Stem cell-derived NANO anticancer drug delivery system having improved targeting ability by using dexamethasone

Abstract

The present invention provides stem cell-derived membrane vesicle-liposome fusion nanoparticles and a use thereof, the nanoparticles exhibiting tumor site targeting ability and anticancer efficacy superior to those of a conventional drug delivery system having targeting ability.

Description

Stem cell-derived nano-anticancer drug delivery system with enhanced targeting ability by dexamethasone

[0001] The present invention relates to stem cell-derived membrane vesicle-liposome fusion nanoparticles, and more particularly, to stem cell-derived membrane vesicle-liposome fusion nanoparticles with enhanced targeting ability to a lesion and their uses.

[0002] In 2018, lung cancer accounted for 11.6% of all cancer incidence and 18.4% of cancer-related deaths worldwide, surpassing other cancer types in mortality (Chen, Y. et al., PLoS One, 14(7), e0220610. 2019). Non-small cell lung cancer (NSCLC) accounts for the majority of lung cancer cases, accounting for 85%, while small cell lung cancer occurs in a minority of patients. Brain metastases are found in 10% to 20% of NSCLC patients at diagnosis, and approximately 40% of this population will experience the onset of brain metastases throughout their disease course (Sung, H., et al., Cancer. J. Clin. 2021. 71(3), 209-249. 2021). The lack of chemotherapy regimens that specifically target lung cancer tissue remains a major challenge in lung cancer treatment. This often leads to excessive nonspecific drug administration and subsequent adverse effects (Huang, CY et al., Biomedicine, 7(4), 23. 2017). To address this issue, the development of drug delivery systems capable of delivering drugs targeting lung cancer is essential. Brain metastases from metastatic lung cancer exhibit more prominent genetic mutations than primary lung cancer (Kang, Y. et al., Front. Oncol, 10, 606300. 2020). The presence of the blood-brain barrier (BBB) ​​protects these metastases, restricting the passage of existing chemotherapeutic drugs across the BBB and resulting in a poor prognosis. To address these issues, active research is underway to develop nanomaterial-based drug delivery systems. Within this category, lipid nanoparticles (LNPs) are recognized as optimal carriers for nucleic acids due to their favorable properties such as biocompatibility and efficient encapsulation (Akinc, A.et al., Nat. Nanotechnol,14(12), 1084-1087. 2019).Specifically, liposomes are composed of an amphipathic phospholipid bilayer encapsulating an inner aqueous core, and the core-shell nanostructure of liposomes is suitable for transporting both hydrophobic and hydrophilic molecules (Nsairat, H, et al., Heliyon, 8(5), e09394. 2022). However, the lipid-based nanoparticles developed above have several problems, including rapid immune system clearance and lack of cell-specific targeting.

[0003] The present invention aims to solve various problems, including those described above, by providing stem cell-derived membrane vesicle-liposome fusion nanoparticles that exhibit superior tumor targeting ability and anticancer efficacy compared to conventional drug delivery systems, and their uses. However, these tasks are exemplary and the scope of the present invention is not limited thereby.

[0004] According to one aspect of the present invention, a fusion nanoparticle is provided, which is formed by fusing stem cell-derived membrane vesicles and liposomes cultured by treating with an anti-inflammatory agent.

[0005] According to another aspect of the present invention, a drug delivery vehicle comprising the fusion nanoparticle as an active ingredient is provided.

[0006] According to another aspect of the present invention, a pharmaceutical composition comprising the drug delivery vehicle and an effective drug is provided.

[0007] 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 in which a membrane vesicle derived from a stem cell cultured by treating an anti-inflammatory agent is fused with a liposome loaded with an anticancer agent therein.

[0008] According to another aspect of the present invention, there is provided a step of isolating membrane vesicles from cultured stem cells by treating them with an anti-inflammatory agent; and

[0009] A method for producing fusion nanoparticles with enhanced targeting ability for tumor tissue is provided, which comprises a step of mixing the membrane vesicles and liposome nanoparticles and fusing the membrane vesicles and liposomes through ultrasonic treatment.

[0010] According to another aspect of the present invention, there is provided a use for manufacturing a medicine for preventing and treating cancer by using a composite fusion nanoparticle having an anticancer agent loaded on the surface or inside of the fusion nanoparticle.

[0011] According to another aspect of the present invention, a method for treating cancer in a subject is provided, comprising administering to the subject a therapeutically effective amount of a composite fusion nanoparticle having an anticancer agent loaded on the surface or interior of the fusion nanoparticle.

[0012] According to another aspect of the present invention, a method for improving the mobility of stem cells is provided, comprising the step of culturing the stem cells in a culture medium containing an anti-inflammatory agent under in vitro conditions.

[0013] According to another aspect of the present invention, there is provided a method for producing a method comprising: culturing stem cells in a culture medium containing an anti-inflammatory agent under in vitro conditions; and

[0014] A method for producing stem cell membrane vesicles with enhanced ability to migrate to tumor tissue is provided, comprising a step of isolating membrane vesicles from the stem cells.

[0015] As described above, the stem cell-derived membrane vesicle-liposome fusion nanoparticles of the present invention exhibit enhanced targeting ability toward tumor sites, which are target diseases. Therefore, they can be utilized as drug delivery vehicles for systemic or local drug release by loading drugs such as anticancer agents, and can be used as therapeutic agents for the treatment of intractable cancers or metastatic tumors. Of course, the scope of the present invention is not limited by these effects.

[0016] Figure 1a is a schematic diagram schematically illustrating a transwell migration assay experiment of educated stem cells (MSCs).

[0017] Figure 1b is a fluorescence micrograph showing the lung cancer cell targeting ability observed through stem cell education.

[0018] Figure 1c is a graph showing the results of analyzing the targeting ability of A549 lung cancer cells through stem cell education and dexamethasone treatment. Data are expressed as mean ± standard error of the mean (SEM) and significance level is **** p<0.0001 is indicated.

[0019] Figure 2a is a schematic diagram schematically showing the chemical structure of liposomes for optimization of liposomes.

[0020] Figure 2b is a schematic diagram showing the stem-liposome optimization parameters.

[0021] Figure 3a is a schematic diagram that analyzes the physicochemical properties of Stem-liposome and schematically shows the manufacturing process of Stem-liposome.

[0022] Figure 3b is a graph showing the results of analyzing the hydrodynamic size distribution of liposomes, Stem-liposomes, Edustem-liposomes, and Dexastem-liposomes.

[0023] Figure 3c is a graph showing the results of analyzing the average hydrodynamic size and zeta potential.

[0024] Figure 4a is a schematic diagram schematically showing the FRET analysis process for FRET analysis of stem-liposome.

[0025] Figure 4b is a graph showing the results of confirming the fusion of stemsomes and liposomes by FRET analysis. DII Ex: 549 / Em: 565. Data are expressed as mean ± standard error of the mean (SEM) and significance level is **** p<0.0001 is indicated.

[0026] Figure 4c is a graph showing the results of confirming the fusion of stemsomes and liposomes by FRET analysis. DID Ex: 664 / Em: 665. Data are expressed as mean ± standard error of the mean (SEM) and significance level is **** p<0.0001 is indicated.

[0027] Figure 4d is a graph showing the results of confirming the fusion of stemsomes and liposomes by FRET analysis. DII / DID Ex: 549 / Em: 665. Data are expressed as mean ± standard error of the mean (SEM) and significance level is **** p<0.0001 is indicated.

[0028] Figure 5a is a schematic diagram showing the structure of CD90, which serves as a marker for stemsomes in immuno-TEM measurements, and explaining the interaction of gold nanoparticles with anti-CD90 antibodies, with respect to EDS mapping of stem-liposomes.

[0029] Figure 5b is an EDS mapping image of Stem-liposome, which is a mapping image showing the structure of Stem-liposome by scanning transmission electron microscopy (STEM) image and electron dispersive spectroscopy (EDS).

[0030] Figure 6a is a schematic diagram showing the physicochemical properties of Stem-lipo-DOX and schematically showing the manufacturing process of stem-lipo-DOX.

[0031] Figure 6b is a graph showing the hydrodynamic size distributions of lipo-DOX, stem-lipo-DOX, Edustem-lipo-DOX, and Dexastem-lipo-DOX.

[0032] Figure 6c is a graph showing the results of the average hydrodynamic size and zeta potential analysis of lipo-DOX, stem-lipo-DOX, Edustem-lipo-DOX, and Dexastem-lipo-DOX.

[0033] Figure 7a is a confocal microscope image observing the degree of uptake of drug-treated A549 lung cancer cells.

[0034] Figure 7b shows fluorescence microscopy images of A549 cells after drug uptake, using confocal analysis to precisely identify uptake channels using various uptake channel inhibitors. All uptake pathways were identified, with microvesicle action emerging as the predominant pathway.

[0035] Figure 7c is a graph showing the results of FACS analysis of A549 cells treated with drugs (left) and the results of analysis of drug uptake levels (right). Data are expressed as mean ± standard error of the mean (SEM), and significance levels are ** p< 0.01, **** p<0.0001 is indicated.

[0036] Figure 7d is a graph showing the results of FACS analysis using EIPA, an absorption channel inhibitor, in drug-treated A549 cells (left) and the results of analyzing drug absorption levels (right).

[0037] Figure 7e is a graph showing the results of an analysis using GEN (Caveolin), an absorption channel inhibitor, in drug-treated A549 cells (left) and the results of an analysis of drug absorption levels (right).

[0038] Figure 7f is a graph showing the results of an analysis using CPZ, an absorption channel inhibitor, in drug-treated A549 cells (left) and the results of an analysis of drug absorption levels (right).

[0039] Figure 8a shows Free DOX, lipo-DOX, stem-lipo-DOX, in A549 cells. Edu This graph shows the results of cell viability analysis using the CCK8 assay after 48 hours of treatment with stem-lipo-DOX and Dexastem-lipo-DOX. Data are expressed as mean ± standard error of the mean (SEM) and significance level is ** p< 0.01, **** p<0.0001 is indicated.

[0040] Figure 8b shows Free DOX, lipo-DOX, stem-lipo-DOX, for 48 hours. Edu stem-lipo-DOX, and Dexa This graph shows the results of cell death analysis using annexin V and fluorescence-activated cell sorting (FACS) after treatment with stem-lipo-DOX.

[0041] Figure 9a is a schematic diagram schematically showing the experimental plan for intravenous injection of stem-liposomes for biodistribution analysis in a lung cancer xenograft model.

[0042] Figure 9b is a bioluminescence imaging (BLI) image after intravenous injection of stem-liposome, showing biodistribution analysis in a lung cancer xenograft model.

[0043] Figure 9c is a graph showing the results of analyzing the fluorescence intensity after intravenous injection of stem-liposome in a lung cancer xenograft model for biodistribution analysis. Data are expressed as mean ± standard error of the mean (SEM) and significance level is *** p< 0.001, **** p<0.0001 is indicated.

[0044] Figure 10a is a schematic diagram schematically showing an experimental plan for intravenous injection of stem-lipo-DOX to analyze targeting efficacy in a lung cancer orthotopic model.

[0045] Figure 10b is a bioluminescence imaging (BLI) image after intravenous injection of stem-lipo-DOX, analyzing the targeting efficacy in a lung cancer orthotopic model.

[0046] Figure 10c is a graph showing the results of analyzing the fluorescence intensity after intravenous injection of stem-lipo-DOX to analyze the targeting efficacy in a lung cancer orthotopic model. Data are expressed as the mean ± standard error of the mean (SEM) and the significance level is ** p< 0.01 is indicated.

[0047] Definition of terms:

[0048] The term "stemsome" used in this document refers to stem cell-derived membrane vesicles used as nanodrug delivery vehicles. Stemsomes contain numerous ligands that bind to unknown targeting factors (receptors) on the membranes of specific cancer cells.

[0049] 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.

[0050] The term "drug delivery carrier" used in this document refers to a material used to deliver a drug to the required lesion and maintain it for an appropriate period of time, and a method of effectively delivering a drug to the lesion using a drug delivery carrier is called a drug delivery system.

[0051] The term "nanoparticle" as used in this document refers to a particle having a size of less than a micrometer, ranging from several to several hundred nanometers (nm). Nanoparticles can be formed from various materials such as metals, phospholipids, biodegradable polymers, carbon nanotubes, fullerenes, carbon nanodots, etc., or a complex of two or more of these. The nanoparticles may have their surfaces coated with biocompatible materials, or may have stem cell surface marker-specific antibodies attached to them for binding to stem cells. In addition, drug complexes may be formed in which drugs are encapsulated inside or attached to the surface by covalent / non-covalent bonds in various ways.

[0052] Detailed description of the invention:

[0053] According to one aspect of the present invention, a fusion nanoparticle is provided, which is formed by fusing stem cell-derived membrane vesicles and liposomes cultured by treating with an anti-inflammatory agent.

[0054] In the above fusion nanoparticle, it may be a spherical vesicle structure made of a lipid bilayer.

[0055] 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.

[0056] In the above fusion nanoparticles, the anti-inflammatory agent may be triamcinolone, hydrocortisone, prednisolone, betamethasone or dexamethasone and may have a size of 100 to 350 nm in diameter, and the membrane vesicles and liposomes may be prepared by mixing at a mass ratio of 1:5 to 1:30, but is not limited thereto.

[0057] According to another aspect of the present invention, a drug delivery vehicle comprising the fusion nanoparticle as an active ingredient is provided.

[0058] According to another aspect of the present invention, a pharmaceutical composition comprising the drug delivery vehicle and an effective drug is provided.

[0059] In the pharmaceutical composition, the active drug may be an anticancer agent, and the anticancer agent may be daunorubicin, doxorubicin, epirubicin, idarubicin, pixantrone, sabarubicin, or valrubicin.

[0060] 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 in which a membrane vesicle derived from a stem cell cultured by treating an anti-inflammatory agent is fused with a liposome loaded with an anticancer agent therein.

[0061] In the above pharmaceutical composition, the anticancer agent may be loaded in a manner such that it is bound to the surface of the fusion nanoparticle by covalent bond or non-covalent bond or is encapsulated inside the fusion nanoparticle.

[0062] In the pharmaceutical composition, the cancer may be a 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, or thyroid cancer.

[0063] According to another aspect of the present invention, there is provided a step of isolating membrane vesicles from cultured stem cells by treating them with an anti-inflammatory agent; and

[0064] A method for producing fusion nanoparticles with enhanced targeting ability for tumor tissue is provided, which comprises a step of mixing the membrane vesicles and liposome nanoparticles and fusing the membrane vesicles and liposomes through ultrasonic treatment.

[0065] In the above manufacturing method, the membrane vesicles and liposomes can be mixed at a mass ratio of 1:5 to 1:30.

[0066] According to another aspect of the present invention, there is provided a use for manufacturing a medicine for preventing and treating cancer by using a composite fusion nanoparticle having an anticancer agent loaded on the surface or inside of the fusion nanoparticle.

[0067] According to another aspect of the present invention, a method for treating cancer in a subject is provided, comprising administering to the subject a therapeutically effective amount of a composite fusion nanoparticle having an anticancer agent loaded on the surface or interior of the fusion nanoparticle.

[0068] According to another aspect of the present invention, a method for improving the mobility of stem cells is provided, comprising the step of culturing the stem cells in a culture medium containing an anti-inflammatory agent under in vitro conditions.

[0069] In the method for improving the above mobility, the stem cells may be embryonic stem cells, mesenchymal stem cells or induced pluripotent stem cells, 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, and the anti-inflammatory agent may be, but is not limited to, triamcinolone, hydrocortisone, prednisolone, betamethasone or dexamethasone.

[0070] According to another aspect of the present invention, there is provided a method for producing a method comprising: culturing stem cells in a culture medium containing an anti-inflammatory agent under in vitro conditions; and

[0071] A method for producing stem cell membrane vesicles with enhanced ability to migrate to tumor tissue is provided, comprising a step of isolating membrane vesicles from the stem cells.

[0072] In the above manufacturing method, the stem cells may be embryonic stem cells, mesenchymal stem cells or induced pluripotent stem cells, 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, and the anti-inflammatory agent may be, but is not limited to, triamcinolone, hydrocortisone, prednisolone, betamethasone or dexamethasone.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] Conventionally developed lipid-based nanoparticles have limitations, such as poor targeting ability. For example, biomimetic coatings composed of stem cells allow nanoparticles to target inflammation or tumors, and mesenchymal stem cells (MSCs) have the ability to focus on them (Ullah, M. et al., iScience, 15, 421-438, 2019). However, precise targeting of cancer cells is difficult, and there is a risk that mutated stem cells can enter the body and cause tumor growth. Therefore, the use of stem cell-derived nanoparticles is desirable to overcome the limitations of existing tumorigenicity and obtain the functional advantages of nanoparticles. The present invention attempts to overcome these limitations by combining the advantages of nanoparticles and stem cell membrane-derived nanovesicles. The development of stem cell membrane-derived nanovesicles maintains tumor-targeting ability while suppressing the potential toxicity associated with direct stem cell use.

[0079] As an alternative approach to enhance the targeting ability of stem cells to cancer cells, previous studies have demonstrated targeting cancer cell surface proteins by introducing antibodies specific for target proteins overexpressed on the stem cell membrane (Park, JY et al., Oncogene, 40(35), 5367-5378, 2021). Despite the enhanced efficacy observed in stem cell-targeting cancer cells through this process, the drawback is the need to generate and culture tumor tissue. To overcome this drawback, we propose a non-invasive technique using dexamethasone (Dexa) for universal tumor targeting. Dexa is frequently used in cancer treatment to alleviate chemotherapy-related side effects such as nausea, vomiting, swelling, and allergic reactions, and to alleviate symptoms associated with advanced cancer.

[0080] Recent studies have shown that Dexa enhances the therapeutic efficacy of various chemotherapeutic regimens in A549 cells (Srivastava, S. et al., Steroids, 198, 109269, 2023). This mechanism of Dexa treatment has been shown to mitigate the tumorigenicity of NSCLC cells by interfering with cell migration and invasion. However, concerns have been raised about its widespread application in solid tumors due to its dual effects. Long-term Dexa treatment induces irreversible senescence and alterations in cell cycle-related genes. Recent studies have shown that Dexa enhances lung metastasis of breast cancer by modulating the PI3K-SGK1-CTGF pathway (Zheng, Y. et al., Mol. Cancer. Ther, 11(12), 2621-32, 2012).

[0081] The present inventors developed a novel drug delivery system targeting lung cancer using stemsomes and liposomes treated with Dexa. Previous studies have demonstrated the ability of Dexa to enhance breast cancer cell migration, leading to the hypothesis that this evidence enhances stem cell migration. Experimental studies have confirmed that Dexa stimulates stem cell migration into cancer cells. Furthermore, the inventors encapsulated the established anticancer drug doxorubicin (DOX) into the system, suggesting a novel and precise treatment for both lung cancer and metastatic brain cancer.

[0082] Liposomes have been extensively studied as effective delivery vehicles for COVID-19 vaccines. However, their inability to selectively target pathophysiological sites compromises their suitability for various disease-targeting therapies. This limitation hinders their use in certain targeted therapeutic modalities. To address these limitations, developing innovative strategies to overcome the limitations of lipid-based nanoparticles is essential. Therefore, designing and optimizing smart drug delivery systems to enhance targeted treatment of metastatic cancer is crucial. To address this, the inventors of this study hybridized stem cell-derived membranes, called stemsomes, with liposomes to develop an anticancer drug complex with potent targeting capabilities.

[0083] The present inventors analyzed the physicochemical properties of a stemsome-liposome hybrid drug delivery system. The results strongly supported its ability to precisely target not only primary tumors but also metastatic tumors. Furthermore, it demonstrated excellent biocompatibility, enhancing in vitro and in vivo antitumor targeting efficacy. Notably, the complex of the present invention exhibited enhanced anticancer efficacy across a wide range of cancer stages, including primary and metastatic tumors. These results suggest a new paradigm shift in chemotherapy aimed at improving outcomes in metastatic cancer.

[0084] As used herein, the term "enhanced targeting to tumor tissue" means a statistically significant increase in targeting to tumor tissue compared to a control fusion nanoparticle (a fusion nanoparticle manufactured from stem cells that has not been treated with an anti-inflammatory agent). Increased targeting is achieved by either an increase in target migration rate or a greater proportion of the fusion nanoparticles recruited to the tumor tissue as a target tissue based on the administered dose.

[0085] 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.

[0086] Example 1: Cell culture

[0087] Human adipose-derived mesenchymal stem cells (MSC) obtained from CEFO (CEFO-ADMSC) AD ) were cultured using human mesenchymal stem cell growth medium (CEFOgro-MSC, CEFO) supplemented with appropriate additives. A549 non-small cell lung cancer (NSCLC) cell line (CCL-185, ATCC) was cultured in high glucose Dulbecco's modified Eagle's medium (11995-065, Gibco). The medium was supplemented with 10% fetal bovine serum (FBS) (16000-044, Gibco) and 1% antibiotics (10378016, Gibco), and cultures were maintained at 37°C in a 5% CO2 atmosphere.

[0088] Example 2: Analysis of tumor cell targeting ability

[0089] The present inventors have demonstrated that stem cells educated with cancer cell or inflammatory cell culture media exhibit enhanced targeting ability toward tumor or inflammatory tissues without using unnecessary processes such as genetic manipulation. They then fused ectosomes derived from the educated stem cells with liposomes, which are spherical vesicle structures composed of a lipid bilayer, to produce novel fusion nanoparticles (Korean Patent Publication No. 10-2023-0154426). Furthermore, the present inventors sought to investigate the effect of dexamethasone (Dexa), an anti-inflammatory agent commonly used to treat inflammatory diseases, on the migration of stem cells toward tumor cells.

[0090] 2-1: Teaching Methods for Stem Cells

[0091] To enhance the targeting ability to lung cancer cells, adipose-derived mesenchymal stem cells (MSCAD) and lung cancer cells (A549) were cultured in a 100π culture plate at a density of 5 × 10 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 cultured, and 10 ml of the medium was dispensed to the stem cells, and cultured for an additional 24 hours (MSCEdu). In addition, adipose-derived mesenchymal stem cells (MSCAD) were cultured in a 100π culture plate at a density of 5 × 10 per well. 5 After sowing the seeds individually, 200 nM dexamethasone and cell culture medium were added at 37°C and cultured for 24 hours (MSCDexa).

[0092] 2-2: Analysis of migration ability toward tumor cells

[0093] A549 cells were seeded at 5 × 10⁴ cells per well in a 24-well culture plate and cultured in 500 μl of medium for 24 h. Then, MSCAD, MSCEdu, and MSCDexa were added to 24-well transwell chambers (polycarbonate membrane, 8 μm pore size, #3422, Corning) at a density of 5 × 10⁴ cells each with 200 μl of medium (Fig. 1a). After 3 h of incubation, cells on the lower surface of the membrane were removed. The membranes were fixed with 100% methanol for 1 min at 4°C. The samples were then washed three times with 1x PBS and stained with DAPI for 30 min. In addition, imaging was performed using an Evos M7000 (High Power Field, HPF) at 1000x magnification, with 10 fields of view per sample (Fig. 1b). A549 cells that migrated through the membrane pores to target cells were quantified by calculating the average number of cells per high-power field (HPF), and the effect of education was compared with the results of dexamethasone treatment experiments. As a result, MSC Edu and MSC Dexa The group is MSC AD Compared to the control group, the ability to migrate toward cancer cells was found to be more than 10-fold enhanced (Figure 1c). These results suggest that dexamethasone treatment alone has the potential to significantly enhance the ability of MSCs to migrate toward cancer cells.

[0094] Example 3: Stemsome-liposome fusion

[0095] 3-1: Preparation of liposome nanoparticles

[0096] Liposomes are biocompatible drug delivery vehicles composed of phospholipids. The inventors of the present invention synthesized liposomes using microfluidic technology. Specifically, 18:1(Δ9-Cis) PC (DOPC) [CAS#: 850375P, Avanti], cholesterol [CAS#: C8667, Sigma], and 18:0 PEG2000 PE [CAS#: 880120, Avanti] were added to ethanol at a molar ratio of 60:35:5, and then vortexed and sonicated for 10 minutes to prepare the liposomes (Figs. 2a and 2b). Subsequently, triple distilled water was prepared for the aqueous phase and synthesized 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, a 100KD filter (Merk, Amicon UFC910024 Amicon ® The remaining liposomes that were not nanoparticleized were removed by centrifugation at 4°C and 4,500 rpm for 20 minutes using a centrifuge (Ultra-15 Centrifugal Filter Unit, 100K).

[0097] 3-2: Isolation of stemsomes from stem cells

[0098] Cytochalasin B (10 μg / μl) was added to 5 μl of adipose-derived mesenchymal stem cells (MSCAD, MSCEdu, MSCDexa) cultured on a plate, and the cells were detached 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 4,500 rpm for 15 minutes to obtain a stemsome pellet, which was resuspended in 10 μl of deionized water and quantified using a nanodrop.

[0099] 3-3: Manufacturing of stemsome-fused liposome nanoparticles

[0100] Stemsomes extracted through Example 3-2 and liposome nanoparticles prepared through Example 3-1 were mixed at a molar ratio of 1:10 and a volume ratio of 1:1. The mixture was sonicated for 10 minutes and then extruded 21 times using an Avanti Research™ Mini-Extruder to produce a homogeneous fusion (stemsome-liposome) (Fig. 3a). Subsequently, the mixture was filtered through a 100KD filter (Merk, Amicon UFC910024 Amicon ® The remaining stemsome-liposome fusion nanoparticles that were not nanoparticleized were removed by centrifugation at 4°C and 4,000 rpm for 20 minutes using an Ultra-15 Centrifugal Filter Unit, 100K, thereby producing stemsome-liposome fusion nanoparticles with a diameter of 80-100 nm. Transmission electron microscopy (TEM) and FRET assay were used to confirm whether liposomes and stemsomes were properly fused.

[0101] 3-4: Physicochemical properties of stemsome-liposome drugs

[0102] The size distribution and potential of the stemsome-liposome fusion nanoparticles of the present invention were analyzed using a nanoparticle size analyzer (Anton Paar LiteSizer 500). As a result, as shown in Fig. 3b, liposomes exhibited a size distribution of 50 nm on average, and stemsome-liposome fusion nanoparticles (Ecto-Liposomes) exhibited a size distribution of 110 nm on average. In addition, liposomes exhibited a charge of -1 mV, stemsomes exhibited a charge of -18.4 mV, and stemsome-liposome fusion nanoparticles exhibited a charge of -20 mV (Fig. 3c).

[0103] Example 4: Confirmation of fusion of stemsomes and liposomes using FRET analysis.

[0104] To confirm the fusion of the stemsome and liposome of the present invention, a FRET (Fluorescence Resonance Energy Transfer) analysis was performed. This analysis is based on the principle of measuring the intermolecular distance (within approximately 10 nm) based on fluorescence. FRET analysis is performed by measuring the energy generated when light emitted from a donor fluorophore is absorbed by an acceptor fluorophore within a distance of approximately 10 nm.

[0105] 4-1: Preparation of stemsome-liposomes labeled with DID, DiI, and DiO

[0106] The inventors of the present invention, in order to produce a fusion product in which a fluorescent dye DiI is attached to a stemsome, treated DiI at 5 μl / ml before Example 3-2 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 3-1, 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 100 KD filter (Merk, Amicon UFC910024 Amicon ®The remaining liposomes were removed by centrifugation at 4℃, 4,500 rpm for 20 minutes using a centrifuge (Ultra-15 Centrifugal Filter Unit, 100K). After that, fusion was confirmed using FRET Assay and Uptake. DiI absorbs light at 549 nm and emits at 565 nm, and DiD absorbs light at 644 nm and emits at 665 nm (Fig. 4a). As a result, the fluorescence of the stem-liposome group at 549 nm / 565 nm was found to decrease compared to the DiI-labeled stemsome group. On the other hand, the fluorescence intensity of the stem-liposome group at 549 nm / 665 nm increased compared to the DiI-labeled stemsome group and the DiD-labeled liposome group (Figs. 4b to 4d). The above results demonstrate that DiI and DiD molecules exist within a distance of approximately 10 nm, indicating successful fusion between stemsomes and liposomes.

[0107] Example 5: Confirmation of fusion of stemsomes and liposomes through EDS mapping analysis.

[0108] 5-1: Preparation of gold nanoparticle (AuNP)-labeled stemsome-liposomes

[0109] Fusion of stemsomes and liposomes was confirmed using an anti-CD90 antibody conjugated to streptavidin using a Streptavidin Conjugation Kit (ab102921, Abcam). Additionally, 5 nm gold nanoparticles (AuNP, 741949, Merck) were coated with biotin-poly(ethylene glycol)-SH for 24 h. First, the stemsome-liposome and liposome samples were reacted with anti-CD90-streptavidin at 4°C for 24 h. Afterwards, the AuNP-biotin was reacted at 20°C for 6 h, and unreacted AuNP-biotin was removed by centrifugation. The obtained samples were stained with a negative staining solution (P4006, Sigma) at a 1:1 ratio for 1 min, applied to a CF-200-Cu-50 grid, and dried. Energy-dispersive X-ray spectroscopy (EDS) mapping and scanning transmission electron microscopy (STEM) techniques were used to analyze stemsome-based nanodrugs at the atomic level. Gold nanoparticles conjugated with anti-CD90 were attached to the fusion of stemsomes and liposomes (Fig. 5a). Analysis revealed yellow gold particles in the stemsome-liposome fusions, which, together with the presence of green oxygen particles, indicated successful fusion of liposomes with stemsomes. In contrast, no gold particles were observed in liposomes that were not fused with stemsomes, and only green oxygen particles were observed (Fig. 5b). These results clearly demonstrate the successful fusion of stemsomes with liposomes.

[0110] Example 6: Physicochemical properties of stemsome-liposome-dox drug

[0111] The present inventors prepared a stemsome-liposome-DOX drug containing doxorubicin. Specifically, the process of loading doxorubicin (DOX) into the liposome nanoparticles prepared through Example 3-1 was performed according to the ammonium sulfate gradient method (Fritze, A. et al., Biochimica. et biophysica. acta, 1758.10, 1633-1640. 2006) (Fig. 6a). 1 mg / ml of DOX solution was added to the empty liposomes and incubated at 50°C for 30 minutes to load DOX into the liposomes. The liposome solution loaded with DOX (Lipo-DOX) was centrifuged at 4,500 rpm for 20 minutes at 4°C, and the Amicon ® Residual liposomes and free doxorubicin were removed using an Ultra 15 mL centrifugal filter. The size distribution and potential of the stemsome-liposome-dox fusion nanoparticles of the present invention were analyzed using a nanoparticle size analyzer (Anton Paar LiteSizer 500). As a result, as shown in Fig. 6b, liposome-dox showed an average size distribution of 70 nm, and stemsome-liposome-dox nanoparticles (Stem-Lipo-DOX) showed an average size distribution of 110 nm. In addition, liposome-dox showed a charge of -5 mV, stemsomes showed -18.4 mV, and stemsome-liposome-dox fusion nanoparticles showed a charge of -28 mV (Fig. 6c).

[0112] Example 7: Analysis of the absorption capacity of stemsome-liposome-dox nanoparticles

[0113] 7-1: Fluorescence analysis using poly-D-lysine coated cover glass

[0114] The inventors of the present invention investigated the tumor cell uptake ability of the stemsome-liposome-dox nanoparticles of the present invention. Specifically, poly-D-lysine coating was initiated by placing cover glasses in a 150 mm dish, treating them with 70% ethanol, and exposing them to ultraviolet (UV) light for 1 day. The cover glasses were then washed four times with distilled water (DW) for 5 minutes each. After washing, the cover glasses were transferred to a 24-well plate and exposed to UV until the PBS in the plate evaporated. The cover glasses were then stored at 4°C. The poly-D-lysine-coated cover glasses were placed in a 24-well plate and preincubated for 30 minutes with DMEM + 10% FBS + 1% penicillin medium. Subsequently, 1 × 10 5 Cells were plated and treated with endocytosis inhibitors (EIPA, CPZ, and GEN) at concentrations of 20 μM, 25 μM, and 200 μM, respectively, 1 h before drug treatment. After pretreatment, Free DOX, Lipo-DOX, Stem-lipo-DOX, Dexastem-lipo-DOX, and Edustem-lipo-DOX were treated at concentrations of 250 ng / ml each and incubated for 2 h. The medium was then removed, the cells were washed three times with 1X PBS, and fixed with 500 μl of 4% paraformaldehyde (PFA) at 4°C for 24 h. Afterwards, mounting solution containing DAPI was added, and the cover glass with the cells was mounted upside down. After incubation at room temperature for 30 min, GFP (DIO), RFP (DOX), and DAPI fluorescence intensities were confirmed using Evos M7000 (high-power field of view, HPF).

[0115] 7-2: Fluorescence analysis using flow cytometry

[0116] A549 cells were seeded at a density of 5 × 10 in 6-well plates. 5Cells were plated, and the next day, the endocytosis inhibitors ethylisopropylamiloride (EIPA, macropinocytosis inhibitor, 20 μM), chlorpromazine (CPZ, clathrin inhibitor, 25 μM), and genistein (GEN, caveolin inhibitor, 200 μM) were pretreated for 1 hour. Then, Free DOX, Lipo-DOX, Stem-lipo-DOX, Dexastem-lipo-DOX, and Edustem-lipo-DOX were treated at a concentration of 250 ng / ml each, and the cells were incubated for 2 hours. For cell harvesting, 200 μl of 1X TE was added, and the cells were isolated by incubating for 5 minutes. The isolated cells were transferred to a 15 ml conical tube containing the medium and centrifuged at 1500 rpm for 5 minutes. After removing the supernatant, the cells were resuspended in 1X Stain Buffer and transferred to a FACS tube for analysis using flow cytometry (FACS). As a result, the DOX fluorescence intensity in the cancer cell nucleus decreased after 4 hours in the DOX and Lipo-DOX groups, whereas the DOX fluorescence intensity in the cancer cell nucleus increased after 4 hours in the Dexastem-lipo-DOX and Edustem-lipo-DOX groups (Fig. 7a). In addition, to investigate the intracellular pathway of nanodrugs, lung cancer cells were treated with endocytosis inhibitors such as Chlorpromazine (CPZ, clathrin pathway), Ethylisopropylamylolide (EIPA, macropinocytosis pathway), and Genistein (GEN, caveolin pathway). As a result, the main intracellular uptake pathway of nanodrugs was identified as the endocytosis pathway via macropinocytosis (Figures 7b to 7f).

[0117] Experimental Example 8: Analysis of lung cancer cell death by stemsome-liposome-dox nanoparticles.

[0118] 8-1: Cell death analysis using the CCK-8 method

[0119] The present inventors investigated the tumor cell killing ability of Stemsome-Liposome-DOX nanoparticles. Specifically, this was performed using Cell Counting Kit-8 (CCK-8, Abcam). First, A549 cells were plated at a density of 5 × 10³ cells per well in a 96-well plate and cultured for 24 hours. After that, Free DOX, Lipo-DOX, Stem-lipo-DOX, Dexastem-lipo-DOX, and Edustem-lipo-DOX were added at concentrations of 0.125 μg / ml, 0.25 μg / ml, and 0.5 μg / ml, respectively, and cultured for 48 hours at 37°C and 5% CO₂ conditions. Afterwards, 10 μl of CCK-8 solution was added to each well and incubated for 2 hours at 37°C. The solution was removed and the absorbance was measured at 460 nm using a microplate reader (UVM 340, Biochrom). As a result, the anticancer effect was shown to be more than twice that of the single DOX group in the Dexastem-lipo-DOX and Edustem-lipo-DOX groups (Fig. 8a).

[0120] 8-2: Apoptosis analysis using flow cytometry (FACS)

[0121] A549 cells were seeded in 6-well plates at a density of 3 × 10 5After seeding at cell / ml, Free DOX, Lipo-DOX, Stem-lipo-DOX, Dexastem-lipo-DOX, and Edustem-lipo-DOX were added at a concentration of 0.5 μg / ml each and cultured at 37°C and 5% CO₂ for 48 h. After that, the culture medium was transferred to a 2 ml e-tube, and the cells were washed once with PBS, added 200 μl of 1X TE, and cultured for 5 minutes to detach them. The detached cells were transferred to a 2 ml e-tube together with the medium and centrifuged at 500 g for 5 minutes at 4°C. After removing the supernatant and washing once with PBS, the cells were centrifuged again at 500 g for 5 minutes at 4 °C, and 100 μl of FACS buffer (1X Annexin V binding buffer 95 μl + Annexin V 5 μl) was added to the pellet for flow cytometry (FACS), suspended, and reacted at room temperature for 10 minutes. After that, the reaction was stopped by adding Annexin V binding buffer (1 ml), centrifuged at 500 g for 5 minutes at 4 °C, the supernatant was removed, and the cells were resuspended in 1X staining buffer. Finally, the cells were transferred to a FACS tube and analyzed by flow cytometry (FACS). As a result, the anticancer effects were found to be more than doubled in the Dexastem-lipo-DOX and Edustem-lipo-DOX groups compared to the single DOX group (Fig. 8b).

[0122] Example 9: Analysis of tumor tissue targeting ability in a lung cancer xenograft model

[0123] The inventors of the present invention analyzed the tumor tissue targeting ability of the stemsome-liposome fusion nanoparticles of the present invention using a lung cancer xenograft model. Specifically, the lung cancer xenograft model was performed by inoculating lung tumor cells (A549-luciferase-GFP, 1 × 10) genetically engineered to express fluorescent protein and bioluminescent enzyme (luciferase) into BALB / c nude mice. 6) was prepared by subcutaneously injecting them into the dorsal area of ​​the mouse. Thereafter, the size of the lung tumor was observed using an in vivo fluorescence imaging device (IVIS imaging system), and after confirming the formation of the lung tumor, the stemsome-liposome nanodrug of the present invention was injected through the tail vein of the lung tumor model mouse twice a week for a total of 7 days, and BLI evaluation was performed on a weekly basis (Fig. 9a). After the experimental animals were sacrificed, the tumors were extracted, and the liposome signals in the cancer site were analyzed through ex vivo imaging to analyze the targeting ability of the stemsome-liposomes to the tumor tissue. As a result, Edustem-liposomes and Dexastem-liposomes showed the strongest signals compared to general liposomes and stemsome-liposomes (Figs. 9b and 9c). The above results clearly suggest that Edustem-liposomes and Dexastem-liposomes have excellent targeting abilities in cancer tissues.

[0124] Example 10: Analysis of tumor tissue targeting ability in an orthotopic lung cancer model

[0125] The present inventors analyzed the tumor tissue targeting ability of stemsome-liposome fusion nanoparticles using a lung cancer xenograft model. Specifically, the lung cancer xenograft model was formed by inoculating lung tumor cells (A549-luciferase-GFP, 1 × 10) genetically engineered to express fluorescent protein and bioluminescent enzyme (luciferase) into BALB / c nude mice. 6) was resuspended in 200 μl of phosphate-buffered saline solution (PBS) and intravenously injected. After that, the size of lung tumors was observed using an IVIS imaging system, and after confirming the formation of lung tumors, DOX-loaded nano-drugs were intravenously injected. To evaluate drug distribution, mice were sacrificed 6 hours after administration (Fig. 10a). As a result, the DOX signal in the lung tissues was significantly increased in the Edustem-liposome and Dexastem-liposome groups. This indicates an enhanced targeting ability compared to the Stem-liposome group without targeting ability (Figs. 10b and 10c). This strongly suggests that Education and Dexa treatment can each significantly enhance the cancer-targeting ability of Stemsome-liposomes.

[0126] 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.

[0127] This invention was supported by the National Research and Development Program (NRDP). This project has the unique number 1711168722. This project is led by the Ministry of Science and ICT (MSIT), a government agency of the Republic of Korea, and the National Research Foundation of Korea (NRF), a public institution, is participating as the specialized management organization. The research project focuses on biomedical technology development, and its goal is to develop a core technology for targeting complex tumors using patient-specific ecto-liposomes. This project has a contribution ratio of 1 / 1, and Gachon University is participating as the implementing organization. The research period is from April 1, 2022, to December 31, 2026.

[0128] This project has the unique number 2420014191 and the project number 00510113. This project is managed by the Ministry of SMEs and Startups, a government agency of the Republic of Korea, and the Korea Technology Information Promotion Agency, a public institution, is participating as the specialized management agency. The research project focuses on the Startup Growth Technology Development Project (Stepping Stone), and its goal is to discover novel stem cell-based pancreatic cancer targets and develop synthetic new drugs. This project has a contribution ratio of 1 / 1, and Gachon University is participating as the implementing agency. The research period is from October 1, 2024, to September 30, 2025.

Claims

1. A fusion nanoparticle formed by fusing stem cell-derived membrane vesicles and liposomes cultured by treating with an anti-inflammatory agent.

2. In paragraph 1, The above fusion nanoparticle is a spherical vesicle structure made of a lipid bilayer.

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

4. In paragraph 3, 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.

5. In paragraph 1, The above anti-inflammatory agent is triamcinolone, hydrocortisone, prednisolone, betamethasone or dexamethasone, fused nanoparticles.

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

7. In paragraph 1, A fusion nanoparticle prepared by mixing the above membrane vesicles and liposomes in a mass ratio of 1:5 to 1:

30.

8. A drug delivery system comprising a fusion nanoparticle of any one of claims 1 to 7 as an active ingredient.

9. A pharmaceutical composition comprising the drug delivery system of Article 8 and an effective drug.

10. In paragraph 9, A pharmaceutical composition wherein the above effective drug is an anticancer agent.

11. In paragraph 10, A pharmaceutical composition wherein the anticancer agent is daunorubicin, doxorubicin, epirubicin, idarubicin, pixantrone, sabarubicin, or valrubicin.

12. A pharmaceutical composition for treating cancer, comprising, as an active ingredient, a fused nanoparticle in which a membrane vesicle derived from stem cells cultured by treating an anti-inflammatory agent is fused with a liposome loaded with an anticancer agent.

13. In paragraph 12, A pharmaceutical composition wherein the anticancer agent is loaded in a manner such that it is bound to the surface of the fusion nanoparticle by covalent bonding or non-covalent bonding or is encapsulated inside the fusion nanoparticle.

14. In paragraph 12, The pharmaceutical composition wherein the cancer is a 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 or thyroid cancer.

15. A step of isolating membrane vesicles from cultured stem cells by treating them with an anti-inflammatory agent; and A method for producing fusion nanoparticles with enhanced targeting ability for tumor tissue, comprising the step of mixing the membrane vesicles and liposome nanoparticles and fusing the membrane vesicles and liposomes through ultrasonic treatment.

16. In paragraph 13, A manufacturing method wherein the above membrane vesicles and liposomes are mixed at a mass ratio of 1:5 to 1:

30.

17. Use of a composite fusion nanoparticle loaded with an anticancer agent on the surface or inside of any one of the fusion nanoparticles of clauses 1 to 7 for the manufacture of a medicine for preventing and treating cancer.

18. A method for treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of a composite fusion nanoparticle having an anticancer agent loaded on the surface or inside of the fusion nanoparticle of any one of claims 1 to 7.

19. A method for improving the mobility of stem cells, comprising a step of culturing the stem cells in a culture medium containing an anti-inflammatory agent under in vitro conditions.

20. In paragraph 19, A method wherein the above stem cells are embryonic stem cells, mesenchymal stem cells or induced pluripotent stem cells.

21. In paragraph 20, The above mesenchymal stem cells 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.

22. In paragraph 19, A method wherein the anti-inflammatory agent is triamcinolone, hydrocortisone, prednisolone, betamethasone or dexamethasone.

23. A step of culturing stem cells in a culture medium containing an anti-inflammatory agent under in vitro conditions; and A method for producing stem cell membrane vesicles with enhanced ability to migrate to tumor tissue, comprising a step of isolating membrane vesicles from the stem cells.

24. In paragraph 23, A method for producing the above stem cells, wherein the stem cells are embryonic stem cells, mesenchymal stem cells or induced pluripotent stem cells.

25. In paragraph 23, A manufacturing method wherein the above mesenchymal stem cells 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.

26. In paragraph 23, A method of manufacturing the anti-inflammatory agent, wherein the anti-inflammatory agent is triamcinolone, hydrocortisone, prednisolone, betamethasone or dexamethasone.

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