CN114948899A

CN114948899A - Bladder targeting exosome and application thereof

Info

Publication number: CN114948899A
Application number: CN202210707551.6A
Authority: CN
Inventors: 姜海涛
Original assignee: Individual
Current assignee: Individual
Priority date: 2021-06-22
Filing date: 2022-06-21
Publication date: 2022-08-30
Also published as: CN113398092A

Abstract

The invention discloses a bladder targeting exosome and application thereof; the invention also discloses a pharmaceutical composition, which is obtained by introducing the medicament for treating bladder diseases into exosomes with bladder tissue targeting property, wherein the exosomes with the bladder tissue targeting property are derived from cells at a kidney part; the bladder disease treatment drug comprises the bladder targeted drug-loaded exosome. Compared with the prior art, the exosome secreted by the cells at the kidney part can be enriched in the bladder tissue without any modification, and the exosome derived from the cells at the kidney part is used, so that the problem of the yield of the exosome is solved, and the exosome has good application prospect; and different drugs or active molecules can be loaded by using exosomes derived from cells at the kidney part, and the treatment effect of bladder diseases is improved and the drug toxic and side effects are reduced by targeted delivery to bladder tissues.

Description

Bladder targeting exosome and application thereof

Technical Field

The invention belongs to the field of biomedicine, and relates to a bladder targeting exosome and application thereof.

Background

The urinary bladder is an organ for storing and discharging urine of a human body, and the urinary bladder loses the normal functions for any reason, namely, the urinary bladder diseases occur due to the abnormality of the times of urination, the urine volume and the urine color. Common bladder disorders include bladder inflammation, bladder polyps, bladder muscle weakness, and bladder cancer. Bladder cancer is the most common malignant tumor of the urinary system and is one of ten common tumors of the whole body. The medicine takes the first place of the disease rate of genitourinary tumors in China, and the disease rate of the medicine is second to that of prostatic cancer in the West and is 2 nd.

Current cancer treatments, whether chemical or biological, involve three problems, the first: the medicine is easy to be degraded in the serum environment in vivo, has small molecular volume and is easy to be excreted; secondly, the method comprises the following steps: the drug is non-specifically distributed in the body to reduce the concentration of the target tissue; thirdly, the method comprises the following steps: the drug needs to overcome the physical barriers of the vessel wall and various tissues to reach the target cells. Therefore, there is a need for a drug delivery system, which has the characteristics of good biocompatibility, high stability, tissue targeting property and strong transmembrane delivery capability.

The targeted drug delivery system is a drug delivery system which selectively enriches drugs in focus or specific parts in vivo by using a carrier. The medicine can play the maximum role in the focus of infection, and the toxic and side effects of the whole body are reduced to the minimum degree. Comprises a passive targeting preparation: insoluble drug-loaded particles (such as liposome, nanocapsule and the like) enable the drug to enter a target region by means of phagocytosis of body cells; active targeting formulation: drug carriers are modified with antibodies and the like as "missiles" to target drug delivery. The targeting preparation can improve the drug effect, reduce the toxicity, improve the safety, effectiveness and reliability of the medicine and the compliance of the patient in the medicine, so the targeting preparation is increasingly paid extensive attention by the medical field at home and abroad. The development of nano-drug delivery systems has solved the above problems to some extent. However, the liposome or albumin-based nano-carrier cannot exist stably in blood, is easy to be eliminated by a reticuloendothelial system or a mononuclear phagocyte system, and lacks tissue targeting; the antibody modified carrier increases the cost of the medicine and is difficult to achieve the ideal targeting effect. In recent years, it has been discovered that exosomes can act as carriers, carrying drugs and delivering them to adjacent or distant cells. Compared with the liposome which is widely researched, the exosome shows greater advantages, and has good biocompatibility, biodegradability, low toxicity, stability and low immunogenicity. The preparation of an exosome drug-loading system with a bladder targeting effect is an ideal solution for treating bladder diseases.

Therefore, the preparation of a high-efficiency bladder targeting exosome drug-carrying system for treating bladder-related diseases is urgently needed.

Disclosure of Invention

The invention aims to solve the defects in the prior art, and provides a bladder targeting exosome, application and a medicament for treating bladder related diseases.

In order to achieve the purpose, the invention is implemented according to the following technical scheme:

the invention provides a bladder tissue targeting exosome in a first aspect, wherein the targeting exosome is derived from cells in a kidney part.

In the present invention, "bladder tissue targeting", "bladder tissue targeting", "bladder targeting" are used interchangeably.

The term "exosome" is a vesicle, which is secreted from a cell to the outside of the cell, or has a membrane structure composed of lipid bilayers existing inside the cell. The exosomes have a diameter of about 30-1000nm and are released from the cell when the multivesicular bodies fuse with the cell membrane, or directly from the cell membrane. Exosomes are known to play a role in transporting intracellular biomolecular proteins, bioactive lipids, and rna (mirna) to achieve their functional role in mediating coagulation, intercellular communication, and cellular immunity.

The concept of exosomes described above includes microvesicles. The marker proteins of exosome are known as CD63, CD81, TSG101 and the like, and besides, cell surface receptor (e.g., EGFR), signal transduction related molecule, cell adhesion related protein, MSC related antigen, heat shock protein, vesicle formation related Alix and the like are known.

In some embodiments, the exosomes are about 30 to about 500 in diameter, about 30 to about 300 in diameter, about 30 to about 250 in diameter, about 30 to about 220 in diameter, about 40 to about 175 in diameter, about 50 to about 150 in diameter, about 30 to about 150 in diameter, or about 30 to about 120nm in diameter.

Further, the cell is selected from a normal cell or a derivative thereof, and a cancer cell.

Further, the cell is derived from a human or non-human mammal.

Further, the non-human mammal includes non-human primates, rodents, cows, pigs, sheep, dogs, rabbits, cats, horses.

Further, the rodent includes a mouse, a rat, a hamster, and a guinea pig.

Further, the primate includes monkey, orangutan, baboon, and ape.

In the present invention, the cells of the kidney site are kidney-derived cells or kidney cells in which stem cells induce differentiation. The stem cells comprise pluripotent stem cells and embryonic stem cells. In the present invention, the pluripotent stem cells are selected from induced pluripotent stem cells.

Induced pluripotent stem cells are artificially derived stem cells that are non-pluripotent cells (usually mature somatic cells) produced by inducing the expression of one or more stem cell-specific genes. Such stem cell specific genes include, but are not limited to, the octamer transcription factor family, i.e., Oct-3/4; the Sox gene family, namely Sox1, Sox2, Sox3, Sox15 and Sox 18; the Klf gene family, i.e., Klf1, Klf2, Klf4, and Klf 5; the Myc gene family, i.e., c-Myc and L-Myc; the Nanog gene family, namely OCT4, NANOG and REX 1.

Further, the cells of the kidney site include embryonic kidney cells or a derivative thereof, kidney cancer cells, induced pluripotent stem cells-induced kidney cells.

Further, the embryonic kidney cells or derivatives thereof comprise HEK-293 or derivatives thereof, CRL-11268, MARC-145; the renal cells or derivatives thereof comprise vero or derivatives thereof, HKb-20, FC33, HK-2C, ProPakA.6, TCMK-1, CV-1, Cos1, Cos3, Cos7, MC53, MRGEC, TCMK-1, mRTEC, BHK-N, BHK-21, BHK-M, RK1, NRK-49F, HBZY-1 and NRK-52E, PK 15.

Further, HEK-293 derivatives include: AD293, HEK293-L, HEK293-T, GP2-293, GP2-293Luc, KiMA, HEK-293-6E, HEK-293A, HEK-293F, HEK-293FT, HEK-293H, 293-L.P, 293-mTLR5, HEK-293T/17, 293XL-hTLR7, 293XL-hTLR9, 2V6.11, AAV-293, GP-293, Phix-293T, HEK-293E, HEK-293ET, 293Ad 5; the vero derivatives include vero-E6, VREO/IgRCD4, VREO/IgR.

In the present invention, cells of the renal site may also be genetically modified or engineered or induced to target bladder tissue, including but not limited to genetic modification, gene overexpression or deletion, molecular modification, and the like; exosomes secreted by cells in the renal site may also be surface modified or engineered to target bladder tissue, including but not limited to surface protein modifications, surface protein alterations, surface small molecule modifications, and the like.

In a second aspect, the present invention provides a method for preparing a targeting exosome according to the first aspect, comprising the following steps:

culturing cells to obtain a cell culture solution;

centrifuging and taking supernatant;

centrifuging for the second time, and taking supernatant;

and centrifuging again, and carrying out resuspension precipitation on the buffer solution to obtain the targeting exosome.

Further, the centrifugation condition of step 2) was 2000g for 10 min.

Further, the centrifugation condition of step 3) is 10000g for 30 min.

Further, the centrifugation condition of the step 4) is 100000g for 2 h.

Further, the buffer solution in step 4) is PBS.

In a third aspect, the present invention provides a pharmaceutical composition comprising a targeted exosome according to the first aspect of the present invention; and therapeutic/prophylactic agents.

Further, the therapeutic or prophylactic agent includes small molecule chemical drugs, peptide or protein drugs, antibodies, enzymes, cytokines, hormones, antibiotics, vaccines, and/or nucleic acid drugs. The therapeutic or prophylactic agent may be any conventional drug as long as it can act to treat a disease derived from the bladder site.

Further, the nucleic acid drugs comprise plasmid DNA, mRNA, microRNA, small interfering RNA, shRNA, sense RNA, antisense oligonucleotide and aptamer.

Further, the antibody includes bevacizumab, cetuximab, panitumumab, nimotuzumab, trastuzumab, pertuzumab.

Further, the small molecule chemical drug comprises gemcitabine hydrochloride, cisplatin, gemcitabine, paclitaxel, carboplatin, etoposide, vincristine, fluorouracil, oxaliplatin, irinotecan, capecitabine, sunitinib, temsirolimus, pazopanib, axitinib, sorafenib, cabozantinib, everolimus, lenvatinib.

Further, the small molecule chemical drugs comprise gemcitabine hydrochloride, cisplatin and gemcitabine.

Further, the small molecule chemical drug is selected from gemcitabine hydrochloride.

Further, the antibiotic includes mitomycin and adriamycin.

Further, the vaccine comprises BCG.

Further, the therapeutic/prophylactic agent is useful for treating or preventing a bladder disease.

In the present invention, a bladder disease refers to any disease occurring at the site of or associated with the bladder.

Further, the bladder diseases include bladder inflammation, bladder polyps, bladder muscle weakness and bladder cancer.

Further, the bladder disease is selected from bladder cancer.

Further, the pharmaceutical composition is obtained by introducing a therapeutic agent or a prophylactic agent into a targeted exosome.

Further, the pharmaceutical composition also comprises a pharmaceutically acceptable carrier.

The term "pharmaceutically acceptable" means that the carrier is generally compatible chemically and/or physically with the other ingredients comprising the formulation, and physiologically with its recipient.

Pharmaceutically acceptable carriers for use in the pharmaceutical compositions of the invention may include, but are not limited to, for example, pharmaceutically acceptable liquid, gel or solid carriers, aqueous vehicles (e.g., sodium chloride injection, ringer's injection, isotonic glucose injection, sterile water injection, or ringer's glucose and lactate injection), non-aqueous vehicles (e.g., non-volatile oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil), antimicrobial agents, isotonic agents (e.g., sodium chloride or dextrose), buffers (e.g., phosphate or citrate buffers), antioxidants (e.g., sodium bisulfate), suspending/dispersing agents (e.g., sodium carboxymethylcellulose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone), chelating agents (e.g., EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid)), emulsifying agents (e.g., polysorbate 80(Tween 80 (Tec)), and the like), Diluents, adjuvants, excipients, or nontoxic auxiliary substances, other components known in the art, or various combinations thereof. Suitable components may include, for example, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavoring agents, thickening agents, coloring agents, or emulsifying agents.

The pharmaceutical composition of the present invention can be formulated into oral dosage forms such as powder, granule, tablet, capsule, suspension, emulsion, syrup, and spray, external preparations, suppositories, and sterile injection solutions according to a conventional method. The carrier, excipient and diluent contained in the pharmaceutical composition may be lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, etc. The formulation can be prepared by using a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant, which is generally used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, which are prepared by mixing one or more excipients, such as starch, calcium carbonate, sucrose or lactose, gelatin and the like, in the pharmaceutical composition of the present invention. In addition, lubricants such as magnesium stearate and talc are used in addition to simple excipients. Liquid preparations for oral administration include suspensions, solutions for internal use, emulsions, syrups and the like, and may include various excipients such as wetting agents, sweeteners, aromatics, preservatives and the like in addition to water and liquid paraffin, which are widely used as simple diluents. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, oils, freeze-dried preparations, suppositories. Non-aqueous solvents, suspending agents, propylene glycol, ethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like may be used. As the base for suppository, witepsol, polyethylene glycol, Tween (tween)61, cacao butter, real resin, glycerogelatin, etc. can be used.

The pharmaceutical composition of the present invention can be administered to mammals such as mice, livestock, humans, etc. in various ways. All modes of administration are contemplated, including but not limited to buccal, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal. Oral or non-oral administration is preferred. The term "non-oral" as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.

The pharmaceutical compositions according to the invention may be administered as the sole therapeutic agent, or in combination with other therapeutic agents, also sequentially or simultaneously with conventional therapeutic agents, and may be administered in single or multiple doses. Taking the above factors together, it is important that the maximum effect be achieved in the minimum amount without side effects, which can be readily determined by one skilled in the art.

The pharmaceutical compositions of the present invention may be administered by any means to deliver the active agent to the target cells. The preferred mode of administration and formulation is injection. The injection can be prepared from an aqueous solvent such as physiological saline, ringer's solution, Hank's (Hank) solution or sterile aqueous solution, a vegetable oil such as olive oil, a higher fatty acid ester such as ethyl oleate, a non-aqueous solvent such as ethanol, benzyl alcohol, propylene glycol, polyethylene glycol or glycerin, and the like, and a non-invasive preparation known in the art suitable for a barrier to be passed through can be used for mucosal permeation, and a pharmaceutically acceptable carrier such as a stabilizer for preventing deterioration such as ascorbic acid, sodium bisulfite, Butylated Hydroxyanisole (BHA), tocopherol, ethylenediaminetetraacetic acid (EDTA), an emulsifier, a buffer for adjusting pH, a preservative for inhibiting microbial growth such as phenylmercuric nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, and the like can be further included.

The dosage of the pharmaceutical composition of the present invention varies depending on the age, sex, body weight of the subject to be treated, the particular disease or pathological state to be treated, the severity of the disease or pathological state, the route of administration, and the judgment of the prescribing personnel. Determination of dosages based on the above factors is within the level of ordinary skill in the art to which this invention pertains.

"treatment" may refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) a targeted pathological condition or disease. Subjects in need of treatment include subjects already with the disorder, as well as subjects predisposed to the disease, or subjects in need of prevention of the disease. Therapeutic benefit may refer to the eradication or amelioration of symptoms or underlying disease being treated. In addition, therapeutic benefit may be achieved by eradicating or ameliorating one or more physiological symptoms associated with the underlying disease such that an improvement is observed in the subject, although the subject may still be afflicted with the underlying disease. A prophylactic effect may include delaying, preventing, or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, stopping, or reversing the progression of a disease or condition, or any combination thereof. For prophylactic benefit, a subject at risk of developing a particular disease or a subject reporting one or more physiological symptoms of a disease can be treated even if a diagnosis of the disease cannot be made.

In a fourth aspect, the invention provides a composition comprising a targeted exosome according to the first aspect of the invention, and a detection marker.

Further, the detectable label comprises a fluorescent protein, biotin, enzyme, tag, radionuclide, luminescent label, or a compound that can be detected by NMR or ESR spectroscopy.

A label according to the present invention is defined as any moiety that can be detected using an assay. Non-limiting examples of reporter molecules include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, photoaffinity molecules, colored particles or ligands, such as biotin. The tags employed in the present invention also include tags such as His tags, Flag tags, and the like. The label comprises biotin, which is a substrate for avidin.

The labeled conjugates are suitable for use as diagnostic agents. Diagnostic agents are generally divided into two classes, one for in vitro diagnosis and the other for in vivo diagnostic protocols, commonly referred to as "directed imaging". Many suitable imaging agents are known in the art. The imaging moiety used may be paramagnetic ions, radioisotopes, fluorescent dyes, NMR detectable substances and X-ray imaging agents.

In the case of paramagnetic ions, ions such as chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and/or erbium (III) may be mentioned by way of example, with gadolinium being particularly preferred. Ions suitable for use in other contexts, such as X-ray imaging, include, but are not limited to, lanthanum (III), gold (III), lead (II), and in particular bismuth (III).

In the case of radioisotopes for therapeutic and/or diagnostic applications, astatine may be mentioned ²¹¹ 、 ¹⁴ Carbon, carbon, ⁵¹ Chromium (b), ³⁶ Chlorine, ⁵⁷ Cobalt, ⁵⁸ Cobalt, copper ⁶⁷ 、 ¹⁵² Eu, Ga ⁶⁷ 、 ³ Hydrogen and iodine ¹²³ Iodine, iodine ¹²⁵ Iodine, iodine ¹³¹ Indium, indium ¹¹¹ 、 ⁵⁹ Iron, iron, ³² Phosphorus, rhenium ¹⁸⁶ Rhenium ¹⁸⁸ 、 ⁷⁵ Selenium, ³⁵ Sulphur, technetium ^99m And/or yttrium ⁹⁰ 。 ¹²⁵ I applies to certain embodiments, and technetium ^99m And/or indium ¹¹¹ It is particularly suitable because of its low energy and suitability for long-range detection. Radiolabeled peptides and polypeptides may be produced according to methods well known in the art. For example, peptides and polypeptides may be iodinated by contact with sodium iodide and/or potassium iodide and a chemical oxidant (e.g., sodium hypochlorite) or an enzymatic oxidant (e.g., lactoperoxidase). Technetium can be used by ligand exchange process ^99m The peptides are labeled, for example, by reducing pertechnetate with a stannous solution, chelating the reduced technetium to a sephadex column, and applying the peptides to the column. Alternatively, direct labeling techniques may be used, for example by incubating pertechnetate, a reducing agent such as SNCl ₂ Buffer solutions such as sodium potassium phthalate solution and peptides. An intermediate functional group commonly used to bind radioisotopes present as metal ions to peptides is diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

Fluorescent labels include Alexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, cascading blue, Cy3, Cy5,6-FAM, fluorescein isothiocyanate, HEX, 6-Joe, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific blue, REG, rhodamine Green, rhodamine Red, contrast agent (Renographin), ROX, TAMRA, PKH67, PKH26, TET, tetramethylrhodamine, and/or Texas Red.

When referring to in vitro diagnostics, linked to a second binding ligand and/or an enzyme (enzyme tag) which will produce a coloured product when contacted with a chromogenic substrate. Examples of suitable enzymes include urease, alkaline phosphatase, (horseradish) catalase or glucose oxidase. Suitable secondary binding ligands are biotin and avidin and streptavidin compounds. The use of such tags is well known to those skilled in the art.

The fifth aspect of the invention provides a preparation method of a drug-loaded bladder tissue targeting exosome, which comprises the following steps:

the medicine for treating bladder diseases and the targeted exosome according to the first aspect of the invention are put into an electrotransfer cup for electrotransfer, and the electrotransfer product is centrifuged to remove free medicine, so that the purified targeted medicine-carrying exosome is obtained.

Further, the voltage used for the electric conversion is selected from 50-300V.

Further, the voltage used for the electric conversion was 250V.

In a specific embodiment of the invention, the preparation method of the drug-loaded bladder tissue targeting exosome comprises the following steps:

1) mixing the bladder targeting exosome with gemcitabine hydrochloride, adding an electrotransformation buffer solution, and transferring the mixture into an electrotransformation cup;

2) performing electrotransformation on the bladder targeting exosome and gemcitabine hydrochloride;

3) the electrotransformation product was ultracentrifuged at 100000g for 120min and the supernatant was collected.

As an alternative embodiment of the present invention, the electrotransfer uses waveforms conventional in the art, including but not limited to exponential waves, square waves.

In a possible embodiment of the present invention, the electrotransfer buffer is selected from the group of electrotransfer buffers conventional in the art, including but not limited to PBS, DMEM, Cytomix, Tris-HCl.

In a sixth aspect, the invention provides a use of a targeting exosome according to the first aspect of the invention for targeting bladder tissue. In the present invention, the targeted exosomes may be targeted to bladder tissue as delivery vehicles or to tracer or detection substances.

In a seventh aspect, the invention provides a use of a targeted exosome according to the first aspect of the invention or a pharmaceutical composition according to the third aspect of the invention in the preparation of a medicament for treating a bladder disease.

Further, the bladder diseases include bladder inflammation, bladder polyps, bladder muscle weakness, bladder cancer.

Further, the bladder disease is selected from bladder cancer.

In an eighth aspect, the invention provides a use of the targeted exosome of the first aspect of the invention or the composition of the fourth aspect of the invention in the preparation of a product for detecting bladder diseases.

Further, the bladder disease is selected from bladder cancer.

The invention has the advantages and beneficial effects that:

the tissue targeting exosome used in the invention can be enriched in the corresponding tissue without any modification, the cell source of the targeting exosome is simple, the problem of exosome yield is solved, and the tissue targeting exosome has good application prospect; the exosome derived from the cells at the kidney part can be loaded with different drugs or active molecules, and the drug delivery is carried out by targeting the bladder tissue, so that the treatment effect of the bladder diseases is improved, and the drug toxic and side effects are reduced.

Drawings

FIG. 1 is a graph of the detection of exosome marker proteins secreted by HEK-293 normal human embryonic kidney cells;

FIG. 2 is a graph showing the particle size distribution of exosomes secreted by HEK-293 cells of normal human embryonic kidney;

FIG. 3 is a distribution diagram of exosomes of different cell origins in different tissues; wherein 3A is HEK-293 cells; 3B is HEK-293F cells; 3C is Cos7 cells; 3D is ACHN cells; 3E is PK15 cells;

FIG. 4 is an in vitro assay of bladder targeting exosome antitumor activity loaded with the drug gemcitabine hydrochloride used for treatment of bladder disease;

FIG. 5 is an in vivo assay of the antitumor activity of bladder targeted exosomes loaded with the drug gemcitabine hydrochloride used to treat bladder diseases.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. The specific embodiments described herein are merely illustrative of the invention and do not limit the invention.

Example 1 preparation and detection of Targeted exosomes

Collecting normal human embryonic kidney cell HEK-293, human embryonic kidney cell HEK-293F, African green monkey kidney cell Cos7, human kidney cancer cell ACHN, and porcine kidney cell PK15, and culturing.

Centrifuging cell culture solution (cultured by exosome-free serum and DMEM or RPMI1640 culture medium) of each cell under 2000g centrifugal force for 10min, and collecting supernatant; centrifuging for 30min under the centrifugal force of 10000g, taking supernatant, and removing cell debris and precipitates; centrifuging the centrifuged cell culture solution under 100000g (g is gravity acceleration) for 2h, re-suspending and collecting the precipitate with sterile PBS, and storing at 4 deg.C for a short period to obtain the target exosome. The obtained targeted exosome surface marker protein by extraction and separation and the particle size distribution are shown in fig. 1 and fig. 2.

The expression quantity of the exosome secreted by the cells is measured, the content of the exosome protein secreted by the cells is detected by a BCA method, and the result shows that the concentration of the exosome secreted by the kidney cells HEK-293 is 2.85 mu g/mu L.

Example 2 Targeted detection of exosomes

The exosomes prepared in example 1 were stained with PKH67 or PKH26, and the distribution of exosomes secreted from cells in the renal region was traced in vivo, and male C57bl/6 mice (4-6 weeks) were purchased from beijing wakaukang biotechnology component limited, all of which were bred in SPF-grade facility. The method comprises the following specific steps:

taking 100 mu g of exosome, incubating the exosome with 1 mu L of PKH67 or PKH 264 ℃ in a dark place overnight, centrifuging for 2h under the centrifugal force of 100000g (g is the gravity acceleration), discarding supernatant, washing with PBS twice, then resuspending the exosome secreted by cells at the kidney part with sterile PBS, and injecting the exosome into a C57bl/6 mouse through tail vein; after 24h, anesthetizing the mice, taking the liver, lung, heart, bladder, kidney, stomach and intestine of the mice to make frozen sections, staining nuclei by Hoechst33342, and observing the biological distribution of exosomes in each organ of the mice.

The results are shown in FIG. 3: exosomes secreted by cells from the kidney, HEK-293F, Cos7, ACHN, PK15, were significantly enriched in bladder tissue (fig. 3A-3E), with targeting efficiency as high as 65% -90%. The target efficiency is calculated by taking 100 cells in a visual field, wherein 65-90 cells have exosomes to take in, and the total exosomes taken in by other tissues is 10-35 per 100 cells.

Example 3 preparation of Targeted drug-loaded exosomes

Introducing a drug for treating a disease into a corresponding targeted exosome to prepare a targeted therapeutic drug, the targeted drug-loaded exosome comprising:

1) 150 mu g of bladder target exosome is mixed with gemcitabine hydrochloride, and the electrotransfer buffer solution can be (PBS, DMEM, Cytomix, Tris-HCl) to supplement the mixture to 150 mu L, and the mixture is transferred to electrotransfers with different specifications (0.2cm and 0.4 cm). Adopting different waveforms (exponential waves and square waves) and using different voltages (50-300V) to respectively carry out electrotransformation on the bladder target exosomes and the gemcitabine hydrochloride;

2) and (4) ultracentrifuging the electrotransformation product for 120min under the centrifugal force of 100000g, and collecting the supernatant to measure the drug loading.

3) As a result: for gemcitabine hydrochloride, 150 μ g of bladder targeting exosomes were electrotransformed at a voltage of 250V with the highest efficiency, which could be 35.7%.

Example 4 detection of therapeutic Effect of Targeted drug-loaded exosomes

This example further examined the therapeutic effect of the targeting exosomes loaded with the drug for treating diseases prepared in example 3 on the diseases by in vivo and in vitro experiments, and Balb/c nude mice (4-6 weeks) used in vivo experiments were purchased from beijing waukee biotechnology limited, and all mice were bred in SPF-level facilities.

The method comprises the following specific steps:

1. in vitro experiments:

the bladder cancer cells BIU-87 were plated in 96-well plates at 5X 10 per well ³ And (3) adding a target exosome (a control group) and a target exosome loaded with a therapeutic drug into each cell, and detecting the killing effect of the target exosome loaded with the therapeutic drug on the corresponding cell by MTT (methyl thiazolyl tetrazolium).

The results are shown in fig. 4, and the targeting exosome loaded with the therapeutic drug can effectively kill the tumor cells compared with the pure targeting exosome.

2. In vivo experiments:

in vivo experiments, a model of bladder cancer was established, and 5X 10 cells were used ⁶ Injecting BIU-87 into Balb/c nude mouse subcutaneously to grow to about 100mm ³ When the bladder target exosome carrying the bladder tumor treatment drug gemcitabine hydrochloride is injected into the tail vein, the injection is performed once every 3 days for 4 times, and then the tumor volume is measured every other day, wherein the tumor volume is 1/2 × a × b ² . a represents a long diameter, and b represents a short diameter.

The results are shown in fig. 5, and the target exosomes loaded with the tumor treatment drugs can significantly reduce the tumor size of the mice compared with the free anti-tumor drug group with the same dose.

It will be evident to those skilled in the art that the embodiments of the present invention are not limited to the details of the foregoing illustrative embodiments, and that the embodiments of the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the embodiments being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.

Claims

1. A bladder tissue-targeting exosome, wherein the target exosome is derived from cells of a renal site;

preferably, the cell is selected from the group consisting of a normal cell or a derivative thereof, a cancer cell;

preferably, the cell is derived from a human or non-human mammal;

preferably, the non-human mammal includes a non-human primate, rodent, bovine, porcine, ovine, canine, rabbit, feline, equine;

preferably, the rodent comprises a mouse, rat, hamster, guinea pig.

2. The targeted exosome according to claim 1, wherein the cells of the renal site comprise embryonic kidney cells or their derivatives, kidney cancer cells, induced pluripotent stem cell-induced kidney cells;

preferably, the embryonic kidney cells comprise HEK-293 or a derivative thereof, CRL-11268, MARC-145; the kidney cells comprise vero or derivatives thereof, HKb-20, FC33, HK-2C, ProPakA.6, TCMK-1, CV-1, Cos7, MC53, MRGEC, TCMK-1, mRTEC, BHK-N, BHK-21, BHK-M, RK1, NRK-49F, HBZY-1 and NRK-52E, PK 15;

preferably, the HEK-293 derivative comprises: AD293, HEK293-L, HEK293-T, GP2-293, GP2-293Luc, KiMA, HEK-293-6E, 293A, 293F, 293FT, 293H, 293-L.P, 293-mTLR5, 293T/17, 293XL-hTLR7, 293XL-hTLR9, 2V6.11, AAV-293, GP-293, Phix-293T, 293E, 293ET, 293Ad 5; the vero derivatives include vero-E6, VREO/IgRCD4, VREO/IgR.

3. The method of preparing a targeted exosome according to claim 1 or 2, comprising the steps of:

1) culturing cells to obtain a cell culture solution;

2) centrifuging and taking supernatant;

3) centrifuging for the second time, and taking supernatant;

4) centrifuging again, and carrying out resuspension precipitation on the buffer solution to obtain a targeting exosome;

preferably, the centrifugation conditions of the step 2) are 2000g for 10 min;

preferably, the centrifugation condition of the step 3) is 10000g for 30 min;

preferably, the centrifugation condition of the step 4) is 100000g for 2 h;

preferably, the buffer solution in step 4) is PBS.

4. A pharmaceutical composition comprising the targeted exosome of claim 1 or 2; and therapeutic/prophylactic agents.

Preferably, the therapeutic or prophylactic agent comprises a small molecule chemical drug, a peptide or protein drug, an antibody, an enzyme, a cytokine, a hormone, an antibiotic, a vaccine, and/or a nucleic acid drug;

preferably, the nucleic acid drug comprises plasmid DNA, mRNA, microRNA, small interfering RNA, shRNA, sense RNA, antisense oligonucleotide and aptamer;

preferably, the antibody comprises bevacizumab, cetuximab, panitumumab, nimotuzumab, trastuzumab, pertuzumab;

preferably, the small molecule chemical drug comprises gemcitabine hydrochloride, cisplatin, gemcitabine, paclitaxel, carboplatin, etoposide, vincristine, fluorouracil, oxaliplatin, irinotecan, capecitabine, sunitinib, temsirolimus, pazopanib, axitinib, sorafenib, cabozantinib, everolimus, lenvatinib;

preferably, the small molecule chemical comprises gemcitabine hydrochloride, cisplatin, gemcitabine;

preferably, the small molecule chemical is selected from gemcitabine hydrochloride;

preferably, the antibiotic comprises mitomycin, doxorubicin;

preferably, the vaccine comprises bcg;

preferably, the therapeutic/prophylactic agent is for treating or preventing a bladder disease;

preferably, the bladder disease includes bladder inflammation, bladder polyps, bladder muscle weakness and bladder cancer;

preferably, the bladder disease is selected from bladder cancer.

5. The pharmaceutical composition of claim 4, wherein the pharmaceutical composition is obtained by introducing a therapeutic or prophylactic agent into a targeted exosome;

preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

6. A composition comprising the targeted exosome of claim 1 or 2, and a detection marker;

preferably, the detectable label comprises a fluorescent protein, biotin, enzyme, tag, radionuclide, luminescent label, or compound that can be detected by NMR or ESR spectroscopy.

7. The preparation method of the drug-loaded bladder tissue targeting exosome is characterized by comprising the following steps:

placing a medicament for treating bladder diseases and the targeted exosome of claim 1 or 2 into an electrotransfer cup for electrotransfer, centrifuging an electrotransfer product to remove free medicament, and obtaining a purified targeted medicament-loaded exosome;

preferably, the voltage used for the electrical conversion is selected from 50-300V;

preferably, the voltage used for the electrical conversion is 250V.

8. Use of the targeted exosome of claim 1 or 2 for targeting bladder tissue.

9. Use of a targeting exosome according to claim 1 or 2 or a pharmaceutical composition according to any one of claims 4 to 5 in the preparation of a medicament for treating a bladder disease;

preferably, the bladder disease comprises bladder inflammation, bladder polyps, bladder muscle weakness, bladder cancer;

preferably, the bladder disease is selected from bladder cancer.

10. Use of the targeted exosome of claim 1 or 2 or the composition of claim 6 in the preparation of a product for detecting bladder disease;

preferably, the bladder disease includes bladder inflammation, bladder polyps, bladder muscle weakness, bladder cancer;

preferably, the bladder disease is selected from bladder cancer.