CN114949233A

CN114949233A - Pancreas targeted drug-loaded exosome, application thereof and drug for treating pancreatic diseases

Info

Publication number: CN114949233A
Application number: CN202210707539.5A
Authority: CN
Inventors: 姜海涛
Original assignee: Individual
Current assignee: Individual
Priority date: 2021-11-05
Filing date: 2022-06-21
Publication date: 2022-08-30
Also published as: CN114081959A

Abstract

The invention discloses a pancreatic targeted drug-loaded exosome and a drug for treating pancreatic diseases; is obtained by introducing a medicament for treating pancreatic diseases into exosomes with pancreatic tissue targeting property, wherein the exosomes with pancreatic tissue targeting property are derived from cells at a stomach part; the pancreatic disease treatment drug comprises the pancreatic targeted drug-loaded exosome. The invention uses the exosome secreted by the cells at the stomach part to enrich in the pancreas without any modification, and uses the exosome derived from the cells at the stomach part to solve the problem of the output of the exosome, thereby having good application prospect; and different drugs or active molecules can be loaded by using exosomes derived from cells at the stomach position, and the treatment effect of pancreatic diseases is improved and the drug toxic and side effects are reduced by targeted pancreatic tissue administration.

Description

Pancreas targeted drug-loaded exosome, application thereof and drug for treating pancreatic diseases

Technical Field

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

Background

Pancreatic diseases mainly include different types of pancreatitis, insulinoma, and pancreatic cancer, and diabetes is also a disease of the endocrine system caused by insufficient secretion of insulin from cells secreting insulin present in pancreatic tissues. In recent years, the incidence of pancreatic cancer has been on the rise, pancreatic cancer is very heterogeneous, and genetic markers include whole genome instability such as mutations, translocations and insertions/deletions and aneuploidy. Genome-wide analysis revealed that all 12 core signaling pathways were genetically altered. The most common genetic alterations include DNA replication, KRAS, TGF- β, apoptosis, and cell cycle pathway alterations, among others. KRAS mutations are present in more than 90% of aggressive pancreatic cancers and are associated with progression of pancreatic intraepithelial tumors to pancreatic cancer.

The traditional treatment method mainly comprises surgery, chemotherapy and radiotherapy, but the curative effect is poor. At present, the targeted drug therapy is greatly advanced, and with the deep research of molecular mechanism of pancreatic cancer, people can treat the pancreatic cancer from gene level.

In recent years, the use of targeted drugs alone and in combination has shown good results in preclinical models and in partial clinical trials. However, how to specifically deliver the molecular targeted drug to pancreatic tissues, increase the curative effect of the drug, and reduce the toxic and side effects of the drug on non-specific tissues is the key to the success of the molecular targeted therapy of pancreatic cancer and other pancreatic related diseases.

Therefore, in the process of treating pancreatic diseases, it is urgently needed to solve the problems that the curative effect of the drug is reduced and the drug has toxic and side effects in non-specific tissues during treatment.

Disclosure of Invention

The invention aims to solve the defects in the prior art and provide a pancreas targeted exosome, application and a medicament for treating pancreatic 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 pancreatic tissue targeting exosome derived from cells of a gastric 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 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 stomach site are stomach cells derived from the stomach or stomach 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 stomach part include stomach cells, stomach cancer cells, induced pluripotent stem cell-induced stomach cells.

Further, the gastric cells include gastric mucosal epithelial cells, human gastric wall acid-secreting cells, and gastric interstitial cells.

Further, gastric mucosal epithelial cells include, but are not limited to GES-1, gastric stromal cells include, but are not limited to, gastric fibroblasts, gastric cancer cells include, but are not limited to, BGC-823, MKN-45, SGC-7901, HGC-27, AGS, MFC, SNU-1, GIST 882.

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

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

Further, the nucleic acid drug is selected from siRNA.

Further, the siRNA is siRNA against KRAS.

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 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 pancreatic diseases. Herein, pancreatic disease refers to any disease occurring in the pancreatic region or associated with the pancreas.

Further, the pancreatic diseases include pancreatitis, insulinoma, pancreatic cancer, diabetes.

Further, the pancreatic disease is selected from pancreatic 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 may include various excipients such as wetting agents, sweeteners, aromatics, preservatives and the like in addition to water and liquid paraffin, which are widely used as simple diluents. Formulations for parenteral administration to the pancreas include sterilized 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 suppositories, witepsol, polyethylene glycol, Tween 61, cacao butter, ground nut, 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, synovial, 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 The peptides are labeled, for example, by reducing pertechnetate with a stannous solution, chelating the reduced technetium to a sephadex column, and applying the peptides to the column. Alternatively, direct labeling techniques may be used, for example by incubating pertechnetate, a reducing agent such as SNCl ₂ Buffer solutions such as sodium potassium phthalate solution and peptides. An intermediate functional group commonly used to bind radioisotopes present as metal ions to peptides is diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

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

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

The fifth aspect of the present invention provides a method for preparing a drug-loaded pancreatic tissue targeting exosome, comprising:

the medicament for treating pancreatic 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 medicament, so that the purified targeted medicament-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, a drug-loaded pancreatic tissue targeting exosome is prepared as follows:

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

2) carrying out electrotransformation on pancreatic tissue targeting exosomes and medicaments for treating pancreatic diseases;

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 agent for treating pancreatic disease is selected from siRNA against KRAS.

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 pancreatic tissue. In the present invention, the targeted exosomes may be targeted to pancreatic 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 treating a pancreatic disease.

Further, the pancreatic diseases include pancreatitis, insulinoma, diabetes, pancreatic cancer.

Further, the pancreatic disease is selected from pancreatic cancer.

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 pancreatic disease.

Further, the pancreatic disease is selected from pancreatic cancer.

In the present invention, "pancreatic tissue targeting," "pancreatic targeting," "targeting pancreatic tissue," "targeting pancreas," 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 exosomes derived from cells at the stomach part can be loaded with different drugs or active molecules, and the drug delivery is carried out by targeting pancreatic tissues, so that the treatment effect of pancreatic diseases is improved, and the drug toxic and side effects are reduced.

Drawings

FIG. 1 is a schematic diagram of a human gastric epithelial mucosal cell GES-1 secreting pancreatic targeted exosome marker protein;

FIG. 2 is a graph showing the size distribution of pancreatic targeting exosomes secreted by human gastric epithelial mucosal cell GES-1;

FIG. 3 is a distribution diagram of exosomes of different cell origins in different tissues; wherein 3A is GES-1 cells; 3B is BGC-823 cells; 3C is MKN-45 cells; 3D is porcine gastric cells;

FIG. 4 is an in vitro assay of pancreatic targeted drug-loaded exosome anti-tumor activity loaded with siRNA-KRAS for treating pancreatic cancer;

FIG. 5 is an in vivo assay for pancreatic targeted drug-loaded exosome anti-tumor activity loaded with siRNA-KRAS for treatment of pancreatic 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 gastric mucosa epithelial cell GES-1, human gastric adenocarcinoma cell BGC-823, human gastric cancer cell MKN-45, and pig gastric 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 gastric cell culture solution comprises the following steps:

after anesthetizing newborn pigs with ketamine (18mg/kg), the organs were removed by laparotomy under sterile conditions, washed 3 times with D-Hank's containing antibiotics to remove blood and remove the connective tissue and lymphoid tissue that were visible to the naked eye. Then cutting the stomach tissue into 0.5-1mm ³ Washing the large and small tissue fragments with D-Hank's solution for 3 times, placing into a 50mL conical flask, adding fresh V-type collagenase with concentration of 0.5-1mg/mLAnd (3) digesting for 10min by shaking in a water bath at 37 ℃, adding 40ml of cold D-Hamk's solution, stopping digestion at 4 ℃, shaking a conical flask to precipitate larger tissue blocks, transferring supernatant into a 50ml centrifuge tube, adding 8ml of calf serum, uniformly mixing, and then centrifuging at a low speed (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 gastric cells GES-1 is 3.35 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 at the gastric site was traced, and male C57bl/6 mice (4-6 weeks) were purchased from beijing wakaukang biotechnology limited, all of which were bred at SPF-level 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 gastric 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 pancreas and the stomach of the mouse to make frozen sections, staining the nucleus with Hoechst33342, and observing the biological distribution of exosomes in each organ of the mouse.

The results are shown in FIG. 3: cells from stomach sites GES-1, BGC-823, MKN-45, and pancreas targeting exosomes secreted by porcine stomach cells appeared to be significantly enriched in pancreas tissues (FIGS. 3A-3D), and targeting efficiency could 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 this example, the drug siRNA-KRAS for treating liver cancer was 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 pancreatic target exosome with siRNA-KRAS, supplementing the electrotransfer buffer solution (PBS, DMEM, Cytomix, Tris-HCl) to 150 μ L, and transferring to different specifications (0.2cm, 0.4cm) electrotransfers. Respectively carrying out electrotransformation on the pancreas targeting exosomes and the siRNA-KRAS by adopting index waves or square waves and using different voltages (50-300V);

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

3) As a result: the pancreas target exosome and the siRNA-KRAS are electrically transformed under the voltage of 250V, the drug loading rate can reach 32.5%, and the siRNA-KRAS is successfully loaded into the pancreas target exosome.

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 pancreatic 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:

pancreatic cancer cells PANC-1 were plated in 96-well plates at 5X 10 per well ³ And respectively adding a target exosome (control group) and a therapeutic drug-loaded target exosome into each cell, and detecting the killing effect of the therapeutic drug-loaded target exosome on pancreatic cancer cells by MTT (methyl thiazolyl tetrazolium).

As shown in fig. 4, the targeted exosome loaded with the therapeutic drug can effectively kill pancreatic cancer cells compared with the simple targeted exosome, and the simple targeted exosome does not have the effect of inhibiting pancreatic cancer cells.

2. In vivo experiments:

in vivo experiments, a pancreatic cancer model was established, which was 4X 10 ⁶ Injecting pancreatic cancer cell PANC-1 into Balb/c nude mouse subcutaneously until it grows to about 100mm ³ In the method, a pancreas target exosome loaded with pancreatic cancer treatment drug siRNA-KRAS is injected into tail vein, the pancreas target exosome is injected 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 (siRNA-KRAS-exosome) loaded with the tumor therapy drug can significantly reduce the tumor size of the mouse compared with the free antitumor drug group (siRNA-KRAS) 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 pancreatic tissue targeting exosome, wherein the targeting exosome is derived from cells of the gastric site;

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

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

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

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

2. The targeted exosome according to claim 1, wherein cells of the gastric site include gastric cells, gastric cancer cells, induced pluripotent stem cell-induced gastric cells;

preferably, the gastric cells comprise gastric mucosal epithelial cells, human gastric parietal secretory cells, gastric interstitial cells;

preferably, the gastric mucosal epithelial cells comprise GES-1, the gastric stromal cells comprise gastric fibroblasts, and the gastric cancer cells comprise BGC-823, MKN-45, SGC-7901, HGC-27, AGS, MFC, SNU-1, GIST 882.

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 supernate;

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;

preferably, the cells are selected from cells of the gastric 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 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 siRNA;

preferably, the siRNA is an siRNA against KRAS;

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 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 pancreatic disease;

preferably, the pancreatic disease comprises pancreatitis, insulinoma, pancreatic cancer, diabetes;

preferably, the pancreatic disease is selected from pancreatic 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 medicine-carrying pancreatic tissue target exosome is characterized by comprising the following steps:

placing a medicament for treating pancreatic 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 in targeting pancreatic 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 pancreatic disease;

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

preferably, the pancreatic disorder is selected from pancreatic cancer.