CN115998893A

CN115998893A - Liver targeting drug-carrying exosome and application and drug for treating liver diseases

Info

Publication number: CN115998893A
Application number: CN202210709497.9A
Authority: CN
Inventors: 姜海涛
Original assignee: Individual
Current assignee: Individual
Priority date: 2021-06-22
Filing date: 2022-06-21
Publication date: 2023-04-25
Also published as: CN113388571A

Abstract

The invention discloses a liver targeting drug-carrying exosome and application and a drug for treating liver diseases; the liver tissue targeting exosome is obtained by introducing a medicine for treating liver diseases into the exosome with liver tissue targeting, and the exosome with liver tissue targeting is derived from cells of gall bladder or bile duct parts; the liver disease therapeutic drug comprises the liver targeting drug-carrying exosome. The method can enrich the liver by using the exosomes secreted by the cells at the gall bladder or bile duct without any modification, solves the problem of exosomes output by using the exosomes from the cells at the gall bladder or bile duct, and has good application prospect; and different drugs or active molecules can be loaded on the exosomes derived from cells of the gall bladder or the bile duct part, and the drug administration is performed by targeting liver tissues, so that the treatment effect of liver diseases is improved, and the toxic and side effects of the drugs are reduced.

Description

Liver targeting drug-carrying exosome and application and drug for treating liver diseases

Technical Field

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

Background

Liver disease is a generic term for all diseases occurring in the liver. Including infectious diseases, neoplastic diseases, vascular diseases, metabolic diseases, toxic diseases, autoimmune diseases, hereditary diseases, calculus of intrahepatic duct, etc. Infectious diseases include viral infection, bacterial infection, parasitic infection, etc., such as viral hepatitis, echinococcosis hepatica, etc. Liver cancer is the third most common malignant tumor with death rate inferior to stomach cancer and esophagus cancer, the initial symptoms are not obvious, and the later stage mainly shows symptoms such as liver pain, hypodynamia, emaciation, jaundice, ascites and the like. In clinic, western medicine operation, radiotherapy and chemotherapy and traditional Chinese medicine combination therapy are generally adopted, but the cure rate of advanced patients is lower due to cancer cell diffusion.

Traditional chemotherapy is difficult or less to reach a target site due to nonspecific distribution of drugs, the blood concentration of the drugs in the body is low, the ideal therapeutic effect is often not achieved, and certain adverse reactions can be generated. The liver active targeting preparation is used as a novel drug delivery system, can target and deliver drugs to liver cells, improves the drug concentration in a target area, and realizes the effects of synergism and attenuation. In recent years, pharmaceutical workers have been working on the development of liver-targeted drug delivery systems. Existing liver targeting systems can be divided into hepatic parenchymal cell targeting delivery systems, hepatic non-parenchymal cell targeting delivery systems, and hepatic tumor cell targeting delivery systems.

For example, liver parenchymal cells contain a plurality of receptors such as asialoglycoprotein receptor, glycyrrhetinic acid receptor and the like on the surface, and the liver active targeting of the medicine can be realized by utilizing the specific combination between the receptors and the corresponding ligands. Mannitol receptors belong to the C-type lectin-like receptor, named for their binding properties to mannose, exist in large numbers on the surface of liver endothelial cells, have a broad binding specificity, and are used for targeted therapy using this mechanism. Monoclonal antibodies, abbreviated as monoclonal antibodies, are secreted by a hybridoma cell line recognizing a specific epitope, have the characteristics of strong specificity, high sensitivity and the like, and have been widely used in the field of biological medicine. Monoclonal antibodies can be combined with radioisotopes, chemotherapeutic drugs, toxins and the like to form immunoconjugates, and are one of the current therapeutic approaches for liver diseases. Monoclonal antibodies are the main components in the antibody-mediated liver targeting drug delivery system, and the strength of the antibodies directly influences the targeting therapeutic effect. The targeting system realizes targeting at the molecular level, the targeting effect is not more than 30%, the targeting effect is low, and the off-target phenomenon exists. Therefore, the preparation of a drug-carrying system with high efficiency targeting liver tissue is an ideal choice for treating liver diseases, especially liver cancer.

There are a wide variety of chemotherapeutic agents currently used in the treatment of liver cancer, such as fluorouracil, doxorubicin, cisplatin, capecitabine, gemcitabine, irinotecan, oxaliplatin, and the like. Chemotherapeutic agents, although having good anti-tumor effects, have clinically strong toxic and side effects due to weaker targeting.

Therefore, it is highly desirable to prepare an efficient liver-targeted exosome drug-carrying system for treating liver-related diseases.

Disclosure of Invention

The invention aims to solve the defects in the prior art and provide a liver targeting exosome, application and a medicament for treating liver diseases, and the medicament can improve the treatment effect of the liver diseases and reduce the toxic and side effects of the medicament by targeting liver tissue administration.

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

in a first aspect the invention provides a liver tissue targeted exosome derived from cells at the gall bladder or bile duct site.

The term "exosome" is a vesicle that is secreted from a cell to the outside of the cell, or has a membrane structure composed of a lipid bilayer that is present within the cell. The exosomes are about 30-1000nm in diameter 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 function to transport intracellular biomolecular proteins, bioactive lipids and RNAs (mirnas) to achieve their functional role in mediating clotting, intercellular communication and cellular immunity.

The above exosome concepts include microbubbles. The known exosome marker proteins include CD63, CD81, TSG101, etc., and cell surface receptors (e.g., EGFR), signaling related molecules, cell adhesion related proteins, MSC related antigens, heat shock proteins, vesicle formation related Alix, etc., are also known.

In some embodiments, the exosomes are about 30 to about 500, about 30 to about 300, about 30 to about 250, about 30 to about 220, 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 cells are selected from normal cells or derivatives thereof, and cancer cells.

Further, the cells are derived from a human or non-human mammal.

Further, the non-human mammal includes a non-human primate, rodent, cow, pig, sheep, dog, rabbit, cat, horse.

Further, the rodents include mice, rats, hamsters, guinea pigs.

Further, the primate includes a monkey, gorilla, baboon, ape.

In the present invention, the cells of the gall bladder or bile duct part refer to gall bladder or bile duct cells derived from the cells of the gall bladder or bile duct or stem cells induced to differentiate. 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 of non-pluripotent cells (typically mature somatic cells) produced by inducing 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, oct-3/4; sox gene families, i.e., sox1, sox2, sox3, sox15 and Sox 18; the Klf gene family, namely Klf1, klf2, klf4 and Klf5; myc gene families, i.e., c-Myc and L-Myc; the Nanog gene family, OCT4, nanog and REX1.

Further, cells of the gall bladder or bile duct part comprise gall bladder cells, bile duct cells, gall bladder cancer cells, bile duct cancer cells, and gall bladder cells or bile duct cells induced by induced pluripotent stem cells.

Further, bile duct cells include HIBEPIC, gallbladder cells include gallbladder epithelial cells and gallbladder epithelial immortalized cells, gallbladder cancer cells include GBC-SD, NOZ, EH-GB1, SGC-996 and OCUG-1, and human bile duct cancer cells include CCLP1, SK-ch-1, RBE, SK-chA-1, FRH-0201 and QBC939.

In the present invention, cells at the gallbladder or bile duct site may also be genetically modified or engineered or induced to be targeted to liver tissue, including, but not limited to, genetic modification, gene overexpression or deletion, molecular modification, etc.; exosomes secreted by cells in the gall bladder or bile duct site may also be surface modified or engineered to target liver tissue, including but not limited to surface protein modifications, surface small molecule modifications, and the like.

The second aspect of the present invention provides a method for preparing the targeted exosome according to the first aspect of the present invention, comprising the steps of:

culturing cells to obtain a cell culture solution;

centrifuging, and taking supernatant;

secondary centrifugation is carried out, and supernatant fluid is taken;

and (5) centrifuging again, and re-suspending and precipitating by using a buffer solution to obtain the targeted exosomes.

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

Further, the centrifugation condition in the step 3) was 10000g for 30min.

Further, the centrifugation condition in step 4) was 100000g for 2 hours.

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

Further, the cells are selected from cells of the gall bladder or bile duct site.

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

Further, the therapeutic or prophylactic agent includes small molecule chemicals, peptides or proteins, 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 treat liver diseases.

Further, the nucleic acid drugs comprise plasmids DNA, mRNA, microRNA, small interfering RNAs, shRNAs, sense RNAs, antisense oligonucleotides and aptamers.

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

Further, the small molecule chemical includes fluorouracil, cisplatin, capecitabine, gemcitabine, irinotecan, oxaliplatin.

Further, the small molecule chemical is selected from fluorouracil.

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

Further, the antibiotics include mitomycin and doxorubicin.

Further, the vaccine includes bacillus calmette-guerin.

Further, the therapeutic/prophylactic agent is for treating or preventing liver diseases. Liver disease refers to any disease that occurs at or associated with the liver. Including infectious diseases, neoplastic diseases, vascular diseases, metabolic diseases, toxic diseases, autoimmune diseases, hereditary diseases, calculus of intrahepatic duct, etc. Infectious diseases include viral infection, bacterial infection, parasitic infection, etc., such as viral hepatitis, echinococcosis hepatica, etc.

Further, the liver diseases include hepatitis, liver cancer, liver cirrhosis, and fatty liver.

Further, the liver disease is selected from liver cancer.

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

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

The term "pharmaceutically acceptable" means that the 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 dextrose injection, sterile water injection, or ringer's dextrose 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 carboxymethyl cellulose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone), chelating agents (e.g., EDTA (ethylenediamine tetraacetic acid) or EGTA (ethylene glycol tetraacetic acid)), emulsifiers (e.g., polysorbate 80 (Tween 80)), 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, flavouring agents, thickening agents, colouring agents or emulsifying agents.

The pharmaceutical composition of the present invention can be formulated into oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, sprays, etc., external preparations, suppositories, and sterile injectable solutions, respectively, according to a usual method. The carrier, excipient and diluent contained in the pharmaceutical composition can be lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talcum, magnesium stearate, mineral oil and the like. The composition 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 are commonly used. Solid formulations 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. Besides the simple excipient, lubricants such as magnesium stearate and talc are used. Liquid preparations for oral administration include suspending agents, oral liquid preparations, emulsions, syrups, and the like, and may include various excipients such as wetting agents, sweeteners, fragrances, preservatives, and the like, in addition to water and liquid paraffin, which are widely used as simple diluents. Formulations for parenteral administration to the liver include sterile aqueous solutions, nonaqueous solvents, suspensions, oils, freeze-dried formulations, suppositories. As the nonaqueous solvent and suspending agent, propylene glycol, ethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. can be used. As a base for suppositories, witepsol, polyethylene glycol, tween (tween) 61, cocoa butter, meat resin, glycerogelatin, etc. can be used.

The pharmaceutical composition of the present invention can be administered to mammals such as mice, livestock, and humans in various ways. All modes of administration can be predicted including, but not limited to, oral, 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, intra-articular, intra-synovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.

The pharmaceutical composition according to the present invention may be administered as a single therapeutic agent, or concurrently with other therapeutic agents, and also sequentially or simultaneously with conventional therapeutic agents, and may be administered in a single or multiple times. Taking the above factors into consideration, it is important that the maximum effect should be achieved in a minimum amount and without side effects, which is easily determined by those skilled in the art.

The pharmaceutical compositions of the invention may be administered by any means to deliver the active agent to the target cell. The preferred mode of administration and formulation are injections. The injection can be formulated with physiological saline, ringer's solution, hank's (Hank) solution or an aqueous solvent such as sterile aqueous solution, vegetable oil such as olive oil, higher fatty acid esters such as ethyl oleate, and nonaqueous solvents such as ethanol, benzyl alcohol, propylene glycol, polyethylene glycol or glycerin, etc., and for mucous membrane permeation, a non-invasive preparation known in the art suitable for the barrier to be passed may be used, and pharmaceutically acceptable carriers such as stabilizer for preventing deterioration, emulsifier, buffer for adjusting pH, phenylmercuric nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc. for preventing deterioration, such as ascorbic acid, sodium bisulfite, butylated Hydroxyanisole (BHA), tocopherol, ethylenediamine tetraacetic acid (EDTA) and the like may be included.

The dosage of the pharmaceutical composition of the present invention varies depending on the age, sex, weight, specific disease or pathological condition to be treated, severity of disease or pathological condition, administration route and judgment of prescribing staff of the subject. Determination of dosages based on the above factors is within the level of ordinary skill in the art to which the 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 having a predisposition to the disease, or subjects in need of disease prevention. Therapeutic benefit may refer to eradication or amelioration of symptoms or the underlying disease being treated. In addition, improvements are observed in a subject by eradicating or ameliorating one or more of the physiological symptoms associated with the underlying disease, although the subject may still be afflicted with the underlying disease and may achieve therapeutic benefits. The prophylactic effect can 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 detection label includes 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 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 tag used in the present invention also includes tags such as His tag, flag tag, etc. 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 diagnostics 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) can be mentioned by way of example, with gadolinium being particularly preferred. Ions suitable for other contexts, such as X-ray imaging, include, but are not limited to, lanthanum (III), gold (III), lead (II), and especially bismuth (III).

In the case of radioisotopes for therapeutic and/or diagnostic applications, astatine may be mentioned ²¹¹ 、 ¹⁴ Carbon (C), ⁵¹ Chromium (Cr), ³⁶ Chlorine (Cl), ⁵⁷ Cobalt (Co), ⁵⁸ Cobalt, copper ⁶⁷ 、 ¹⁵² Eu, ga ⁶⁷ 、 ³ Hydrogen, iodine ¹²³ Iodine ¹²⁵ Iodine ¹³¹ Indium (indium) ¹¹¹ 、 ⁵⁹ Iron (Fe), ³² Phosphorus, rhenium ¹⁸⁶ Rhenium (Re) ¹⁸⁸ 、 ⁷⁵ Selenium (Se), ³⁵ Sulfur, technetium ^99m And/or yttrium ⁹⁰ 。 ¹²⁵ I is applicable to some embodiments, technetium ^99m And/or indium ¹¹¹ It is particularly suitable because of its low energy and suitability for long distance 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 and/or potassium iodide, a chemical oxidant (e.g., sodium hypochlorite) or an enzymatic oxidant (e.g., lactoperoxidase). Technetium can be used by ligand exchange procedures ^99m Labeling the peptide, for example by reducing pertechnetate with a stannous solution, chelating the reduced technetium to a sephadex column, and applying the peptide to this column. Alternatively, direct labelling 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. The intermediate functional group commonly used to bind the radioisotope present as a metal ion to the peptide is diethylenetriamine pentaacetic acid (DTPA) or ethylenediamine tetraacetic acid (EDTA).

The fluorescent label comprises Alexa 350, alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, cascade blue, cy3, cy5,6-FAM, fluorescein isothiocyanate, HEX, 6-Joe, oreg green 488, oreg green 500, oreg green 514, pacific blue, REG, rhodamine green, rhodamine red, contrast agent (Renographin), ROX, TAMRA, PKH, PKH26, TET, tetramethylrhodamine, and/or Texas red.

When in vitro diagnostics are involved, it is linked to a second binding ligand and/or an enzyme (enzyme tag) which will produce a colored product upon contact 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.

In a fifth aspect, the present invention provides a method for preparing a drug-loaded liver tissue-targeted exosome, comprising:

placing the medicine for treating liver diseases and the targeted exosome according to the first aspect of the invention into an electrorotating cup for electrorotating, centrifuging the electrorotating product to remove free medicine, and obtaining the purified targeted medicine carrying exosome.

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 liver tissue targeted exosome comprises the following steps:

1) Mixing liver tissue targeted exosome with medicine for treating liver diseases, adding electrotransport buffer solution, and transferring to electrotransport cup;

2) Electrotransport of liver tissue targeted exosomes and drugs for treating liver disease;

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

Further, the electrical transitions employ waveforms conventional in the art, including but not limited to exponential, square.

In a preferred embodiment of the invention, the drug for treating liver diseases is selected from the small molecule chemical fluorouracil.

In an embodiment of the invention, the electrotransport buffer is selected from electrotransport 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 for targeting liver tissue. In the present invention, the targeted exosomes may be targeted to liver tissue as delivery vehicles or tracer or detection substances.

The seventh aspect of the present invention provides the use of the targeted exosome according to the first aspect of the present invention or the pharmaceutical composition according to the third aspect of the present invention in the manufacture of a medicament for the treatment of liver disease.

Further, the liver disease is selected from liver cancer.

In an eighth aspect, the invention provides the use of a targeted exosome according to the first aspect of the invention or a composition according to the fourth aspect of the invention in the manufacture of a product for detecting liver disease.

Further, the liver disease is selected from liver cancer.

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

The invention has the advantages and beneficial effects that:

the tissue targeted exosome used in the invention can be enriched in corresponding tissues without any modification, the cell source of the targeted exosome is simple, the problem of exosome yield is solved, and the tissue targeted exosome has good application prospect; the exosomes from cells at the gall bladder or bile duct can be loaded with different drugs or active molecules, and the drug administration is performed by targeting liver tissues, so that the treatment effect of liver diseases is improved, and the toxic and side effects of the drugs are reduced.

Drawings

FIG. 1 is a diagram showing detection of exosome marker proteins secreted by human gallbladder cancer cells GBC-SD;

FIG. 2 is a graph showing the measurement of the size distribution of exosomes secreted by human gallbladder cancer cells GBC-SD;

FIG. 3 is a graph showing the distribution of exosomes of different cell origins in different tissues; wherein 3A is GBC-SD cells; 3B is a CCLP1 cell; 3C is HIBEpiC cells; 3D is porcine cholecystocytes;

FIG. 4 is an in vitro assay of liver targeted exosome antitumor activity loaded with the drug fluorouracil for treatment of liver disease;

FIG. 5 is an in vivo assay of liver targeted exosome antitumor activity loaded with the drug fluorouracil for treatment of liver disease.

Detailed Description

The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. The specific embodiments described herein are for purposes of illustration only and are not intended to limit the invention.

Example 1 preparation and detection of Targeted exosomes

Culturing human gallbladder cancer cells GBC-SD, human bile duct cancer cells CCLP1, human intrahepatic bile duct epithelial cells HIBEpi C and pig gallbladder cells.

Taking cell culture solution of each cell (each cell culture solution is cultured without exosome serum and DMEM culture medium or RPMI1640 culture medium), centrifuging for 10min under a centrifugal force of 2000g, and taking supernatant; centrifuging under 10000g centrifugal force for 30min, collecting supernatant, and removing cell debris and precipitate; centrifuging the centrifuged cell culture solution under 100000g (g is gravity acceleration) centrifugal force for 2h, re-suspending and collecting precipitate with sterile PBS, and preserving at 4deg.C for short term to obtain targeted exosomes. The targeted exosome surface marker proteins obtained by extraction and separation and the particle size distribution are shown in figure 1 and figure 2.

The preparation method of the pig gall bladder cell culture solution comprises the following steps:

after anesthetizing a newborn pig with ketamine (18 mg/kg), organs were removed by laparotomy under aseptic conditions, and blood was removed by washing 3 times with D-Hank's containing antibiotics and macroscopic connective and lymphoid tissues were carefully removed. Cutting gallbladder tissue into 0.5-1mm ³ Washing large and small tissue fragments with D-Hank's solution for 3 times, placing into a 50mL conical flask, adding freshly prepared V-type collagenase with concentration of 0.5-1mg/mL, shake-digesting in 37 deg.C water bath for 10min, adding 40mL cold D-Hamk's solution, stopping digestion at 4deg.C, shaking the conical flask, transferring supernatant into a 50mL centrifuge tube after larger tissue mass is precipitated, adding 8mL calf serum, mixing, and centrifuging at low speed (800 r/min) to obtain precipitated cellsCulturing. D-Hank's solution is added into the sediment of the residual tissue blocks, and the supernatant is sucked and centrifuged after the sediment is sufficiently vibrated. The process is repeated for 2-3 times to separate the cells which can be separated in the first digestion as much as possible, so as to avoid entering the second digestion process. Isolated cells were incubated in 20% calf serum, penicillin 100U/mL, glutamine 100mg/L supplemented RPMI 1640. The medium was changed the next day, and then once every other day. The exosome-free serum medium was changed on day 5 and the cell culture broth was collected on day 7.

The amount of the exosome protein secreted by the cells was measured, and the BCA method was used to measure the content of the exosome protein secreted by the cells, and the result shows that the concentration of the exosome protein secreted by the GBC-SD cells was 2.96 mug/. Mu.L.

Example 2 Targeted detection of exosomes

The exosomes prepared in example 1 were stained with PKH67 or PKH26, the distribution of the exosomes secreted by cells in the gall bladder or bile duct site in vivo was tracked, male C57bl/6 mice (4-6 weeks) were purchased from beggar fukang biotechnology, inc, beijing, and all mice were grown in SPF grade facilities. The method comprises the following specific steps:

taking 100 mug of exosomes, incubating with 1 mug of PKH67 or PKH26 at 4 ℃ for overnight in a dark place, centrifuging for 2 hours under the centrifugal force of 100000g (g is gravitational acceleration), discarding the supernatant, washing twice with PBS, re-suspending exosomes secreted by cells at the gall bladder or bile duct part by using sterile PBS, and injecting into a C57bl/6 mouse through a tail vein; after 24h, the mice were anesthetized, frozen sections were taken from the heart, liver, spleen, lung, kidney, stomach and intestine of the mice, and after staining the nuclei with Hoechst33342, the biodistribution of the exosomes in the individual organs of the mice was observed.

The results are shown in fig. 3: the exosomes secreted by cells GBC-SD, CCLP1, HIBEpi C and pig gall bladder cells from gall bladder or bile duct parts are obviously enriched in liver tissues (figures 3A-3D), and the targeting efficiency can reach 65% -90%. The calculation method of the targeting efficiency is to take 100 cells in the visual field, wherein exosomes are taken in 65-90 cells, and the total exosomes taken in by other tissues is 10-35 cells/100 cells.

EXAMPLE 3 preparation of Targeted drug-carrying exosomes

In this embodiment, fluorouracil, a drug for treating liver cancer, is selected.

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

1) 150 μg of liver-targeting exosomes were mixed with fluorouracil, and the electrotransport buffer, which can be (PBS, DMEM, cytomix, tris-HCl), was made up to 150 μl and transferred to electrotransport cups of different format (0.2 cm, 0.4 cm). Adopting exponential waves or square waves, and respectively carrying out electrotransformation on liver targeted exosomes and fluorouracil by using different voltages (50-300V);

2) The electrokinetic transfer product was ultracentrifuged under 100000g centrifugal force for 120min, and the supernatant was collected to measure the drug loading.

3) Results: for fluorouracil, the highest electrotransformation efficiency of liver-targeted exosomes and fluorouracil under the voltage of 250V can reach 33.8%; fluorouracil was successfully loaded into exosomes secreted by GBC-SD cells.

Example 4 therapeutic Effect detection of Targeted drug-carrying exosomes

This example further examined the therapeutic effect of the targeted exosomes loaded with the drugs for treating liver diseases prepared in example 3 by in vitro and in vivo experiments using Balb/c nude mice (4-6 weeks) purchased from beggar's biotechnology co. The method comprises the following specific steps:

1. in vitro experiments:

spreading liver cancer cell HEPG2 in 96-well plate with each well being 5×10 ³ And (3) adding the target exosomes (control group) and the target exosomes loaded with the therapeutic drugs into the individual cells, and detecting the killing effect of the target exosomes loaded with the therapeutic drugs on the liver cancer cells by MTT.

As shown in fig. 4, the targeted exosomes loaded with therapeutic drugs can effectively kill liver cancer cells compared with the pure targeted exosomes, and the pure targeted exosomes do not have the effect of inhibiting liver cancer cells.

2. In vivo experiments:

in-vivoIn the internal experiment, a liver cancer model was established, 5×10 ⁶ HEPG2 of individual hepatoma cells was injected subcutaneously into Balb/cnude mice until they grew to about 100mm ³ At this time, the liver-targeted exosome loaded with the liver cancer therapeutic drug fluorouracil was injected into the tail vein once every 3 days for 4 times, and then the tumor volume was measured every other day, tumor volume=1/2×a×b ² . a represents a long diameter and b represents a short diameter.

As shown in fig. 5, the targeted exosomes (fluorouracil-exosomes) loaded with the tumor therapeutic drugs significantly reduced the tumor size in mice compared to the free antitumor drug group (fluorouracil) at an equivalent dose.

It will be evident to those skilled in the art that the embodiments of the 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 characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of 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 disclosure describes embodiments, not every embodiment is provided with a separate embodiment, and that this description is provided for clarity only, and that the disclosure is not limited to the embodiments described in detail below, and that the embodiments described in the examples may be combined as appropriate to form other embodiments that will be apparent to those skilled in the art.

Claims

1. A liver tissue targeted exosome, wherein the targeted exosome is derived from cells at the gall bladder or bile duct site;

preferably, the cell is selected from normal cells or a derivative thereof, cancer cells;

preferably, the cells are derived from a human or non-human mammal;

preferably, the non-human mammal comprises a non-human primate, rodent, cow, pig, sheep, dog, rabbit, cat, horse;

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

2. A targeted exosome according to claim 1, wherein cells of the gall bladder or bile duct site comprise gall bladder cells, bile duct cells, gall bladder cancer cells, bile duct cancer cells, induced pluripotent stem cell-induced gall bladder cells or bile duct cells;

preferably, the cholangiocytes comprise HIBEPIC, the cholecystocytes comprise gallbladder epithelial cells, and gallbladder epithelial immortalized cells, the cholecyst cancer cells comprise GBC-SD, NOZ, EH-GB1, SGC-996, and OCUG-1, and the human cholangiocarcinocytes comprise CCLP1, SK-ch-1, RBE, SK-chA-1, FRH-0201, and QBC939.

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

1) Culturing cells to obtain a cell culture solution;

2) Centrifuging, and taking supernatant;

3) Secondary centrifugation is carried out, and supernatant fluid is taken;

4) Re-centrifuging, and re-suspending and precipitating with buffer solution to obtain targeted exosomes;

preferably, it is characterized in that the centrifugation conditions of step 2) are 2000g for 10min;

preferably, the centrifugation conditions of step 3) are 10000g for 30min;

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

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

preferably, the cells are selected from cells of the gall bladder or bile duct site.

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, 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 drugs comprise plasmids DNA, mRNA, microRNA, small interfering RNAs, shRNAs, sense RNAs, antisense oligonucleotides and aptamers;

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

preferably, the small molecule chemical comprises fluorouracil, cisplatin, capecitabine, gemcitabine, irinotecan, oxaliplatin;

preferably, the small molecule chemical is selected from fluorouracil;

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

preferably, the antibiotic comprises mitomycin and doxorubicin;

preferably, the vaccine comprises bacillus calmette-guerin;

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

preferably, the liver disease comprises hepatitis, liver cancer, liver cirrhosis, fatty liver;

preferably, the liver disease is selected from liver cancer.

5. The pharmaceutical composition according to 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 claims 1 or 2, and a detection label;

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

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

placing a drug for treating liver diseases and the targeted drug-carrying exosome according to claim 1 or 2 into an electrorotating cup for electrorotating, centrifuging the electrorotating product to remove free drug, and obtaining the purified targeted drug-carrying exosome;

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

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

8. Use of a targeted exosome according to claim 1 or 2 for targeting liver tissue.

9. Use of a targeted exosome according to claim 1 or 2 or a pharmaceutical composition according to any one of claims 4-5 for the preparation of a medicament for the treatment of liver diseases;

preferably, the liver disease is selected from liver cancer.

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

preferably, the liver disease is selected from liver cancer.