Stem cell-derived ecto-liposome fusion nanoparticles and use thereof

Abstract

The present invention relates to novel ecto-liposome nanoparticles. More specifically, the present invention provides novel ecto-liposome nanoparticles exhibiting tumor site targeting ability superior to that of a conventional drug carrier having targeting ability, and thus can be used as a drug carrier that loads a drug such as an anticancer drug so as to release the drug systemically or locally, and can be used as a therapeutic agent for treating refractory cancer or metastatic tumor.

Description

Stem cell-derived ecto-liposome fusion nanoparticles and their uses

[0001] The present invention relates to novel ecto-liposome fusion nanoparticles, and more particularly, to stem cell-derived ecto-liposome fusion nanoparticles with enhanced targeting ability to tumor sites.

[0002] Ectosomes are vesicles of various sizes (0.1–1 mm in diameter) that bud off directly from the plasma membrane and are secreted into the extracellular space. Unlike living cells, ectosomes have phosphatidylserine, a phospholipid, on their surface. Although ectosomes are produced and secreted by direct budding of the cell membrane, the cargo components contained within them are not identical to those of the parent cell and are known to vary depending on the state of the cell (Cocucci and Meldolesi, Curr. Biol. 21(23): R940-R940, 2011). Although ectosomes differ from exosomes, which are secreted from endosomes through the multivesicular body (MVB) pathway, in terms of their production method and the substances contained within, their interactions in the extracellular space and with target cells are known to be similar to those of exosomes. However, while exosomes have received attention for their functional aspects, such as disease treatment and diagnosis, relatively little research has been conducted on ectosomes.

[0003] Meanwhile, liposomes are spherical vesicle structures made of a lipid bilayer, composed of phospholipids, which can be manufactured artificially or found inside cells. Because they have both hydrophobic and hydrophilic properties, they can selectively absorb substances from the surrounding environment through the membrane and grow in size. Most liposomes are spherical or oval in shape, and because there is space inside the liposome, they are used as drug delivery vehicles to deliver drugs such as anticancer agents into the body. In this regard, Korean U.S. Patent Publication No. 2021-0063254 discloses a composition for adjuvant anticancer treatment comprising a liposome composition and a drug delivery method using the same.

[0004] However, in the case of the above prior art, there is a problem of low intracellular delivery rate as it relates to immunotherapy that treats cancer by neutralizing the cancer's evasion function against immune cells.

[0005] The present invention aims to solve various problems, including the aforementioned ones, by providing a novel stem cell-derived ecto-liposome fusion that exhibits superior tumor targeting ability compared to conventional drug delivery systems. However, these tasks are exemplary and the scope of the present invention is not limited thereby.

[0006] According to one aspect of the present invention, a fusion nanoparticle formed by fusing stem cell-derived ectosomes and liposomes is provided.

[0007] According to another aspect of the present invention, a drug delivery vehicle comprising the fusion nanoparticle as an active ingredient is provided.

[0008] According to another aspect of the present invention, a pharmaceutical composition comprising the drug delivery vehicle and an effective drug is provided.

[0009] According to another aspect of the present invention, a pharmaceutical composition for treating cancer is provided, which comprises, as an active ingredient, a fused nanoparticle formed by fusing a stem cell-derived ectosome with a liposome loaded with an anticancer agent therein.

[0010] According to another aspect of the present invention, there is provided a step of isolating ectosomes from stem cells cultured in a culture medium of tumor tissue; and

[0011] A method for producing a fusion nanoparticle having a targeting ability for tumor tissue is provided, which comprises a step of mixing the ectosome and liposome nanoparticles and fusing the ectosome and liposome through ultrasonic treatment.

[0012] According to another aspect of the present invention, there is provided a use for manufacturing a medicament for preventing and treating cancer by using a fusion nanoparticle formed by fusing a stem cell-derived ectosome with a liposome loaded with an anticancer agent therein.

[0013] According to another aspect of the present invention, a method for treating cancer in a subject is provided, comprising administering to the subject a fused nanoparticle formed by fusing a therapeutically effective amount of stem cell-derived ectosome with a liposome loaded with an anticancer agent therein.

[0014] As described above, the stem cell-derived ecto-liposome fusion of the present invention exhibits enhanced targeting ability toward tumor sites, which are target diseases. Therefore, it can be utilized as a drug delivery vehicle that can be loaded with drugs such as anticancer agents and release the drugs systemically or locally, and can be used as a therapeutic agent for the treatment of intractable cancer or metastatic tumors. Of course, the scope of the present invention is not limited by these effects.

[0015] Figure 1 is a schematic diagram schematically illustrating a manufacturing process of an ecto-liposome fusion nanoparticle in which an ectosome and a liposome derived from adipose-derived mesenchymal stem cells (AD-MSC) of the present invention are fused.

[0016] Figure 2 is a microscopic photograph showing the results of observing the fusion of ectosomes and liposomes using a transmission electron microscope (TEM) after manufacturing AD-MSC-derived ecto-liposome fusion nanoparticles of the present invention, and a graph showing the results of analyzing the same.

[0017] Figure 3 is a graph showing the results of analyzing the hydrodynamic sizes and electrical potentials of liposomes, AD-MSC-derived ectosomes, and ecto-liposome fusion nanoparticles.

[0018] Figure 4 is a graph showing the results of analyzing the size distribution of the ecto-liposome fusion nanoparticles of the present invention using nanoparticle tracking (NTA).

[0019] Figure 5 is a gel photograph showing the results of confirming the fusion of liposomes, ectosomes, and ecto-liposomes using polyacrylamide gel electrophoresis (SDS-PAGE).

[0020] Figure 6a is a schematic diagram schematically illustrating the principle of confirming the fusion of the ecto-liposome fusion nanoparticles of the present invention through FRET (fluorescence resonance energy transfer) analysis.

[0021] Figure 6b is a graph showing the results of analyzing the fluorescence intensity of the ecto-liposome fusion nanoparticles of the present invention. DiI and DiD fluorescence were attached to the ectosome, and the FRET experiment was performed using a Victor 1420 Multilabel Counter (PerkinElmer, Waltham, MA, USA) at an excitation wavelength of 549 nm and an excitation wavelength and emission wavelength of 665 nm for DiI fluorescence.

[0022] Figure 7 is a fluorescence microscope photograph showing the targeting ability of the ecto-liposome fusion nanoparticles of the present invention toward lung cancer cells (A549). To evaluate the cancer targeting ability of the ecto-liposome fusion nanoparticles, the uptake of liposomes stained with a DIO fluorescent substance, ectosomes extracted from adipose-derived mesenchymal stem cells (AD-MSCs) stained with a DiI fluorescent substance, liposomes stained with a DIO fluorescent substance, and ecto-liposome fusion nanoparticles stained with a DiI fluorescent substance were visualized by fluorescence.

[0023] Figure 8a shows the targeting ability of the ecto-liposome fusion nanoparticles of the present invention. The upper part is a fluorescence photograph observed using a brain tumor animal model. Brain cancer cells (U87MG) were injected into the brain of an experimental mouse, and a xenograft tumor experimental animal model was prepared, and the tumor size of the animal was photographed using in vivo fluorescence imaging. The lower part is a fluorescence photograph observing the distribution of the ecto-liposome fusion nanoparticles of the present invention in tumor tissue extracted from a brain tumor animal model. The distribution of the tumor signal (upper part) and the drug signal (lower part) of the ecto-liposome fusion nanoparticles for the brain tumor extracted from the experimental animal model were photographed using in vivo fluorescence imaging.

[0024] Figure 8b shows the targeting ability of the ecto-liposome fusion nanoparticles of the present invention. The upper part is a fluorescence photograph observed using a lung tumor animal model. After injecting lung cancer cells (A549) into the lungs of experimental mice, a xenograft tumor experimental animal model was prepared, and the tumor size of the animal was photographed using in vivo fluorescence imaging. The lower part is a fluorescence photograph observing the distribution of the ecto-liposome fusion nanoparticles of the present invention in tumor tissues extracted from the lung tumor animal model. The distribution of the tumor signal (upper part) and the drug signal (lower part) of the ecto-liposome fusion nanoparticles for the lung tumor extracted from the experimental animal model were photographed using in vivo fluorescence imaging.

[0025] Definition of terms:

[0026] The term "ectosome" used in this document refers to a nano-vesicle with a phospholipid membrane structure having a diameter of 100 to 500 nm, which is produced by budding of the plasma membrane, unlike 'exosomes' among extracellular vesicles.

[0027] The term "liposome" used in this document refers to a spherical or oval vesicle structure made of lipids. As a structure, molecules such as phospholipids contain both hydrophobic (water-hating) and hydrophilic (water-loving) portions within a single molecule. Liposomes can encapsulate drugs such as anticancer agents or reconstitute membrane proteins within their membranes, similar to biological membranes, to replicate various functions. Therefore, they are being developed as drug delivery vehicles for intracellular delivery in the form of microcapsules.

[0028] The term "drug delivery carrier" used in this document refers to a material used to deliver a drug to a required site and maintain it for an appropriate period of time, and a method of effectively delivering a drug to a site using a drug delivery carrier is called a drug delivery system.

[0029] Detailed description of the invention:

[0030] According to one aspect of the present invention, a fusion nanoparticle formed by fusing stem cell-derived ectosomes and liposomes is provided.

[0031] In the above fusion nanoparticles, the stem cells may be embryonic stem cells, mesenchymal stem cells, or induced pluripotent stem cells, and the mesenchymal stem cells may be bone marrow-derived stem cells, cord blood-derived stem cells, adipose-derived stem cells, dental pulp-derived stem cells, or peripheral blood-derived stem cells.

[0032] In the above fusion nanoparticle, the stem cells may be stem cells educated as lesion-derived cells, the lesion may be tumor tissue, and the tumor tissue may be primary or metastatic.

[0033] In the above fusion nanoparticles, the educated stem cells may be stem cells cultured by contacting the culture medium in which the lesion-derived cells are cultured, and may have a diameter of 100 to 350 nm, and the ectosomes and liposomes may be prepared by mixing them at a mass ratio of 1:20. Also, preferably, the ectosomes and liposomes may be prepared by mixing them at a mass ratio of 1:10.

[0034] According to another aspect of the present invention, a drug delivery vehicle comprising the fusion nanoparticle as an active ingredient is provided.

[0035] According to another aspect of the present invention, a pharmaceutical composition comprising the drug delivery vehicle and an effective drug is provided.

[0036] In the above pharmaceutical composition, the effective drug may be an anticancer agent. At this time, the anticancer drugs include mechloethamine, chlorambucil, phenylalanine, mustard, cyclophosphamide, ifosfamide, carmustine (BCNU), lomustine (CCNU), streptozotocin, busulfan, thiotepa, cisplatin, carboplatin, doxorubicin, daunorubicin, idarubicin, mitoxantrone, plicamycin, mitomycin, C bleomycin, vincristine, vinblastine, It may be selected from the group consisting of paclitaxel, docetaxel, etoposide, teniposide, topotecan, and iridotecan.

[0037] The above anticancer agent is described in more detail as follows:

[0038] (i) asparaginase;

[0039] (ii) methotrexate;

[0040] (iii) pyrimidine analogs

[0041] Examples: 5-fluorouracil, gemcitabine, and arabinosylcytosine;

[0042] (iv) hydroxyurea;

[0043] (v) purine analogs

[0044] mercaptopurine and thioguanine;

[0045] (vi) alkylating agents

[0046] nitrogen mustard and cyclosporamide;

[0047] (vii) Anthracycline anticancer drugs

[0048] Anthracyclines, doxorubicin, daunorubicin, idarubicin, mitoxantrone, and actinomycin D;

[0049] (viii) mitotic inhibitors

[0050] vincristine and taxol;

[0051] (ix) Antiangiogenic agents

[0052] Antibodies specific for VEGF, combretastatin A4, Fumagillin, herbimycin A, 2-methoxyestradiol, OGT 2115, TNP 470, tranilast, XRP44X, thalidomide, endostatin, salmosin, angiostatin or the kringle domain of plasminogen or apolipoprotein;

[0053] (x) intercalating agents

[0054] carboplatin and cisplatin; and

[0055] (xi) radionuclides

[0056] 18 F, 90 Y, 188 Re, 32 P, 89 Sr, 165 Dy, 186 Re, 198 Au, 153 Sm, 131 I, 169 Er, 125 I, 99 Tc and 166 Ho, etc.

[0057] According to another aspect of the present invention, a pharmaceutical composition for treating cancer is provided, which comprises, as an active ingredient, a fused nanoparticle formed by fusing the stem cell-derived ectosome with a liposome loaded with an anticancer agent therein.

[0058] In the pharmaceutical composition, the stem cells may be educated stem cells that have been educated to be cancer cells to be treated, and the anticancer agent may be loaded by being bound to the surface or interior of the fusion nanoparticle by covalent or non-covalent bonding, or by being encapsulated inside the nanoparticle having a core-shell structure. In addition, more specifically, the anticancer agent may be loaded into the liposome nanoparticle by being bound to the surface or interior of the nanoparticle by covalent or non-covalent bonding, or by being encapsulated inside the nanoparticle having a core-shell structure. In the case of being loaded by covalent bonding, the anticancer agent may be covalently bound to the terminal or side chain of a biodegradable polymer. Alternatively, the anticancer agent may be loaded by being covalently or non-covalently bound to the surface of the ectosome membrane and presented on the surface.

[0059] In the above pharmaceutical composition, the cancer may be selected from the group consisting of brain tumor, lung cancer, stomach cancer, liver cancer, bone cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, skin cancer, head and neck cancer, skin melanoma, uterine cancer, ovarian cancer, rectal cancer, colon cancer, colon cancer, breast cancer, uterine sarcoma, esophageal cancer, laryngeal cancer, small intestine cancer, and thyroid cancer, but is not limited thereto.

[0060] According to another aspect of the present invention, a method for treating cancer in a subject is provided, comprising administering to the subject a fusion nanoparticle formed by fusing a stem cell-derived ectosome and a liposome.

[0061] According to another aspect of the present invention, there is provided a step of isolating ectosomes from stem cells cultured in a culture medium of tumor tissue; and

[0062] A method for producing a fusion nanoparticle having a targeting ability for tumor tissue is provided, which comprises a step of mixing the ectosome and liposome nanoparticles and fusing the ectosome and liposome through ultrasonic treatment.

[0063] In the above manufacturing method, the ectosome and liposome nanoparticles can be mixed at a mass ratio of 1:20.

[0064] According to another aspect of the present invention, there is provided a use for manufacturing a medicament for preventing and treating cancer by using a fusion nanoparticle formed by fusing a stem cell-derived ectosome with a liposome loaded with an anticancer agent therein.

[0065] According to another aspect of the present invention, a method for treating cancer in a subject is provided, comprising administering to the subject a fused nanoparticle formed by fusing a therapeutically effective amount of stem cell-derived ectosome with a liposome loaded with an anticancer agent therein.

[0066] The pharmaceutical composition of the present invention may vary depending on the type of the patient's affected area, application site, number of treatments, treatment time, formulation, patient's condition, type of adjuvant, etc. The dosage is not particularly limited, but may be 0.01 μg / kg / day to 10 mg / kg / day. The above daily dose may be administered once a day, or divided into 2 to 3 times a day at appropriate intervals, or intermittently at intervals of several days.

[0067] In the pharmaceutical composition of the present invention, the compound can be administered orally or parenterally, and preferably parenterally, by intravenous injection, subcutaneous injection, intracerebroventricular injection, intracerebrospinal fluid injection, intramuscular injection, and intraperitoneal injection.

[0068] The pharmaceutical composition of the present invention may further include suitable carriers, excipients, and diluents commonly used in the manufacture of pharmaceutical compositions. In addition, solid or liquid formulation additives may be used in the manufacture of the pharmaceutical composition. The formulation additives may be either organic or inorganic. Examples of excipients include lactose, sucrose, sucrose, glucose, cornstarch, starch, talc, sorbitol, crystalline cellulose, dextrin, kaolin, calcium carbonate, and silicon dioxide. Examples of binders include polyvinyl alcohol, polyvinyl ether, ethyl cellulose, methyl cellulose, gum arabic, tragacanth, gelatin, shellac, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, calcium citrate, dextrin, and pectin. Examples of lubricants include magnesium stearate, talc, polyethylene glycol, silica, and hydrogenated vegetable oil. Any colorant that is generally approved for addition to pharmaceuticals can be used. These tablets and granules can be appropriately coated with sugar, gelatin, or other agents as needed. In addition, preservatives, antioxidants, and the like can be added as needed. In addition, when the pharmaceutical composition is a drug, it can additionally contain one or more selected from fillers, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers, or preservatives. Meanwhile, the formulation of the pharmaceutical composition of the present invention may be in a desirable form depending on the method of use, and in particular, it is preferable to formulate it by adopting a method known in the art so as to provide rapid, sustained, or delayed release of the active ingredient after administration to a mammal.Examples of specific dosage forms include: PLASTERS, GRANULES, LOTIONS, LINIMENTS, LEMONADES, POWDERS, SYRUPS, LIQUIDS AND SOLUTIONS, AEROSOLS, EXTRACTS, ELIXIRS, FLUIDEXTRACTS, EMULSIONS, SUSPENSIONS, DECOCTIONS, INFUSIONS, TABLETS, SUPPOSITORIES, INJECTIONS, SPIRITS, CATAPLSMA, CAPSULES, TROCHES, TINCTURES, PASTES, PILLS, SOFT or any one of hard gelatin capsules.

[0069] The pharmaceutical composition of the present invention may further include additional ingredients commonly used in the composition, such as conventional auxiliary agents such as stabilizers, solubilizers, and flavoring agents, and carriers.

[0070] The above pharmaceutical composition or intra-articular injection may contain various carriers suitable for direct injection into the affected area.

[0071] Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are those commonly used in formulations, and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above components, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweetening agents, flavoring agents, emulsifiers, suspending agents, preservatives, etc. Suitable pharmaceutically acceptable carriers and formulations are described in Remington's Pharmaceutical Sciences (19 th Ed., 1995) is described in detail.

[0072] The present inventors have previously discovered through research that stem cells educated with cancer cell or inflammatory cell culture medium have enhanced targeting ability for tumor tissue or inflammatory tissue without using unnecessary processes such as genetic manipulation (Korean Patent Publication No. 2022-0117474). Subsequently, the present inventors hypothesized that ectosomes secreted from the above-mentioned educated stem cells would also have targeting ability for lesions such as tumor tissue, and to verify the hypothesis, the ectosomes were isolated from the educated stem cells and mixed with PLGA nanoparticles, a biodegradable polymer, thereby producing ectosome-biodegradable fusion nanoparticles coated with ectosome membranes. The fusion nanoparticles were examined to see whether they were well targeted to tumor tissue. As a result, they confirmed that the fusion nanoparticles exhibited significantly higher tumor cell targeting ability compared to non-educated stem cell-derived ectosome-biodegradable polymer fusion nanoparticles, and that the fusion nanoparticles exhibited further enhanced targeting ability to tumor tissue.

[0073] Furthermore, the inventors of the present invention isolated ectosomes from educated stem cells and then fused them with liposomes, which are spherical vesicle structures made of an artificially manufactured lipid bilayer, to produce novel fusion nanoparticles (see Fig. 1). Subsequently, the tumor tissue targeting ability of the ecto-liposome fusion nanoparticles of the present invention was analyzed using lung cancer and brain tumor animal models. As a result, it was confirmed that the ecto-liposome fusion nanoparticles derived from educated stem cells exhibited excellent targeting ability to brain tumor and lung cancer cell tissues by treating the culture medium of cancer cells. The above results provide a basis for overcoming the technical difficulty of limiting anticancer treatment due to the presence of the blood-brain barrier in the past, and suggest that the ecto-liposome fusion nanoparticles of the present invention can be usefully used as drug delivery vehicles for effective drugs for lesions in intractable cancers such as lung cancer by increasing the targeting ability to the relevant lesions.

[0074] Hereinafter, the present invention will be described in more detail through examples. However, the present invention is not limited to the examples disclosed below, but can be implemented in various different forms. The following examples are provided to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the invention.

[0075] Example 1: Extraction of ectosomes derived from adipose-derived mesenchymal stem cells (AD-MSCs)

[0076] The present inventors isolated ectosomes (nanoevesicles) to manufacture fusion nanoparticles in which ectosomes (nanoevesicles) and liposomes (liposomes) are fused from adipose-derived mesenchymal stem cells (AD-MSCs) that have enhanced targeting ability by being trained as cancer cells.

[0077] 1-1: Teaching Methods for Stem Cells

[0078] To enhance the targeting ability to lung cancer cells, adipose-derived mesenchymal stem cells (AD-MSCs) and lung cancer cells (A549) were seeded at 5x10 per well in a 100π culture plate. 5 After dividing into individual cells, they were cultured at 37°C for 24 hours. In addition, to enhance the targeting ability to brain cancer cells, adipose-derived mesenchymal stem cells (AD-MSC) and brain cancer cells (U87MG) were cultured in a 100π culture plate at a density of 5x10 per well. 5 After dividing into individual cells, they were cultured at 37°C for 24 hours. Afterwards, the culture medium (medium) was obtained from the plate where the lung cancer cells were being cultured, and 10 ml of the medium was dispensed to each stem cell, and then cultured for an additional 24 hours (education).

[0079] 1-2: Isolation of ectosomes from educated stem cells

[0080] 10 μg / μl of cytochalasin B (5 μl) was added to the plate cultured with adipose-derived mesenchymal stem cells (AD-MSC) cultured with the above lung cancer cell or brain cancer cell culture medium, and the cells were separated and vortexed for 3 minutes. Subsequently, the cells were centrifuged at 1,000 rpm for 10 minutes using a centrifuge, the supernatant was collected, and the supernatant was removed by centrifugation at 4,000 rpm for 15 minutes to obtain an ectosome pellet, which was resuspended in 10 μl of DW and quantified using a nanodrop.

[0081] Example 2: Ecto-liposome fusion

[0082] 2-1: Preparation of liposome nanoparticles

[0083] Liposomes are biocompatible drug delivery vehicles composed of phospholipids. The inventors of the present invention synthesized liposomes using microfluidic technology. Specifically, the lipid phase was prepared by adding 18:1(Δ9-Cis) PC (DOPC) [CAS#: 850375P, Avanti], cholesterol [CAS#: C8667, Sigma], and 18:0 PEG2000 PE [CAS#: 880120, Avanti] to ethanol at a molar ratio of 60:35:5, followed by vortexing and sonication for 10 minutes. Subsequently, triple-distilled water was prepared for the aqua phase, and synthesis was performed at a flow rate of 8 ml / min for aqua and 0.8 ml / min for lipid using a microfluidic chip with a pore size of 5 μm. Afterwards, centrifugation was performed for 20 minutes at 4°C and 3000xg using a centrifuge through a 100KD filter (Merk, Amicon UFC910024 Amicon® Ultra-15 Centrifugal Filter Unit, 100K) to remove residual liposomes that were not nanoparticleized.

[0084] 2-2: Fabrication of ectosome-fused liposome nanoparticles

[0085] The ectosomes extracted through Example 1 and the liposome nanoparticles prepared through Example 2-1 were mixed at a mass ratio of 1:10 and sonicated for 10 minutes in a bath sonicator. Thereafter, 3 cycles were performed, each consisting of 10 minutes in liquid nitrogen and 10 minutes at 37°C incubation. Subsequently, centrifugation was performed at 4°C and 3000×g for 20 minutes using a 100KD filter (Merk, Amicon UFC910024 Amicon® Ultra-15 Centrifugal Filter Unit, 100K) to remove any remaining ecto-liposome fused nanoparticles that were not nanoparticleized. Transmission electron microscopy (TEM) and FRET assay were used to confirm whether the liposomes and ectosomes were properly fused (Fig. 2).

[0086] Example 3: Preparation of ecto-liposomes labeled with DID, DiI, and DiO

[0087] The inventors of the present invention, in order to prepare a fusion product in which a fluorescent dye DiI is attached to an ectosome, treated DiI at 5 μl / 1 ml before Example 1-2 and incubated for 15 minutes. Then, in order to attach a fluorescent dye DiD or DiO to the liposome, liposomes were synthesized as in Example 2-1, and then treated with a fluorescent dye DiD or DiO at 5 μl / 1 ml, followed by vortexing and sonication for 10 minutes. Subsequently, the mixture was filtered through a 100KD filter (Merk, Amicon UFC910024 Amicon ® The residual liposomes were removed by centrifugation at 4°C and 3000xg for 20 minutes using a centrifuge (Ultra-15 Centrifugal Filter Unit, 100K). Afterwards, fusion was confirmed using FRET Assay and Uptake (Fig. 6a and 6b).

[0088] Example 4: Polyacrylamide gel electrophoresis (SDS-PAGE)

[0089] The inventors of the present invention performed SDS-PAGE to confirm the fusion of the ectosome-liposome fusion nanoparticles prepared above. Specifically, proteins in the ectosome-liposome fusions were separated and purified from SDS-PAGE, and each liposome, ectosome, and ecto-liposome were mixed with lysis buffer and incubated on ice for 30 minutes. Thereafter, centrifugation was performed at 4°C and 3000xg for 20 minutes to obtain the supernatant, and the proteins were quantified through Coomassie quantification. Subsequently, 30 μg of the liposomes, ectosomes, and ecto-liposomes were mixed with 4X SDS sample buffer, incubated at 95°C for 5 minutes, and then incubated on ice for 5 minutes. Then, centrifugation was performed at 4℃, 13,000 rpm for 5 minutes to obtain the supernatant, and electrophoresis (SDS-PAGE) was performed by loading the gel at 5 mV for 20 minutes. After that, staining with Coomassie blue was performed for 1 hour, destaining was performed for 30 minutes to remove the remaining staining reagent, and protein bands were confirmed through scanning. As a result, protein bands were not observed in liposomes, and protein bands with similar shapes were confirmed in ectosomes and ecto-liposomes, confirming ecto-liposome fusion (Fig. 5).

[0090] Experimental Example 1: Physical and chemical properties

[0091] The size distribution and potential of the ecto-liposome fusion nanoparticles of the present invention were analyzed using a nanoparticle size analyzer (Anton Paar LiteSizer 500), and NTA analysis was performed using Nanosight NS300 (Nanosight, UK). As a result, as shown in FIGS. 3 and 4, liposomes exhibited a size distribution of an average of 50 nm, ectosomes an average of 184 nm, and ectosome-liposome fusion nanoparticles (Ecto-Liposome) an average of 90 nm. In addition, liposomes exhibited a charge of -1 mV, ectosomes an average of -7.3 mV, and ectosome-liposome fusion nanoparticles an average of -2.38 mV. The above NTA analysis confirmed that the ectosome-liposome fusion nanoparticles were a substance with a size of 121.9±2.7 nm and a particle count of 3.21e+08±1.88e+07 per ml.

[0092] Experimental Example 2: Analysis of the absorption capacity of ecto-liposome fusion nanoparticles.

[0093] The inventors of the present invention investigated the uptake ability of the ectosome-liposome fusion nanoparticles of the present invention toward tumor cells. Specifically, lung cancer cells (A549) were seeded (5 x 10) on poly-d-lysine-coated cover slips. 4Cells) and cultured overnight. Thereafter, DiO-labeled liposome nanoparticles, DiI-labeled ectosomes isolated from lung cancer cells, and DiI-labeled ectosome-liposome fusion nanoparticles isolated from lung cancer cells (Ecto-Liposome) were added to the cultured cells, and cultured at 37°C for 6 hours. Afterwards, all of the above samples were fixed overnight with 4% paraformaldehyde in medium at 4°C, and DiO and DiI fluorescence within the tumor cells was photographed using an LSM700 confocal microscope (Carl Zeiss). As a result, as shown in Fig. 7, only DiO fluorescence was observed in tumor cells treated with simple liposome nanoparticles, and only DiI fluorescence was observed in tumor cells treated with ectosomes, whereas both DiO and DiI fluorescence were observed inside tumor cells treated with ecto-liposome fusion nanoparticles.

[0094] Example 3: Analysis of tumor tissue targeting ability in a brain tumor model

[0095] The inventors of the present invention analyzed the tumor tissue targeting ability of the ecto-liposome fusion nanoparticles of the present invention using a brain tumor animal model. Specifically, the brain tumor animal model was a BALB / c nude mouse in which brain tumor cells genetically engineered to express fluorescent protein and bioluminescent enzyme (luciferase) (U87MG-luciferase-GFP, 3x10 5 The cells) were prepared by directly injecting them into the brain through surgery. After that, the size of the brain tumor was observed using a biofluorescence imaging device (IVIS imaging system), and after confirming that the brain tumor had formed, ecto-liposomes (1x10) extracted from the educated AD-MSCs were injected. 6 cells), educated AD-MSCs (1x10 6Cells), and liposomes were intravenously injected through the tail vein of the brain tumor model mice once a day for a total of 3 days (Fig. 8a). Then, on the 4th day, whole-body bioimaging was performed on the experimental animals, and after the experimental animals were sacrificed, the brains where tumors were formed were removed and the targeting ability of the ecto-liposomes to tumor tissues was analyzed through ex vivo imaging.

[0096] As a result, as shown in Fig. 8a, it was confirmed that the targeting ability to brain tumor tissue was enhanced in the case of ecto-liposomes trained by treating the culture medium of brain cancer cells. The above results were achieved by intravenously injecting stem cells into experimental animals, and it was proven that stem cells accurately migrate through the blood-brain barrier to brain tumors. This suggests that this provides an important clue to solving the technical difficulty in brain tumor treatment, in which anticancer treatment was limited due to the existence of the blood-brain barrier.

[0097] Example 4: Analysis of tumor tissue targeting ability in a lung cancer model

[0098] The inventors of the present invention also analyzed the tumor tissue targeting ability of the ecto-liposome fusion nanoparticles of the present invention using a lung cancer animal model. Specifically, the lung cancer animal model was a BALB / c nude mouse in which lung cancer cells genetically engineered to express fluorescent protein and bioluminescent enzyme (luciferase) (A549-luciferase-RFP, 1x10 6 ) was prepared by injecting it through the tail vein. After that, the size of lung cancer was observed for 4 weeks using a biofluorescence imaging device (IVIS imaging system) (Fig. 8b), and after confirming that lung cancer was formed, ecto-liposomes (1x10) extracted from the educated AD-MSCs were injected. 6 cells), educated AD-MSCs (1x10 6Cells) and liposomes were intravenously injected once through the tail vein of the lung cancer model mice. After 6 hours, whole-body bioimaging was performed on the experimental animals, and after the experimental animals were sacrificed, the lungs where tumors were formed were removed and the targeting ability of the ecto-liposomes to tumor tissues was analyzed through ex vivo imaging.

[0099] As a result, as shown in Figure 8b, the targeting ability to tumor tissue was enhanced in the case of educated ecto-liposomes treated with lung cancer cell culture medium. These results suggest that educated stem cells can be used very effectively for cancer cell-specific drug delivery, particularly for lung cancer, a representative intractable malignant tumor.

[0100] While the present invention has been described with reference to the above-described embodiments, these are merely exemplary, and those skilled in the art will understand that various modifications and equivalent alternative embodiments are possible. Therefore, the true scope of technical protection of the present invention should be determined by the technical spirit of the appended claims.

[0101] (National Research and Development Project that supported this invention)

[0102] (Project ID) 2022398018

[0103] (Task Number) 2022M3A9G8018189

[0104] (Ministry Name) Ministry of Science and ICT

[0105] (Name of Project Management (Specialist) Institution) National Research Foundation of Korea

[0106] (Research Project Name) Bio-Medical Technology Development Project (Bio-Source Convergence Technology Development)

[0107] (Research Project Name) Development of a Composite Tumor Targeting Source Technology Using Patient-Specific Ecto-Liposomes

[0108] (Contribution rate) 1 / 1

[0109] (Name of the project performing organization) Gachon University Industry-Academic Cooperation Foundation

[0110] (Research Period) January 1, 2023 - December 31, 2023

Claims

1. A fusion nanoparticle formed by fusing stem cell-derived ectosomes and liposomes.

2. In paragraph 1, The above stem cells are embryonic stem cells, mesenchymal stem cells or induced pluripotent stem cells, fusion nanoparticles.

3. In paragraph 2, The above mesenchymal stem cells are fusion nanoparticles that are bone marrow-derived stem cells, umbilical cord blood-derived stem cells, adipose-derived stem cells, dental pulp-derived stem cells, or peripheral blood-derived stem cells.

4. In paragraph 1, The above stem cells are fusion nanoparticles that are stem cells educated from lesion-derived cells.

5. In paragraph 4, The above lesion is a tumor tissue, a fusion nanoparticle.

6. In paragraph 5, The above tumor tissue is primary or metastatic, fusion nanoparticle.

7. In paragraph 4, The above-mentioned educated stem cells are cultured stem cells that are in contact with the culture medium in which the above-mentioned lesion-derived cells are cultured, and are fusion nanoparticles.

8. In paragraph 1, A fused nanoparticle having a diameter of 100 to 350 nm.

9. In paragraph 1, A fusion nanoparticle manufactured by mixing the above ectosome and liposome at a mass ratio of 1:

20.

10. A drug delivery system comprising a fusion nanoparticle of any one of claims 1 to 9 as an active ingredient.

11. A pharmaceutical composition comprising the drug delivery system of Article 10 and an effective drug.

12. In paragraph 10, A pharmaceutical composition wherein the above effective drug is an anticancer agent.

13. In paragraph 12, The above anticancer drugs include mechloethamine, chlorambucil, phenylalanine, mustard, cyclophosphamide, ifosfamide, carmustine (BCNU), lomustine (CCNU), streptozotocin, busulfan, thiotepa, cisplatin, carboplatin, doxorubicin, daunorubicin, idarubicin, mitoxantrone, plicamycin, mitomycin, C bleomycin, vincristine, vinblastine, A pharmaceutical composition selected from the group consisting of paclitaxel, docetaxel, etoposide, teniposide, topotecan and iridotecan.

14. A pharmaceutical composition for treating cancer, comprising, as an active ingredient, a fused nanoparticle formed by fusing a stem cell-derived ectosome with a liposome loaded with an anticancer agent.

15. In paragraph 14, A pharmaceutical composition wherein the above stem cells are educated stem cells that have been trained to become cancer cells to be treated.

16. In paragraph 14, A pharmaceutical composition wherein the anticancer agent is loaded by being bound to the surface or interior of the fused nanoparticle by covalent or non-covalent bonding or by being encapsulated inside the nanoparticle having a core-shell structure.

17. In paragraph 14, A pharmaceutical composition wherein the cancer is selected from the group consisting of brain tumor, lung cancer, stomach cancer, liver cancer, bone cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, skin cancer, head and neck cancer, skin melanoma, uterine cancer, ovarian cancer, rectal cancer, colon cancer, colon cancer, breast cancer, uterine sarcoma, esophageal cancer, laryngeal cancer, small intestine cancer, and thyroid cancer.

18. A step of isolating ectosomes from stem cells cultured in a culture medium of tumor tissue; and A method for producing a fusion nanoparticle having a targeting ability for tumor tissue, comprising a step of mixing the ectosome and liposome nanoparticles and fusing the ectosome and liposome through ultrasonic treatment.

19. In paragraph 18, A manufacturing method in which the above ectosome and liposome nanoparticles are mixed at a mass ratio of 1:

20.

20. Use of a fusion nanoparticle formed by fusing a stem cell-derived ectosome with a liposome loaded with an anticancer agent inside for the manufacture of a medicine for preventing and treating cancer.

21. A method for treating cancer in a subject, comprising administering to the subject a fused nanoparticle formed by fusing a therapeutically effective amount of stem cell-derived ectosome with a liposome loaded with an anticancer agent therein.

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