 6	125	15％	0.5	20％	-20mV
						Example 7	125	15％	0.5	20％	-20mV
Example 8	125	15％	1	10％	-20mV
						Example 9	125	15％	1	10％	-20mV
Example 10	125	0％	0.5	20％	-20mV
						Example 11	125	5％	1	20％	-20mV
Comparative example 1	125	15％	1	20％	-20mV
						Comparative example 2	125	15％	1	20％	-20mV
Comparative example 3	125	5％	Is composed of	10	-20mV

As can be seen from the results in table 2, as the total amount of extracellular vesicles obtained increased, the drug loading and modification efficiency also increased, and the zeta potential did not change significantly.

It is understood from a comparison of example 1 with examples 6 to 7 that increasing the reaction time or increasing the reaction temperature leads to vesicle fragmentation and thus to a decrease in the yield of vesicles.

As is clear from comparison of example 1 with examples 8 to 9, the modification efficiency is insufficient by lowering the reagent concentration, and the modification efficiency is decreased by breaking the vesicles by increasing the reagent concentration.

It can be seen from a comparison of example 1 with examples 10-11 that lower voltages do not penetrate the vesicles and result in no more drug loading, and that higher voltages break up the vesicles and result in no drug loading.

As can be seen from the comparison between example 1 and comparative example 3, the drug loading efficiency is low due to the low modification efficiency caused by the self-contained amino group of the liposome and the inability of using the electroporation method for drug loading.

Test example 3

The in vivo distribution effect of the bone-targeting extracellular vesicles described in example 1 and the extracellular vesicles in comparative example 2 was examined.

100 mu L of the bone targeting extracellular vesicles are stained for 30min by Cy5.5 according to the instruction, free dye is removed by using a clean 100kDa ultrafiltration centrifugal tube according to the instruction after the reaction time is up, the bone targeting extracellular vesicles are injected into a mouse body through tail veins, after 6h, the heart, liver, spleen, lung, kidney and leg bones are taken out, the distribution of the fluorescent dye in each organ is photographed under living body imaging equipment, the distribution of the bone targeting extracellular vesicles in each tissue of the mouse is shown in figures 6A and 6B, and the result shows that the targeting performance of the extracellular vesicles after the bone targeting polypeptide is modified in the bone tissue is effectively improved, and the fluorescence intensity value is 4 times that of the extracellular vesicles in a control group (the extracellular vesicles in comparative example 2).

Test example 4

Test the in vitro osteogenesis promoting effect test of the bone targeting extracellular vesicles described in example 1. The detection method comprises the following steps:

human fetal bone marrow mesenchymal stem cells are arranged according to the proportion of 1 multiplied by 10 per hole ⁵ The amount of (a) was inoculated in a six-well plate, culture was performed for 21 days using an osteogenesis-promoting induction medium, the medium was changed every three days, bone-targeting extracellular vesicles of 100. mu.g per well were added with the medium change, and alizarin red staining for osteogenic differentiation was performed after 21 days, with the results of staining shown in FIGS. 7A and 7B. The redder the alizarin red staining result shows that the osteogenic differentiation effect is better, and it is seen from fig. 7A and 7B that the in vitro stem cell osteogenic differentiation can be remarkably promoted after the vesicle is added.

In summary, the bone targeting polypeptide in the bone targeting extracellular vesicle provided by the invention has good targeting effect, and is beneficial to delivering the extracellular vesicle to the surface of bone tissue so as to improve the treatment effect, the bone promoting drug loaded in the bone targeting extracellular vesicle can further enhance the treatment effect, and the defects of poor treatment effect and weak targeting property of the original stem cell extracellular vesicle are overcome, and the bone targeting extracellular vesicle has important application value in preparing products for treating orthopedic diseases.

The applicant declares that the above description is only a specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and it should be understood by those skilled in the art that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are within the scope and disclosure of the present invention.

Sequence listing

<110> Shenzhen advanced technology research institute of Chinese academy of sciences

<120> bone-targeting extracellular vesicle and preparation method and application thereof

<130> 2022

<160> 5

<170> PatentIn version 3.3

<210> 1

<211> 13

<212> PRT

<213> Artificial sequence

<400> 1

Thr Pro Leu Ser Tyr Leu Lys Gly Leu Val Thr Val Gly

1 5 10

<210> 2

<211> 22

<212> RNA

<213> Artificial sequence

<400> 2

uucaaguaau ccaggauagg cu 22

<210> 3

<211> 23

<212> RNA

<213> Artificial sequence

<400> 3

uagcaccauu ugaaaucagu guu 23

<210> 4

<211> 21

<212> RNA

<213> Artificial sequence

<400> 4

uucacagugg cuaaguuccg c 21

<210> 5

<211> 51

<212> RNA

<213> Artificial sequence

<400> 5

gaagugguuc agaagaugac gcccaaaccc aaacccaaac ccaaacccaa a 51

Claims

1. A bone-targeting extracellular vesicle, comprising an extracellular vesicle modified on the outer surface with a bone-targeting molecule and a bone-promoting drug loaded in the extracellular vesicle.

2. The bone-targeting extracellular vesicle according to claim 1, wherein the bone-targeting molecule is modified on the surface of the extracellular vesicle loaded with bone-promoting drugs by generating a triazole linkage through a click chemistry reaction with a bifunctional molecule; the bifunctional molecule is grafted on the outer surface of the extracellular vesicle loaded with the bone-promoting drug through forming a covalent bond with an amino group on the surface of the extracellular vesicle; the bifunctional molecule is a molecule simultaneously carrying diphenyl cyclooctyne and N-hydroxysuccinimide;

preferably, the bifunctional molecule is selected from any one of dibenzylcyclooctyl-sulfo-N-hydroxysuccinimide ester, aza-dibenzocyclooctyne-succinimide ester, diphenyl cyclooctyne-PEG 4-hydrogenated succinimide ester or diphenyl cyclooctyne-C6-succinimide ester, preferably dibenzylcyclooctyl-sulfo-N-hydroxysuccinimide ester;

preferably, the bone targeting molecule is modified with azide groups, and comprises polypeptide bone targeting molecules and chemical bone targeting molecules;

preferably, the amino acid sequence of the polypeptide bone targeting molecule comprises any one of (D) n, SDSSD, (DSS)6 or TPLSYLKGLVTVG, or a combination of at least two thereof, wherein n in (D) n is 6 to 10, and n is a positive integer; the polypeptide bone targeting molecule is connected with an azide group through lysine;

preferably, the chemical bone targeting molecule comprises any one of or a combination of at least two of bisphosphonates, alendronate, zoledronate or pamidronate;

preferably, the molar ratio of the bone targeting molecule to the bifunctional molecule in the bone targeting extracellular vesicle is 1 (5-20), preferably 1 (12-18);

preferably, the load of the bone targeting molecule on the bone targeting extracellular vesicles is 0.1-0.3 μmol per gram of vesicle;

preferably, the loading of bone-targeting extracellular vesicles with bone-contributing drugs is 5-50%;

preferably, the structure of the bone-targeting extracellular vesicle is a double phospholipid layer vesicle;

preferably, the bone-targeting extracellular vesicles have a particle size of 30-200 nm;

preferably, the zeta potential of the bone-targeting extracellular vesicles is between-5 and-40 mV.

3. The bone-targeting extracellular vesicle according to claim 1 or 2, wherein the osteogenic drug comprises any one of or a combination of at least two of nucleic acids, proteins, peptide chains or small molecule chemical drugs;

preferably, the nucleic acid comprises 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;

preferably, the protein comprises any one of bone morphogenic protein, osteopontin, catenin, collagen or silk fibroin or a combination of at least two thereof;

4. The bone-targeting extracellular vesicle according to any one of claims 1-3, wherein the source of the extracellular vesicle is human-derived stem cells;

5. The bone-targeting extracellular vesicle according to any one of claims 1 to 4, wherein the extracellular vesicle is prepared by the following preparation method:

(a) culturing human stem cells until the confluence degree is 80-90%, culturing in a complete culture medium, and collecting the culture medium;

(b) centrifuging the culture medium collected in the step (a) for three times by increasing centrifugal force to obtain the extracellular vesicles;

preferably, in step (a), the specific steps of culturing to reach 80-90% of confluence are: mixing human stem cells and complete culture medium, and culturing at 37 deg.C for 2-3 days until the confluency is 80-90%; wherein the content of the first and second substances,the amount of human stem cells added per 1mL of complete medium was 5X 10 ⁵ -1×10 ⁶ A cell;

preferably, in the step (a), the cultivation in complete medium comprises the following specific steps: transferring the human stem cells with 80-90% confluency to a complete culture medium prepared by extracellular vesicle-free serum, and culturing at 37 ℃ for 36-72 h; wherein the amount of human stem cells added per 1mL of complete medium is 5X 10 ⁵ -1×10 ⁶ (ii) individual cells;

preferably, in step (a), the complete medium comprises alpha-MEM medium or DMEM medium, and 8-12% fetal bovine serum and 0.5-2% diabody are added to the complete medium;

preferably, in step (b), the centrifugation process with three increasing centrifugal forces comprises the following specific steps: centrifuging the culture collected in the step (a) for 10-30min under the centrifugal force of 800-1200g at 0-10 ℃, and collecting the supernatant; centrifuging the supernatant at 0-10 deg.C and 8000-; finally, centrifuging the supernatant for 90-120min at the centrifugal force of 120000g at the temperature of 0-10 ℃ and 80000-;

preferably, in step (b), the extracellular vesicles are preserved without resuspension in sterile PBS buffer;

preferably, the mass ratio of the extracellular vesicles to the sterile PBS buffer is 1 (1-5).

6. A method for preparing the bone-targeting extracellular vesicles according to any one of claims 1 to 5, wherein the method for preparing the bone-targeting extracellular vesicles comprises the following steps:

(2) loading of bone-promoting drugs: loading bone-promoting drugs into the extracellular vesicles of which the outer surfaces are modified with bone-targeting molecules through electroporation treatment to obtain the bone-targeting extracellular vesicles.

7. The method for preparing bone-targeting extracellular vesicles according to claim 6, wherein the modification in step (1) comprises the following specific steps:

(B) resuspending the obtained extracellular vesicles with the surface grafted with the bifunctional molecules in a PBS buffer solution, mixing the extracellular vesicles with the bone targeting molecules, and performing coupling reaction to obtain extracellular vesicles with the outer surfaces modified with the bone targeting molecules;

preferably, the bifunctional molecule is selected from any one of dibenzylcyclooctyl-sulfo-N-hydroxysuccinimide ester, aza-dibenzocyclooctyne-succinimide ester, diphenyl cyclooctyne-PEG 4-hydrogenated succinimide ester, or diphenyl cyclooctyne-carbon 6-succinimide ester, preferably dibenzylcyclooctyl-sulfo-N-hydroxysuccinimide ester;

preferably, in the step (A), the concentration of the bifunctional molecule in the mixed solution obtained after mixing is 10-50 μ M, and the concentration of the extracellular vesicles is 0.5-1 mg/mL;

preferably, in the step (A), the temperature of the grafting reaction is 30-40 ℃, and the time of the grafting reaction is 3-6 h;

preferably, in step (a), the non-grafted bifunctional molecule is removed by ultrafiltration and centrifugation after the grafting reaction;

preferably, in step (B), the concentration of the extracellular vesicles with the bifunctional molecules grafted on the surface in the solution obtained after resuspension is 0.5-5 mg/mL;

preferably, in the step (B), the concentration of the bone targeting molecule in the mixed solution obtained after mixing is 10-50 μ M, and the concentration of the extracellular vesicle with the bifunctional molecule grafted on the surface is 0.5-5 mg/mL;

preferably, in the step (B), the temperature of the coupling reaction is 0-10 ℃, and the time of the coupling reaction is 8-12 h;

preferably, in the step (B), after the coupling reaction is finished, the unmodified bone targeting molecule is removed by ultrafiltration and centrifugation;

preferably, in step (B), the extracellular vesicles modified with the bone targeting molecules on the outer surface are stored in sterile PBS buffer in a resuspension manner;

preferably, the mass ratio of the extracellular vesicles modified with the bone targeting molecules on the outer surfaces to the sterile PBS buffer solution is 1 (1-5).

8. The method for preparing bone-targeting extracellular vesicles according to claim 6 or 7, wherein the loading in step (2) comprises the following specific steps: mixing the extracellular vesicles the outer surfaces of which are modified with bone targeting molecules, bone promoting drugs and electroporation buffer solution, performing electroporation, and standing after the electroporation is completed to obtain the bone targeting extracellular vesicles;

preferably, the concentration of the extracellular vesicles with the bone targeting molecules modified on the outer surfaces in the mixed solution obtained after mixing is 0.5-5mg/mL, and the concentration of the bone promoting drugs is 0.01-0.5 mug/muL;

preferably, the electroporation buffer comprises a mixed solution of iodixanol and potassium dihydrogen phosphate-potassium chloride solution in a volume ratio of (18-23): (77-82); the mass concentration of the potassium dihydrogen phosphate in the potassium dihydrogen phosphate-potassium chloride solution is 1-1.2mM, the mass concentration of the potassium chloride is 23-27mM, and the solvent is water;

preferably, the voltage of the electroporation is 100-400V, and the time of the electroporation is 1-20 ms;

preferably, the standing temperature is 30-40 ℃, and the standing time is 25-35 min;

preferably, the non-loaded bone-contributing drugs are removed by ultrafiltration centrifugation after the standing.

9. A pharmaceutical composition comprising the bone-targeting extracellular vesicle of any one of claims 1-5;

10. Use of any one of the bone-targeting extracellular vesicles as defined in any one of claims 1 to 5, a process for the preparation of the bone-targeting extracellular vesicles as defined in any one of claims 6 to 8, or a pharmaceutical composition as defined in claim 9, or a combination of at least two thereof, for the preparation of a product for the treatment of an orthopaedic disorder.