CN114949234A

CN114949234A - Gallbladder-targeted drug-loaded exosome, application thereof and drug for treating gallbladder diseases

Info

Publication number: CN114949234A
Application number: CN202210707554.XA
Authority: CN
Inventors: 姜海涛
Original assignee: Individual
Current assignee: Individual
Priority date: 2021-12-01
Filing date: 2022-06-21
Publication date: 2022-08-30
Also published as: CN114129734A

Abstract

The invention discloses a gallbladder targeted drug-loaded exosome, application and a drug for treating gallbladder diseases, wherein the drug for treating the gallbladder diseases is obtained by introducing the drug into the exosome with gallbladder tissue targeting property, and the exosome with the gallbladder tissue targeting property is derived from cells at a liver part; the medicine for treating the biliary diseases comprises the biliary targeting medicine-carrying exosome. The exosome secreted by the cells at the liver part can be enriched in the gallbladder without any modification, and the exosome derived from the cells at the liver 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 of the liver, and the drug delivery is carried out by targeting the biliary tissue, so that the treatment effect of biliary diseases is improved, and the drug toxic and side effects are reduced.

Description

Gallbladder-targeted drug-loaded exosome, application thereof and drug for treating gallbladder diseases

Technical Field

The invention belongs to the field of biomedicine, and relates to a biliary targeting drug-loaded exosome, application thereof and a drug for treating biliary diseases.

Background

Biliary tissue disease is a common disease. Such as cholecystitis, cholecystolithiasis, gallbladder polyp, gallbladder cancer, and bile duct cancer. Gallbladder cancer is the most common tumor of biliary system in clinic. Early-stage gallbladder cancer has hidden symptoms, is usually classified in middle and late stages when clinically confirmed, is easy to cause local invasion and distant metastasis, and is the most invasive biliary system tumor. The overall prognosis of gallbladder cancer is poor, and the 5-year survival rate of patients is less than 5%. In recent years, the diagnosis and treatment of the gallbladder cancer is changed from single surgical treatment to a comprehensive diagnosis and treatment mode which is dominated by multidisciplinary cooperation group evaluation and operation combined auxiliary treatment, and the prognosis of a gallbladder cancer patient is effectively improved.

Chemotherapy is widely used in the treatment of a variety of neoplastic diseases as an adjunctive treatment to surgery. The current common chemotherapeutic drugs used for gallbladder cancer are cisplatin, gemcitabine, fluorouracil, adriamycin and the like. Most of the traditional chemotherapy schemes are combined chemotherapy mainly by 5-fluorouracil, and the traditional chemotherapy schemes are limited in clinical application because the traditional chemotherapy schemes often cause severe toxic and side effects. Gemcitabine is a chemotherapeutic drug widely used in neoplastic diseases, and the clinical efficacy of gemcitabine in treating advanced gallbladder cancer is gradually recognized, and gemcitabine becomes a first-line drug for patients with advanced gallbladder cancer who lose the chance of surgery. Researches show that gemcitabine of a patient with advanced gallbladder cancer is combined with platinum chemotherapeutic drugs, and the treatment effective rate can reach 17%. However, some patients with gallbladder cancer are not sensitive to chemotherapeutic drugs, and the common side effects include anorexia, nausea, vomiting, diarrhea, impaired liver function, alopecia, bone marrow suppression, and the like.

How to specifically deliver the medicine to the biliary tissue increases the curative effect of the medicine, improves the sensitivity of the patient to the medicine, reduces the toxic and side effect of the medicine on non-specific tissues is the key to the success of the treatment of the gallbladder cancer and the bile duct cancer.

Therefore, in the process of treating gallbladder and bile duct diseases, the problems that the curative effect of the medicine is reduced and the medicine has toxic and side effects on non-specific tissues in the treatment process need to be solved urgently.

Therefore, the preparation of a biliary tissue targeted drug-loaded exosome for treating biliary tissue-related diseases is urgently needed.

Disclosure of Invention

The invention aims to solve the defects in the prior art and provide a biliary targeting exosome, application and a medicine for treating biliary diseases.

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

in a first aspect, the invention provides a biliary tissue targeting exosome derived from cells in a liver 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 are released 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. Known as the marker proteins of exosomes are CD63, CD81, TSG101 and the like, and known are cell surface receptors (e.g., EGFR), signaling-related molecules, cell adhesion-related proteins, MSC-related antigens, heat shock proteins, vesicle formation-related Alix and the like.

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 liver site refer to liver cells derived from the liver or liver cells induced by stem cells. 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 liver part comprise liver cells, hepatic stellate cells, liver immortalized cells, fetal liver cells, liver cancer cells and liver cells induced by induced pluripotent stem cells.

Further, hepatocytes include NCTC1469, AML-12, QSG-7701, HL-7702, WRL-68, hepatic stellate cells include LX-2, hepatic immortalized cells include THLE-3, fetal hepatocytes include HL-2, L-02, CCC-HEL-1, BNL CL.2, HHL-5, hepatoma cells include HepG2, Hep3B, SMMC-7721, HHCC, PLC/PRF/5, HB611, BEL-7402, BEL-7404, BEL-7405.

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

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.

Further, the cells are selected from cells of the liver site.

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 a biliary disorder.

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

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 is selected from cisplatin.

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

Further, the antibiotic includes mitomycin and adriamycin.

Further, the vaccine comprises BCG.

Further, the therapeutic/prophylactic agent is used for treating or preventing a biliary disease. The term "biliary diseases" refers to any diseases occurring in or associated with the biliary tract.

Further, the biliary diseases include cholecystitis, cholelithiasis, gallbladder polyp, gallbladder cancer, and cholangiocarcinoma.

Further, the biliary disease is selected from gallbladder cancer and bile duct cancer.

In a particular embodiment of the invention, the biliary disorder is selected from gallbladder 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" indicates that the specified carrier is generally chemically and/or physically compatible with the other ingredients comprising the formulation, and physiologically compatible with its recipient.

Pharmaceutically acceptable carriers for use in the pharmaceutical compositions of the present 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)), sodium salts, sodium alginate, sodium or sodium alginate, sodium or sodium alginate, or sodium (sodium alginate, or sodium (sodium alginate, or sodium (such as a mixture of a salt, or sodium (such as a salt, or a carrier, or a pharmaceutically acceptable carrier, or a carrier, such as a carrier, or a carrier, a, 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. The preparation for parenteral administration comprises sterilized aqueous solution, non-aqueous solvent, suspending agent, oil, lyophilized preparation, and suppository. 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, as well as 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 label.

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 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,Reducing agents 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 biliary tissue targeted exosome, which comprises the following steps:

the medicine for treating the biliary disease 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 is 250V.

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

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

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

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

Further, the electrical transfer uses waveforms conventional in the art, including but not limited to exponential waves, square waves.

In a preferred embodiment of the invention, the drug for treating biliary disorders is selected from the small molecule chemical drugs cisplatin.

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 targeting exosome according to the first aspect of the invention in targeting biliary tissue. In the present invention, the targeted exosomes may be targeted to biliary tissue as delivery vehicles or to 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 a biliary disorder.

In an eighth aspect, the invention provides a use of the targeting 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 a biliary disorder.

In the present invention, "biliary tissue targeting", "biliary targeting", "targeting biliary tissue", "targeting bile" 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 from the cell source of the liver part can be loaded with different drugs or active molecules, and the drug delivery is carried out by targeting the biliary tissue, so that the treatment effect of biliary diseases is improved, and the drug toxic and side effects are reduced.

Drawings

FIG. 1 is a diagram of the detection of a biliary tissue targeted exosome marker protein secreted by human liver immortalized cell THLE-3;

FIG. 2 is a graph showing the particle size distribution of exosomes targeted by the bile tissues secreted by human liver immortalized cells THLE-3;

FIG. 3 is a distribution diagram of exosomes of different cell origins in different tissues; wherein 3A is THLE-3 cells; 3B is Hep3B cells; 3C is HHL-5 cells; 3D is porcine hepatocytes;

FIG. 4 is an in vitro assay of the antitumor activity of biliary tissue targeted drug-loaded exosomes loaded with cisplatin for the treatment of gallbladder cancer;

figure 5 is an in vivo assay of cisplatin loaded biliary tissue targeted drug-loaded exosome anti-tumor activity for treatment of gallbladder cancer.

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

Culturing human liver immortalized epithelial cell THLE-3, human liver cancer cell Hep3B, human embryo liver cell HHL-5, and pig liver 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 liver 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 liver tissue into 0.5-1mm ³ Washing the large and small tissue fragments with D-Hank's solution for 3 times, placing the small and large 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, fully shaking, sucking supernatant and centrifuging. The process is repeated for 2-3 times to separate the cells separated by the first digestion as much as possible, and avoid 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 human hepatocyte THLE-3 is 3.63 mug/muL.

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 liver site was traced in vivo, and male C57bl/6 mice (4-6 weeks) were purchased from beijing waukang 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 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 a liver part with sterile PBS, and injecting the exosome into a C57bl/6 mouse through tail vein; after 24h, anesthetizing the mouse, taking the heart, the liver, the spleen, the lung, the kidney, the gallbladder and the stomach of the mouse to make frozen sections, staining the cell nucleus by Hoechst33342, and observing the biological distribution of exosomes in each organ of the mouse.

The results are shown in FIG. 3: the cells from the liver, THLE-3, Hep3B, HHL-5, and bile target exosomes secreted by pig hepatocytes are obviously enriched in the bile tissues (fig. 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, cisplatin, a medicine for treating liver 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) mixing 150 μ g of bile target exosome with cisplatin, supplementing 150 μ L of electrotransfer buffer solution (PBS, DMEM, Cytomix, Tris-HCl), and transferring to different specifications (0.2cm, 0.4cm) of electrotransfer cups. Carrying out electrotransformation on the gall bladder targeting exosome and cisplatin respectively by adopting index waves or square waves and using different voltages (50-300V);

2) subjecting the electrotransformation product to ultracentrifugation at 100000g centrifugal force for 120min, collecting supernatant, and measuring drug loading

3) As a result: the bile tissue target exosome and cisplatin are subjected to electrotransformation under the voltage of 250V, and the drug loading rate can reach 26.5%; cisplatin was successfully loaded into biliary tissue targeting exosomes.

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

This example further examined the therapeutic effect of the target exosome loaded with the drug for treating biliary 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:

spreading gallbladder cancer cell GBC-SD in 96-well plate at 5 × 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 gallbladder cancer cells by MTT (methyl thiazolyl tetrazolium).

The results are shown in fig. 4, the targeted exosome loaded with the therapeutic drug can effectively kill the gallbladder cancer cells compared with the pure targeted exosome, and the pure targeted exosome has no effect of inhibiting the gallbladder cancer cells.

2. In vivo experiments:

in vivo experiments, a gallbladder cancer model is established, and 4 × 10 is used ⁶ Injecting GBC-SD into Balb/c nude mouse subcutaneously until it grows to about 100mm ³ In the course of treatment, the tail vein is injected with gallbladder cancer therapeutic medicine cisplatin-carrying biliary target exosome, every 3 days, and 4 times of total injection, then every other day the tumor volume is measured, and its 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 targeted exosomes (cisplatin-exosomes) loaded with the tumor therapeutic drugs can significantly reduce the tumor size of the mice compared with the free antitumor drug group (cisplatin) with equal 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 biliary tissue targeting exosome, wherein the targeting exosome is derived from cells of a liver 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 includes a mouse, rat, hamster, guinea pig.

2. The targeted exosome according to claim 1, wherein cells of the liver site comprise hepatocytes, hepatic stellate cells, hepatic immortalized cells, fetal hepatocytes, hepatoma cells, induced pluripotent stem cells induced hepatocytes;

preferably, the hepatocytes comprise NCTC1469, AML-12, QSG-7701, HL-7702, WRL-68, the hepatic stellate cells comprise LX-2, the hepatic immortalized cells comprise THLE-3, the fetal hepatocytes comprise HL-2, L-02, CCC-HEL-1, BNL CL.2, HHL-5, the hepatoma cells comprise HepG2, Hep3B, SMMC-7721, HHCC, PLC/PRF/5, HB611, BEL-7402, BEL-7404, BEL-7405.

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 conditions of the step 4) are 100000g for 2 h;

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

preferably, the cells are selected from cells of the hepatic region.

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 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 is selected from cisplatin;

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

preferably, the antibiotic comprises mitomycin, doxorubicin;

preferably, the vaccine comprises bcg;

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

preferably, the biliary disorder comprises cholecystitis, cholelithiasis, gallbladder polyp, gallbladder cancer, cholangiocarcinoma;

preferably, the biliary disease is selected from gallbladder cancer, bile duct 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 biliary tissue targeted exosome is characterized by comprising the following steps:

placing a medicament for treating biliary 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 biliary tissue.

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

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 biliary disease;