CN115029297A

CN115029297A - Kidney targeting drug-loading exosome, application thereof and drug for treating kidney diseases

Info

Publication number: CN115029297A
Application number: CN202210709469.7A
Authority: CN
Inventors: 姜海涛
Original assignee: Individual
Current assignee: Individual
Priority date: 2021-06-22
Filing date: 2022-06-21
Publication date: 2022-09-09
Also published as: CN113846047A

Abstract

The invention discloses a kidney targeting drug-loaded exosome and application thereof and a drug for treating kidney diseases; the medicine for treating kidney diseases is introduced into exosome with kidney tissue targeting property, and the exosome with the kidney tissue targeting property is derived from cells at a bladder part; the kidney disease treatment drug comprises the kidney targeting drug-loaded exosome. Compared with the prior art, the exosome secreted by the cells at the bladder part can be enriched in the kidney without any modification, and the exosome derived from the cells at the bladder 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 bladder part, and the drug delivery is carried out by targeting kidney tissues, so that the treatment effect of kidney diseases is improved, and the drug toxic and side effects are reduced.

Description

Kidney targeted drug-loaded exosome, application thereof and drug for treating kidney diseases

Technical Field

The invention belongs to the field of biomedicine, and relates to a kidney targeted drug-loading exosome, application thereof and a drug for treating kidney diseases.

Background

Kidney disease is a general term for common diseases seriously harming human health, and mainly comprises different types of nephritis, acute renal failure, kidney stones, renal cysts and the like. Renal tumors are one of the most common tumors in the human urogenital system, and the general therapeutic principle of renal cell carcinoma is that patients who do not have metastasis in the early stage or only locally progress are treated by surgery, patients who have no surgical chance of metastatic renal cancer in the late stage have surgery, and comprehensive treatment mainly based on internal medicine should be adopted. In the aspect of internal medicine treatment, a targeted treatment method of renal cell carcinoma is adopted, so that the overall life cycle and the like are greatly prolonged.

Currently, the FDA has approved 9 targeted therapeutic drugs for advanced renal cell carcinoma, which are: sunitinib, temsirolimus, pazopanib, axitinib, sorafenib, cabozantinib, everolimus, lenvatinib, and bevacizumab. The targeted therapy is a standard treatment scheme for the advanced renal clear cell carcinoma, and the median disease control time of a patient can reach about 30 months by alternately using a plurality of targeted drugs. The target medicine for treatment mainly acts through two signal pathways of VHL/HIF/VEGF and PI 3K/AKT/mTOR. The targeted drugs of kidney cancer are two classes, one is an anti-angiogenic tyrosine kinase inhibitor and the other is an mTOR inhibitor, and the sequential treatment is the rotation of the two classes of drugs. A large number of researches prove that the expression reduction of the microRNA-1271 is closely related to kidney cancer, and the microRNA-1271 can inhibit the proliferation and the metastasis of kidney cancer cells by inhibiting an mTOR signal pathway.

Although the kidney cancer targeting preparation has been developed to a certain extent in recent years, the kidney cancer targeting preparation has a therapeutic effect on a signal pathway and has strong toxic and side effects on normal cells and tissues. Poor targeting for the treatment of kidney cancer and other kidney-related diseases. Exosomes are used as drug-carrying systems, and can carry drugs and deliver the drugs to adjacent or distant cells due to good biocompatibility, biodegradability, low toxicity, stability and low immunogenicity.

Therefore, it is highly desirable to prepare a highly efficient drug-loaded system of kidney targeting exosomes for treating kidney tissue-related diseases.

Disclosure of Invention

The invention aims to solve the defects in the prior art, provides a kidney targeting exosome, application and a medicament for treating kidney related diseases, and improves the treatment effect of the kidney diseases and reduces the toxic and side effects of the medicament by targeted delivery to kidney tissues.

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

in a first aspect, the invention provides a kidney tissue targeting exosome, the targeting exosome being derived from cells of a bladder site.

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 500nm in diameter, about 30 to about 300 in diameter, about 30 to about 250nm in diameter, about 30 to about 220nm in diameter, about 40 to about 175, about 50 to about 150, about 30 to about 150, 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 at the bladder site refer to bladder cells derived from the bladder or bladder 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 bladder site include bladder cells, bladder epithelial immortalized cells, bladder epithelial cells, embryonic bladder tissue cells, bladder cancer cells, induced pluripotent stem cell-induced bladder cells.

Further, the bladder epithelial immortalized cell comprises SV-HUC-1, the bladder epithelial cell comprises HCV-29, MBEC, the embryonic bladder tissue cell comprises CCC-HB-2, the bladder cancer cell comprises SCaBER, T24, RT4, SW780, BIU-87, H/RB-CL2, MB-49, EJ-1, UM-UC-3, 5637, RT 112/84.

In the present invention, the cells at the bladder site may also be genetically modified or engineered or induced to target kidney tissue, including but not limited to genetic modification, gene overexpression or deletion, molecular modification, and the like; exosomes secreted by cells at the bladder site may also be surface modified or engineered to target kidney 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 the targeted 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 supernate;

and centrifuging again, and resuspending the precipitate by using the buffer solution to obtain the target 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 targeting 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 kidney disease.

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

Further, the nucleic acid drug is selected from microRNA.

Further, the microRNA is selected from the group consisting of microRNA-1271 micic.

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 drug comprises sunitinib, temsirolimus, pazopanib, axitinib, sorafenib, cabozantinib, everolimus, and lenvatinib.

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

Further, the antibody comprises bevacizumab.

Further, the antibiotic includes mitomycin and adriamycin.

Further, the vaccine comprises BCG.

Further, the therapeutic/prophylactic agent is used for treating or preventing kidney diseases. In the present invention, kidney disease refers to any disease occurring in the area of or associated with the kidney.

Further, the kidney diseases include nephritis, acute renal failure, renal calculus, renal cyst, and renal cancer.

Further, the kidney disease is selected from kidney cancer.

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

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 besides water and liquid paraffin which are widely used as simple diluents, various excipients such as wetting agents, sweeteners, aromatics, preservatives and the like can be included. 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 into consideration, it is important that the maximum effect be achieved in the minimum amount without side effects, which can be easily determined by those 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 (II), 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 Labelling of peptides, e.g. by reduction of pertechnetate with stannous solutionThe original technetium is chelated to a sephadex column and the peptide is applied to this 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, BODIPY650/665, 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 kidney tissue targeted exosome, which comprises the following steps:

the medicine for treating kidney diseases and the targeted exosome of 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 drug-loaded kidney tissue targeted exosomes are prepared as follows:

1) mixing the kidney tissue target exosome with a medicament for treating kidney diseases, adding an electrotransformation buffer solution, and transferring the mixture into an electrotransfer cup;

2) carrying out electric transformation on the kidney tissue targeting exosome and the medicament for treating the kidney disease;

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

As an alternative embodiment, the electrical transfer uses waveforms conventional in the art, including but not limited to exponential waves, square waves.

In a specific embodiment of the invention, the drug for treating kidney disease is selected from microRNA-1271 mimic.

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

In a seventh aspect, the invention provides the use of a targeting 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 the treatment of renal disease.

Further, the kidney disease is selected from kidney 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 kidney disease.

Further, the kidney disease is selected from kidney cancer.

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

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 bladder part can be loaded with different drugs or active molecules, and the drug delivery is carried out by targeting kidney tissues, so that the treatment effect of kidney diseases is improved, and the toxic and side effects of the drugs are reduced.

Drawings

FIG. 1 is a graph of the detection of exosome-tagged proteins secreted by human bladder epithelial immortalized cells SV-HUC-1;

FIG. 2 is a graph showing the measurement of the size distribution of exosomes secreted by SV-HUC-1, an immortalized cell of human bladder epithelium;

FIG. 3 is a distribution diagram of exosomes of different cell origins in different tissues; wherein 3A is SV-HUC-1 cells; 3B is T24 cells; 3C is a CCC-HB-2 cell; 3D is porcine bladder cells;

FIG. 4 is an in vitro assay for the antitumor activity of renal targeting exosomes loaded with microRNA-1271mimic drug for the treatment of renal disease;

FIG. 5 is an in vivo assay for kidney targeting exosome anti-tumor activity loaded with microRNA-1271mimic drug for treatment of kidney disease.

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

Human bladder tissue epithelial cell FHC, human bladder tissue fiber cell CCD-18Co, human bladder cancer cell HCT-116 and pig bladder cell.

Centrifuging cell culture solution (cultured by exosome-free serum and DMEM medium or RPMI1640 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 preparation method of the pig bladder cell culture solution comprises the following steps:

after anesthetizing newborn pigs with ketamine (18mg/kg), organs were removed by laparotomy under sterile conditions, blood was removed by 3 washes with D-Hank's containing antibiotics and macroscopic connective and lymphatic tissues were carefully removed. Then cutting bladder tissue into 0.5-1mm ³ Washing the large and small tissue fragments with D-Hank's solution for 3 times, placing the tissue fragments into a 50mL conical flask, adding freshly prepared V-type collagenase with the concentration of 0.5-1mg/mL, carrying out shock digestion in water bath at 37 ℃ for 10min, adding 40mL of cold D-Hamk's solution, stopping digestion at 4 ℃, shaking the conical flask to precipitate larger tissue blocks, transferring the supernatant into a 50mL centrifuge tube, adding 8mL of calf serum, uniformly mixing, and then carrying out low-speed centrifugation (800r/min) to obtain precipitated cells to be cultured. Adding D-Hank's solution into the residual tissue mass precipitate, shaking thoroughly, sucking supernatant, and centrifuging. The process is repeated for 2-3 times to separate the cells which can be separated in the first digestion process as much as possible, and the cells are prevented from entering the second digestion process. The isolated cells were cultured in RPMI1640 medium supplemented with 20% calf serum, penicillin 100U/mL, and glutamine 100 mg/L. The culture medium is changed the next day, and the culture medium is changed every other day. The exosome-free serum medium was changed on day 5, and the cell culture fluid was collected on day 7.

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 protein secreted by the bladder cells FHC is 2.46 mu g/mu L.

Example 2 Targeted detection of exosomes

The exosomes prepared in example 1 were stained with PKH67 or PKH26, and the in vivo distribution of exosomes secreted from cells in the bladder site was traced, and male C57bl/6 mice (4-6 weeks) were purchased from beijing wakaukang biotechnology 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 2 hours 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 bladder part with sterile PBS, and injecting the exosome into a C57bl/6 mouse through tail vein; after 24h, anesthetizing the mice, taking the heart, liver, spleen, lung, 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: the cells SV-HUC-1, T24, CCC-HB-2 from the bladder part and exosomes secreted by porcine bladder cells are obviously enriched in kidney tissues (figures 3A-3D), and the targeting efficiency can reach 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

In the embodiment, the drug microRNA-1271 mice for treating kidney cancer is selected.

Introducing a drug for treating diseases into a corresponding targeted exosome to prepare a targeted therapeutic drug, wherein the preparation of the targeted drug-loaded exosome comprises the following steps:

1) 150 mu g of kidney targeting exosome is mixed with microRNA-1271 mice, and the mixture is complemented to 150 mu L by electrotransfer buffer solution (PBS, DMEM, Cytomix and Tris-HCl), and transferred to electrotransfer cups with different specifications (0.2cm and 0.4 cm). Adopting different waveforms (exponential waves or square waves) and using different voltages (50V-300V) to respectively carry out electrotransformation on the kidney target exosomes and the microRNA-1271 mices;

2) the electrotransformation product is ultracentrifuged for 120min under 100000g of centrifugal force, and the supernatant is collected to measure the drug loading.

3) As a result: for microRNA-1271 micic, 150. mu.g of kidney targeting exosomes and

the microRNA-1271 micic is subjected to electrotransformation under the voltage of 250V, and the efficiency is highest and can reach 24.9%;

indicating that the microRNA-1271 micic is successfully loaded into an exosome secreted by the SV-HUC-1 cell.

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:

renal carcinoma cells ACHN 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).

As shown in fig. 4, the targeting exosome loaded with the therapeutic drug can effectively kill tumor cells compared with the simple targeting exosome, and the simple targeting exosome has no tumor-inhibiting effect.

2. In vivo experiments:

in vivo experiments, a kidney cancer model was established, and 4 × 10 was used ⁶ Injecting the kidney cancer cell ACHN into Balb/cNuded mouse subcutaneously until it grows to about 100mm ³ In the preparation process, a kidney target exosome loaded with a kidney tumor treatment drug microRNA-1271 micic is injected into the tail vein, the kidney target exosome is injected 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 exosome (microRNA-1271 mimic-exosome) loaded with the tumor treatment drug can significantly reduce the tumor size of the mouse compared with the free anti-tumor drug group (microRNA-1271mimic) 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 are capable of being 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 kidney tissue targeting exosome, wherein the targeting exosome is derived from cells at the bladder 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. A targeted exosome according to claim 1, wherein cells of the bladder site include bladder cells, urothelial immortalised cells, urothelial cells, embryonic bladder tissue cells, bladder cancer cells, induced pluripotent stem cell induced bladder cells;

preferably, the bladder epithelial immortalized cells comprise SV-HUC-1, the bladder epithelial cells comprise HCV-29, MBEC, the embryonic bladder tissue cells comprise CCC-HB-2, the bladder cancer cells comprise SCaBER, T24, RT4, SW780, BIU-87, H/RB-CL2, MB-49, EJ-1, UM-UC-3, 5637, RT 112/84.

3. A method of producing 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 on the precipitate by using a buffer solution to obtain a target exosome;

preferably, the centrifugation condition of the step 2) is 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 a 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 nucleic acid drug is selected from microRNA;

preferably, the microRNA is selected from the group consisting of microRNA-1271 micic;

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 drug comprises sunitinib, temsirolimus, pazopanib, axitinib, sorafenib, cabozantinib, everolimus, lenvatinib;

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

preferably, the antibody comprises bevacizumab;

preferably, the antibiotic comprises mitomycin, doxorubicin;

preferably, the vaccine comprises bcg;

preferably, the therapeutic/prophylactic agent is for the treatment or prevention of kidney disease;

preferably, the kidney disease includes nephritis, acute renal failure, kidney stones, kidney cysts, kidney cancer;

preferably, the kidney disease is selected from kidney 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 a compound detectable by NMR or ESR spectroscopy.

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

placing a medicament for treating kidney 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 kidney tissue.

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

preferably, the kidney disease is selected from kidney 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 kidney disease;

preferably, the renal disease includes nephritis, acute renal failure, kidney stone, kidney cyst, kidney cancer;

preferably, the kidney disease is selected from kidney cancer.