CN115025233A

CN115025233A - Lung targeting drug-loaded exosome, application thereof and drug for treating lung diseases

Info

Publication number: CN115025233A
Application number: CN202210709470.XA
Authority: CN
Inventors: 姜海涛
Original assignee: Individual
Current assignee: Individual
Priority date: 2021-06-22
Filing date: 2022-06-21
Publication date: 2022-09-09
Also published as: CN113476615A

Abstract

The invention discloses a lung targeting drug-loaded exosome and a drug for applying and treating lung diseases; the medicine for treating the lung diseases is introduced into an exosome with lung tissue targeting property, and the exosome with the lung tissue targeting property is obtained from cells at a colorectal part; the lung disease treatment drug comprises the lung targeting drug-loaded exosome. Compared with the prior art, the invention can enrich in the lung by using the exosome secreted by the cells at the colorectal part without any modification, solves the problem of the output of the exosome by using the exosome derived from the cells at the colorectal part, and has good application prospect; and different drugs or active molecules can be loaded by using exosomes derived from cells at the colorectal part, and the treatment effect of lung diseases is improved and the drug toxic and side effects are reduced by targeted lung tissue administration.

Description

Lung targeting drug-loaded exosome, application thereof and drug for treating lung diseases

Technical Field

The invention belongs to the field of biomedicine, and relates to a lung targeting drug-loading exosome and a drug for applying and treating lung diseases.

Background

The lung is one of the important organs of the human body, and abnormalities in the lung cause various lung diseases. Including bronchitis, pulmonary fibrosis, emphysema, pulmonary abscess, pulmonary embolism, chronic obstructive pulmonary disease, pneumonia, tuberculosis, pneumothorax, bullous alveoli, pulmonary nodule, pulmonary tumor, etc. Lung cancer is the most common primary malignant tumor of lung, and most of lung cancers originate on the bronchial mucosa, so the lung cancer is also called bronchogenic lung cancer. For over 50 years, the incidence and the fatality rate of lung cancer rapidly rise in countries of the world, especially in industrially developed countries, and lung cancer is the leading cause of cancer among men who die of cancer. Despite advances in cancer diagnosis and treatment, lung cancer is the first cancer to develop and have a high incidence and mortality in cancer cases.

A targeted drug delivery system refers to a therapeutic means that specifically directs a therapeutic drug or active substance to an organ or site where it is desired to act, with little or no interaction with non-target tissues. The targeting preparation can improve the drug effect, reduce the toxicity, improve the safety, effectiveness and reliability of the medicine and the compliance of the patient in the medicine, so the targeting preparation is increasingly paid extensive attention by the medical field at home and abroad. Current cancer treatments, whether chemical or biological, involve three problems, the first: the medicament has a killing effect on normal tissues due to lack of targeting property, so that the medicament has high toxic and side effects and poor tolerance of patients. Secondly, the method comprises the following steps: many drug molecules are difficult to be taken up by cells due to water solubility, and effective drug treatment concentration in the cells is difficult to achieve; thirdly, the method comprises the following steps: the stability of drug molecules in vivo is problematic, a plurality of enzyme systems in the body degrade the drug molecules before the drug molecules reach a focus, and biological macromolecules have poor stability and are easily degraded in vivo. Therefore, there is a need for a drug delivery system, which has the characteristics of good biocompatibility, strong transmembrane delivery capability, tissue targeting property and high stability.

The development of nano-drug delivery systems has solved the above problems to some extent. However, the liposome or albumin-based nanocarriers cannot exist stably in blood, are easily removed by the reticuloendothelial system (or mononuclear phagocyte system), lack tissue targeting, and are difficult to achieve ideal therapeutic effects. In recent years, exosomes have received increasing attention from researchers as emerging drug delivery vehicles. Exosomes may serve as carriers, carrying drugs and delivered to adjacent or distant cells. Compared with the liposome which is widely researched, the exosome has the advantages of better biocompatibility, biodegradability, low toxicity, stability and low immunogenicity. Therefore, the preparation of the exosome drug-loading system with the lung targeting effect is an ideal solution for treating lung diseases.

Survivin is a new member of apoptosis inhibiting protein family, has multiple functions of resisting tumor apoptosis, promoting mitosis, regulating cell cycle, participating in angiogenesis and chemoradiotherapy tolerance and the like, and is widely concerned by scholars at home and abroad. Survivin is tumor specific, it is expressed specifically in almost all kinds of tumor tissues. The detection of survivin expression in early embryonic cells indicates that survivin is involved in the development of normal embryonic tissues, however, when the tissues are mature, survivin expression is difficult to detect and hardly expressed in most adult tissues. Thus, anti-tumor therapy for survivin can minimize toxic side effects.

Therefore, it is highly desirable to prepare a highly effective lung targeting exosome drug-carrying system for treating lung-related diseases.

Disclosure of Invention

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

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

in a first aspect, the invention provides a lung tissue targeting exosome derived from cells at the colorectal 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 from about 30 to about 500 in diameter, from about 30 to about 300 in diameter, from about 30 to about 250 in diameter, from about 30 to about 220 in diameter, from about 40 to about 175, from about 50 to about 150, from about 30 to about 150, or from about 30 to about 120nm in diameter.

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

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

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

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

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

In the present invention, the cells at the colorectal region refer to colorectal cells derived from the colorectal or colorectal 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 at the colorectal region include colorectal cells, colorectal cancer cells, induced pluripotent stem cell-induced colorectal cells.

Further, the colorectal cells comprise colorectal tissue epithelial cells and colorectal tissue fibroblasts.

Further, the colorectal tissue epithelial cells include NCM460, HCoEpiC, FHC, CCD841CoN, NCOL-1, NCM356, the colorectal tissue fiber cells include CCD-18Co, BNCC342560, CCD-33Co, CCD-112CoN, and the colorectal cancer cells include GPC-16, MC38, HCT-116, Caco-2, SW620, Lovo, CW-2, HT-29, COLO-394, COLO-205, SW 480.

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

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, so long as it functions to treat a pulmonary disease.

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

Further, the nucleic acid drug is selected from siRNA.

Further, the sequence of the siRNA is shown in SEQ ID NO. 1-6.

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, and lenvatinib.

Further, the small molecule chemical drug is selected from paclitaxel, carboplatin, etoposide and vincristine.

Further, the antibody comprises 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 pulmonary disease.

In the present invention, a pulmonary disease refers to any disease occurring at or associated with the lung.

Further, the pulmonary diseases include bronchitis, pulmonary fibrosis, emphysema, pulmonary abscess, pulmonary embolism, chronic obstructive pulmonary disease, pneumonia, pulmonary tuberculosis, pneumothorax, bullous lung, pulmonary nodule, and lung cancer.

Further, the lung disease is selected from lung cancer.

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

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

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

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

The pharmaceutical composition of the present invention can be formulated into oral dosage forms such as powder, granule, tablet, capsule, suspension, emulsion, syrup, spray, etc., 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, for example, starch, calcium carbonate, sucrose or lactose, gelatin and the like, in the pharmaceutical composition of the present invention. In addition, lubricants such as magnesium stearate and talc are used in addition to simple excipients. Liquid preparations for oral administration include suspensions, solutions for internal use, emulsions, syrups and the like, and may include various excipients such as wetting agents, sweeteners, aromatics, preservatives and the like in addition to water and liquid paraffin, which are widely used as simple diluents. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, oils, freeze-dried preparations, suppositories. Non-aqueous solvents, suspending agents, propylene glycol, ethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like may be used. As the base for suppository, witepsol, polyethylene glycol, Tween (tween)61, cacao butter, real resin, glycerogelatin, etc. can be used.

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

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

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

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

The fifth aspect of the invention provides a preparation method of a drug-loaded lung tissue targeted exosome, comprising the following steps:

the drug for treating lung 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 drug, so that the purified targeted drug-loaded 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 drug-loaded lung tissue targeted exosomes are prepared as follows:

1) mixing the lung tissue target exosome with siRNA, adding electrotransfer buffer solution, and transferring the mixture into an electrotransfer cup;

2) carrying out electrotransfer on the lung tissue target exosome and siRNA;

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

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

In an embodiment of the present invention, the electrotransfer buffer is selected from the group consisting 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 lung tissue. In the present invention, the targeted exosomes may be targeted to lung tissue as delivery vehicles or to trace or detect 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 pulmonary disease.

Further, the pulmonary diseases include bronchitis, pulmonary fibrosis, emphysema, lung abscess, pulmonary embolism, chronic obstructive pulmonary disease, pneumonia, pulmonary tuberculosis, pneumothorax, bullous alveoli, pulmonary nodules, and lung cancer.

Further, the lung disease is selected from lung cancer.

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

Further, the lung disease is selected from lung cancer.

In the present invention, "lung tissue targeting", "lung targeting", "targeting lung tissue", "targeting lung" 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 at the colorectal part can be loaded with different medicines or active molecules, and the medicine is delivered by targeting lung tissues, so that the treatment effect of lung diseases is improved, and the toxic and side effects of the medicines are reduced.

Drawings

FIG. 1 is a graph of the detection of exosome marker proteins secreted by FHC from epithelial cells of normal human colorectal tissue;

FIG. 2 is a graph showing the particle size distribution of exosomes secreted by epithelial cells FHC in normal human colorectal tissue;

FIG. 3 is a distribution diagram of exosomes of different cell origins in different tissues; wherein 3A is FHC cells; 3B is a CCD-18Co cell; 3C is HCT-116 cells; 3D is porcine colorectal cells;

FIG. 4 is an in vitro assay of lung targeting exosome anti-tumor activity loaded with drug siRNA for treating lung diseases; wherein 4A is a lung targeting exosome loaded with siRNA-survivin; 4B is a lung targeting exosome carrying siRNA-survivin-2; 4C is a lung targeting exosome loaded with siRNA-survivin-3;

FIG. 5 is an in vivo assay for lung targeting exosome anti-tumor activity loaded with siRNA-survivin for treatment of lung disease.

Detailed Description

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

Example 1 preparation and detection of Targeted exosomes

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

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

The preparation method of the pig colorectal 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 colorectal tissue into 0.5-1mm ³ Washing the large and small tissue fragments with D-Hank's solution for 3 times, placing the tissue fragments into a 50mL conical flask, adding freshly prepared V-type collagenase with the concentration of 0.5-1mg/mL, carrying out shock digestion in water bath at 37 ℃ for 10min, adding 40mL of cold D-Hamk's solution, stopping digestion at 4 ℃, shaking the conical flask to precipitate larger tissue blocks, transferring the supernatant into a 50mL centrifuge tube, adding 8mL of calf serum, uniformly mixing, and then carrying out low-speed centrifugation (800r/min) to obtain precipitated cells to be cultured. Adding D-Hank's solution into the residual tissue mass precipitate, 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.

And measuring the expression quantity of the exosome secreted by the cells, and detecting the content of the exosome protein secreted by the cells by using a BCA method, wherein the result shows that the concentration of the exosome protein secreted by the colorectal cell FHC is 2.46 mu g/mu L.

Example 2 Targeted detection of exosomes

The exosomes prepared in example 1 were taken, stained with PKH67 or PKH26, and the in vivo distribution of exosomes secreted from cells at the colorectal site was followed, and male C57bl/6 mice (4-6 weeks) were purchased from beijing waukee biotech inc, 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 the dark overnight, centrifuging for 2h under the centrifugal force of 100000g (g is the gravity acceleration), discarding the supernatant, washing with PBS twice, then resuspending the exosome secreted by the cells at the rectal part with sterile PBS, and injecting the exosome into a C57bl/6 mouse through the tail vein; after 24h, anesthetizing the mice, taking the heart, liver, spleen, lung, kidney, stomach and intestine of the mice to make frozen sections, staining nuclei by Hoechst33342, and observing the biological distribution of exosomes in each organ of the mice.

The results are shown in FIG. 3: the exosomes secreted by FHC, CCD-18Co, HCT-116 and pig colorectal cells from the colorectal part are obviously enriched in lung tissues (figures 3A-3D), and the targeting efficiency can reach 65% -90%. The target efficiency is calculated by taking 100 cells in a visual field, wherein 65-90 cells have exosomes to take in, and the total exosomes taken in by other tissues is 10-35 per 100 cells.

Example 3 preparation of Targeted drug-loaded exosomes

The siRNA-survivin for treating lung diseases is selected, has fluorescence and is synthesized in Shanghai Jima company, and the sequence is as follows:

the siRNA-survivin sequence is as follows:

5’-GAAUUUGAGGAAACUGCGATT-3’(SEQ ID NO.1)；

5’-UCGCAGUUUCCUCAAAUUCTT-3’(SEQ ID NO.2)；

the siRNA-survivin-2 sequence is as follows:

5’-CUGAGAACGAGCCAGACUUTT-3’(SEQ ID NO.3)；

5’-AAGUCUGGCUCGUUCUCAGTT-3’(SEQ ID NO.4)；

the siRNA-survivin-3 sequence is as follows:

5’-CACCGCAUCUCUACAUUCATT-3’(SEQ ID NO.5)；

5’-UGAAUGUAGAGAUGCGGUGTT-3’(SEQ ID NO.6)

the preparation method comprises the following steps of (1) introducing a medicament for treating diseases into a corresponding targeted exosome to prepare a targeted therapeutic medicament, wherein the preparation of the targeted medicament-carrying exosome comprises the following steps:

1) 150 mu g of lung targeting exosome is mixed with siRNA with fluorescence, and the mixture is complemented to 150 mu L by electrotransfer buffer solution (PBS, DMEM, Cytomix and Tris-HCl), and transferred to electrotransfers with different specifications (0.2cm and 0.4 cm). Adopting different waveforms (exponential waves and square waves) and using different voltages (50-300V) to respectively carry out electrotransfer on the lung targeting exosomes and the siRNA-survivin;

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

3) As a result: for siRNA, 150 μ g of lung targeting exosome and 150pmol of siRNA-survivin were subjected to electrotransformation at a voltage of 250V, with the highest efficiency reaching 25.7%, indicating that siRNA-survivin was successfully loaded into lung targeting 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 diseases prepared in example 3 on the diseases by in vitro and in vivo experiments, Balb/c nude mice (4-6 weeks) used in vivo experiments were purchased from beijing waukancon biotechnology, inc, and all mice were bred in SPF-level facilities.

The method comprises the following specific steps:

1. in vitro experiments:

a549 Lung cancer cells were plated in 96-well plates at 5X 10 per well ³ And (3) adding a target exosome (a control group) and a target exosome loaded with a therapeutic drug into each cell, and detecting the killing effect of the target exosome loaded with the therapeutic drug on the corresponding cell by MTT (methyl thiazolyl tetrazolium).

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

2. In vivo experiments:

in vivo experiments, a lung cancer model was established, and 5X 10 was used ⁶ Injecting lung cancer cell A549 to Balb/c nude mouse subcutaneously until it grows to about 100mm ³ When the lung targeting exosome carrying lung tumor therapeutic drug siRNA-survivin is injected into tail vein, the lung targeting exosome is injected once every 3 days for 4 times, and then the tumor volume is measured every other day, wherein the tumor volume is 1/2 × a × b ² . a represents a long diameter, and b represents a short diameter.

The results are shown in fig. 5, and the targeted exosome (exosome-loading siRAN-survivin) loaded with the tumor therapy drug can significantly reduce the tumor size of the mouse compared with the free antitumor drug group (siRNA-survivin) 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.

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Claims

1. A lung tissue targeting exosome, wherein the targeting exosome is derived from cells at the colorectal 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 colorectal site comprise colorectal cells, colorectal cancer cells, induced pluripotent stem cell induced colorectal cells;

preferably, the colorectal cells comprise colorectal tissue epithelial cells, colorectal tissue fibroblasts;

preferably, the colorectal tissue epithelial cells include NCM460, HCoEpiC, FHC, CCD841CoN, NCOL-1, NCM356, the colorectal tissue fibroblasts include CCD-18Co, BNCC342560, CCD-33Co, CCD-112CoN, and the colorectal cancer cells include GPC-16, MC38, HCT-116, Caco-2, SW620, Lovo, CW-2, HT-29, COLO-394, COLO-205, SW 480.

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

1) culturing cells to obtain a cell culture solution;

2) centrifuging and taking supernatant;

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

4) centrifuging again, and carrying out resuspension on the precipitate by using a buffer solution to obtain a target exosome;

preferably, the centrifugation condition of the step 2) is 2000g for 10 min;

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

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

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

4. A pharmaceutical composition comprising 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 sequence of the siRNA is shown in SEQ ID NO. 1-6;

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

preferably, the small molecule chemical drug comprises paclitaxel, carboplatin, etoposide, doxorubicin, vincristine;

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 pulmonary disease;

preferably, the pulmonary disease comprises bronchitis, pulmonary fibrosis, emphysema, pulmonary abscess, pulmonary embolism, chronic obstructive pulmonary disease, pneumonia, tuberculosis, pneumothorax, bullous alveoli, pulmonary nodules, lung cancer;

preferably, the lung disease is selected from lung 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 lung tissue targeted exosome is characterized by comprising the following steps:

placing a drug for treating lung diseases and the targeted exosome of claim 1 or 2 into an electrotransfer cup for electrotransfer, and centrifuging electrotransfer products to remove free drug to obtain a purified targeted drug-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 lung 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 manufacture of a medicament for treating a pulmonary disease;

preferably, the pulmonary disease comprises bronchitis, pulmonary fibrosis, emphysema, pulmonary abscess, pulmonary embolism, chronic obstructive pulmonary disease, pneumonia, tuberculosis, pneumothorax, bullous lung, pulmonary nodule, lung cancer;

preferably, the pulmonary disease is selected from lung cancer.

10. Use of a targeting exosome according to claim 1 or 2 or a composition according to claim 6 in the manufacture of a product for detecting a pulmonary disease;

preferably, the lung disease comprises bronchitis, pulmonary fibrosis, emphysema, pulmonary abscess, pulmonary embolism, chronic obstructive pulmonary disease, pneumonia, tuberculosis, pneumothorax, bullous lung, pulmonary nodule, lung cancer;

preferably, the lung disease is selected from lung cancer.