A method fo preparing an ectosome-PLGA complex whose targeting ability to lesions is enhanced

Abstract

The present invention relates to a method for manufacturing a novel ectosome-biodegradable polymer nanoparticle composite, and more specifically, provides a method for manufacturing an ectosome-biodegradable polymer nanoparticle composite with enhanced targeting ability against a lesion, comprising the steps of: manufacturing trained stem cells by training stem cells with lesion-derived cells; separating ectosomes from the trained stem cells; and mixing the ectosome with biodegradable polymer nanoparticles to manufacture biodegradable polymer nanoparticles coated with ectosomes.

Description

Technology Field

[0001] The present invention relates to a method for manufacturing a novel ectosome-biodegradable polymer nanoparticle composite, and more specifically, to a method for manufacturing an ectosome-biodegradable polymer nanoparticle composite with enhanced targeting ability for tumors and inflammatory sites. Background Technology

[0002] Ectosomes are vesicles of various sizes (diameter 0.1–1 mm) that budding directly from the plasma membrane and are secreted into the extracellular space. Unlike living cells, ectosomes possess the phospholipid phosphatidylserine on their surface. Although ectosomes are produced and secreted through the direct budding of the cell membrane, it is known that the cargo components contained within them are not identical to those of the parent cell and that the internal composition varies depending on the cell's state (Cocucci and Meldolesi, Curr. Biol . 21(23): R940-R940, 2011). Although ectosomes differ from exosomes—which are secreted extracellularly via the endosome → MVB pathway—in terms of their generation method and the substances contained within, they are known to be similar to exosomes in their interactions within the extracellular space and with target cells. 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] Related prior art includes Patent No. 2162727, which relates to an anticancer cell therapy composition comprising natural killer cells and extracellular vesicles derived from said cells. The problem to be solved

[0004] However, in the case of the aforementioned prior art, there is a problem in that it is unclear whether the extracellular vesicle is an ectosome or an exosome, and it has almost no anticancer therapeutic effect on its own.

[0005] The present invention aims to solve various problems, including those mentioned above, and provides a method for manufacturing a novel ectosome-derived drug delivery system having superior targeting ability for tumors and inflammatory sites. However, these problems are exemplary and do not limit the scope of the present invention. means of solving the problem

[0006] According to one aspect of the present invention, a step of producing trained stem cells by training stem cells with lesion-derived cells;

[0007] A step of isolating ectosomes from the above-mentioned trained stem cells; and

[0008] A method for manufacturing an ectosome-biodegradable polymer nanoparticle composite with enhanced targeting ability for lesions is provided, comprising the step of mixing the ectosome with biodegradable polymer nanoparticles to produce biodegradable polymer nanoparticles coated with the ectosome. Effects of the invention

[0009] The method for manufacturing a stem cell-derived ectosome-biodegradable polymer nanoparticle composite according to the present invention, as described above, can be utilized in the manufacture of drug delivery systems having enhanced targeting capabilities for lesions. Of course, the scope of the present invention is not limited by these effects. Brief explanation of the drawing

[0010] FIG. 1 is a schematic diagram illustrating the process of manufacturing an ectosome-biodegradable polymer (PLGA), which is a biodegradable polymer (PLGA) coated with ectosomes (nanovesicles) derived from adipose-derived mesenchymal stem cells (AD-MSC) targeted for pancreatic cancer or brain tumors according to one embodiment of the present invention. Figure 2 is a micrograph showing the results of confirming that ectosomes are well coated on PLGA using a transmission electron microscope (TME) after preparing an ectosome-biodegradable polymer (PLGA) coated with ectosomes (nanovesicles) isolated from tumor-targeting adipose-derived mesenchymal stem cells (AD-MSC) trained with tumor cells according to one embodiment of the present invention. Figure 3 is a series of graphs showing the results of analyzing the hydrodynamic sizes and electrical potentials of PLGA, PLGA-DID, AD-MSC-derived ectosomes, AD-MSC-derived ectosome-coated PLGA-DID, AD-MSC-derived ectosome-educated with brain tumor cells (U87MG), and AD-MSC-derived ectosome-coated PLGA-DID-educated with brain tumor cells (U87MG). Figure 4 is a graph showing the results of analyzing the fluorescence intensity of PLGA-DID at an excitation wavelength of 646 nm and an emission wavelength of 661 nm using a Victor 1420 Multilabel Counter (PerkinElmer, Waltham, MA, USA). Figure 5 is a series of fluorescence microscope images visualizing the uptake of PLGA (PLGA-DID) stained with DiO, a fluorescent substance in the GFP wavelength range, PLGA coated with ectosomes isolated from adipose-derived mesenchymal stem cells (AD-MSC) stained with DiO, a fluorescent substance in the GFP wavelength range, and PLGA coated with ectosomes extracted from AD-MSC stained with DID fluorescent substance, in order to evaluate the migration ability of ectosome-PLGA to pancreatic cancer cells (PANC-1) and brain tumor cells (U87MG). FIG. 6a is a photograph showing the results of whole-body in vivo fluorescence imaging of the in vivo distribution of a polymer (PLGA) coated with ectosomes (nanovesicles) isolated from tumor-targeting adipose-derived stem cells trained as tumor cells according to one embodiment of the present invention, after injecting the polymer into a pancreatic cancer cell (PANC-1) xenograft tumor model animal. Figure 6b is a series of photographs showing the distribution of cancer cells (top) and ectosome-PLGA (bottom) in tumor tissue extracted from the above experimental model using in vivo fluorescence imaging. Figure 7 is a graph showing the results of analyzing the hydrodynamic sizes and electrical potentials of PLGA, PLGA-DOX, and ectosome-coated PLGA-DOX isolated from AD-MSCs trained with pancreatic cancer cells (PANC-1). FIG. 8a is a photograph showing the results of whole-body in vivo fluorescence imaging of the in vivo distribution of a polymer (PLGA) coated with ectosomes (nanovesicles) isolated from tumor-targeting adipose-derived stem cells trained as tumor cells according to one embodiment of the present invention, after injecting the polymer into a brain cancer cell (U87MG) xenograft tumor model animal. Figure 8b is a series of photographs showing the distribution of cancer cells (top) and ectosome-PLGA (bottom) in tumor tissue extracted from the above experimental model using in vivo fluorescence imaging. Figure 9 is a photograph showing the results of whole-body in vivo fluorescence imaging of the in vivo distribution of a polymer (PLGA) coated with ectosomes (nanovesicles) isolated from trained tumor-targeting adipose-derived stem cells to evaluate the tumor-targeting ability and anticancer effect of an ectosome-PLGA-DOX complex according to one embodiment of the present invention, after injecting the polymer into pancreatic cancer cells (PANC-1) and brain cancer cells (U87MG) xenograft tumor model animals. FIG. 10a is a schematic diagram briefly illustrating an experimental method for evaluating the targeting ability of an educated stem cell-derived ectosome-PLGA nanoparticle complex to arthritis cells according to one embodiment of the present invention. FIG. 9b is a whole-body in vivo fluorescence imaging photograph (left) obtained to evaluate the targeting ability of an educated stem cell-derived ectosome-PLGA nanoparticle complex according to one embodiment of the present invention in an animal model of arthritis, a confocal microscope image (upper right) to evaluate the uptake ability in inflammatory synovial cells (FLSs) and inflammatory macrophages (J774), and a graph (lower right) showing the quantification of fluorescence intensity in the confocal microscope image. Specific details for implementing the invention

[0011] Definition of Terms:

[0012] The term "ectosome" as used in this document refers to a nanovesicle with a phospholipid membrane structure having a diameter of 100 to 500 nm, which is produced by the budding of a cell membrane (plasmamembrane), unlike an "exosome" among extracellular vesicles.

[0013] As used in this document, the term "biodegradable polymer" refers to a polymer that is physiologically harmless and degradable through enzymatic action within the body. Biodegradable polymers include natural polymers such as starch, chitin, cellulose, polyalginate, and collagen, as well as artificial polymers such as PLGA (poly(lactic-co-glycolic) acid), PGA (poly(glycolic acid)), PLA (poly(lactic acid)), PCL (poly(caprolactone)), and PHA (polyhydroxyalkanoate).

[0014] As used in this document, the term "drug delivery carrier" refers to a substance used to effectively deliver a drug to the required lesion and maintain it for an appropriate period of time, and the method of effectively delivering a drug to a lesion using a drug delivery carrier is called a drug delivery system.

[0015] Detailed description of the invention:

[0016] According to one aspect of the present invention, a step of producing trained stem cells by training stem cells with lesion-derived cells;

[0017] A step of isolating ectosomes from the above-mentioned trained stem cells; and

[0018] A method for manufacturing an ectosome-biodegradable polymer nanoparticle composite with enhanced targeting ability for lesions is provided, comprising the step of mixing the ectosome with biodegradable polymer nanoparticles to produce biodegradable polymer nanoparticles coated with the ectosome.

[0019] In the above manufacturing method, the stem cells may be embryonic stem cells, mesenchymal stem cells, or induced pluripotent stem cells.

[0020] In the above manufacturing method, the mesenchymal stem cells may be bone marrow-derived stem cells, umbilical cord blood-derived stem cells, adipose-derived stem cells, pulp-derived stem cells, or peripheral blood-derived stem cells.

[0021] In the above manufacturing method, the lesion-derived cells may be cancer cells or inflammatory cells.

[0022] In the above manufacturing method, the education can be performed by culturing the lesion-derived cells in contact with a culture medium in which they are being cultured.

[0023] In the above manufacturing method, the biodegradable polymer may be a natural biodegradable polymer or an artificial biodegradable polymer, and the natural biodegradable polymer may be starch, chitin, cellulose, polyalginate, or collagen, and the artificial biodegradable polymer may be PLGA (poly(lactic-co-glycolic) acid), PGA (poly(glycolic acid)), PLA (poly(lactic acid)), PCL (poly(caprolactone)), or PHA (polyhydroxyalkanoate).

[0024] In the above manufacturing method, the nanoparticles may have a diameter of 100 to 350 nm.

[0025] In the above manufacturing method, the nanoparticles may be loaded with an effective drug.

[0026] In the above manufacturing method, the active drug may be an anticancer agent or an anti-inflammatory agent.

[0027] The above anticancer agent is described exemplarily as follows:

[0028] (i) asparaginase;

[0029] (ii) Methotrexate;

[0030] (iii) Pyrimidine analogs

[0031] e.g., 5-fluorouracil, gemcitabine, and arabinosylcytosine;

[0032] (iv) hydroxyurea;

[0033] (v) Purine analogs

[0034] mercaptopurine and thioguanine;

[0035] (vi) alkylating agents

[0036] Nitrogen mustard and cyclosporamide;

[0037] (vii) Anthracyclines

[0038] Anthracycline, doxorubicin, daunorubicin, idarubicin, and actinomycin D;

[0039] (viii) Mitotic inhibitors

[0040] Vincristine and Taxol;

[0041] (ix) Anti-angiogenic agents

[0042] Antibodies specific to VEGF, combretastatin A4, fumagilin, herbimycin A, 2-methoxyestradiol, OGT 2115, TNP 470, tranilast, XRP44X, thalidomide, endostatin, salmosin, angiostatin, or the kringle domain of plasminogen or apolipoprotein;

[0043] (x) Intercalating agents

[0044] Carboplatin and cisplatin; and

[0045] (?) radionuclides (radionuclides)

[0046] 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.

[0047] In the above manufacturing method, the anti-inflammatory agent may be a glucocorticoid or a non-steroidal anti-inflammatory agent.

[0048] In the above manufacturing method, the glucocorticoid is hydrocortisone, hydrocortisone acetate, cortisone, cortisone acetate, thixocortol pivalate, hydrocortisone-17-valate, halomethasone, alclometasone dipropionate, betamethasone valerate, betamethasone dipropionate, prednicarbate, clobetasone-17-butyrate, clobetasol-17-propionate, and fluorocortolone Caproate (fluocortolone caproate), fluocortolone pivalate, fluprednidene acetate, prednisone, prednisolone, methylprednisolone, dexamethasone, dexamethasone sodium phosphate, betamethasone, bethamethasone sodium phosphate, fluocortolone, triamcinolone, triamcinolone acetonide, mometasone, amcinonide, desonide, Fluocinonide,It may be fluocinolone acetonide, hacinonide, beclomethasone, fludrocortisone acetate, hydrocortisone-17-butyrate, hydrocortisone-17-aceponate, hydrocortisone-17-buteprate, ciclesonide, or prednicarbate.

[0049] In the above manufacturing method, the non-steroidal anti-inflammatory agent may be a cyclooxygenase (COX) inhibitor. Additionally, the cyclooxygenase inhibitor may be a non-selective COX-1 / COX-2 inhibitor, a selective COX-1 inhibitor, or a selective COX-2 inhibitor, and the selective COX-2 inhibitor may be apricoxib, celecoxib, rofecoxib, parecoxib, lumiracoxib, etoricoxib, or firocoxib.

[0050] In addition, in the above manufacturing method, the active drug may be loaded onto the biodegradable polymer nanoparticles by means of covalent or non-covalent bonding on the surface or interior of the nanoparticles, or by encapsulation within the core-shell structured nanoparticles. In the case of loading by covalent bonding, the active drug may be covalently bonded to the terminal or side chain of the biodegradable polymer. Optionally, the active drug may also be loaded by means of covalent or non-covalent bonding to the surface of an ectosome membrane coated on the outside, thereby being presented on the surface.

[0051] The ectosome-biodegradable polymer nanoparticle complex derived from the trained stem cell produced by the above manufacturing method may vary depending on the type of affected area of ​​the patient, the site of application, the frequency of treatment, the treatment time, the formulation, the patient's condition, the type of adjuvant, etc. The amount used is not particularly limited, but may be 0.01 μg / kg / day to 10 mg / kg / day. The above daily amount may be administered once a day, divided into 2 to 3 doses per day at appropriate intervals, or administered intermittently at intervals of several days.

[0052] The drug delivery system produced by the manufacturing method of the present invention can be administered orally or parenterally along with a loaded active drug, and preferably, by parenteral administration, it can be administered via intravenous injection, subcutaneous injection, intraventricular injection, intracerebrospinal fluid injection, intramuscular injection, and intraperitoneal injection.

[0053] A pharmaceutical composition comprising a drug delivery system and an active drug prepared by the method of the present invention may further include a suitable carrier, excipient, and diluent commonly used in the preparation of pharmaceutical compositions. Additionally, solid or liquid formulation additives may be used in the preparation of pharmaceutical compositions. The formulation additives may be either organic or inorganic. Examples of excipients include lactose, sucrose, white sugar, glucose, cornstarch, starch, talc, sorbitol, crystalline cellulose, dextrin, kaolin, calcium carbonate, and silicon dioxide. Examples of binders include polyvinyl alcohol, polyvinyl ether, ethyl cellulose, methylcellulose, gum arabic, tragacanth, gelatin, shellac, hydroxypropyl cellulose, hydroxypropylmethylcellulose, calcium citrate, dextrin, and pectin. Examples of lubricants include magnesium stearate, talc, polyethylene glycol, silica, hydrogenated vegetable oil, etc. As coloring agents, any that are generally permitted to be added to pharmaceuticals may be used. These tablets and granules may be coated with sugar, gelatin, or other appropriate coatings as needed. Additionally, preservatives, antioxidants, etc., may be added as needed. Furthermore, if the pharmaceutical composition is a drug, it may additionally include one or more selected from fillers, anticoagulants, lubricants, humectants, fragrances, emulsifiers, or preservatives. Meanwhile, the formulation of the pharmaceutical composition of the present invention may be in a preferred form depending on the method of use, and it is particularly preferable to formulate it by adopting a method known in the art to provide rapid, sustained, or delayed release of the active ingredient after administration to mammals.Specific examples of dosage forms include plaques, granules, lotions, liniments, limonades, powders, syrups, liquids and solutions, aerosols, extracts, elixirs, fluid extracts, emulsions, suspensions, decoctions, infusions, tablets, suppositories, injections, spirits, cataplasmas, capsules, troches, tinctures, pastes, pills, soft or hard It may be any one of the gelatin capsules selected.

[0054] A pharmaceutical composition comprising a drug delivery system and an active drug prepared by the method of the present invention may further include components commonly used in formulations of pharmaceutical compositions, such as, for example, conventional auxiliary agents like stabilizers, solubilizers, and flavorings, and a carrier.

[0055] The above pharmaceutical composition may include various carriers suitable for direct injection into the affected area.

[0056] 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, methylcellulose, 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, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. Suitable pharmaceutically acceptable carriers and formulations are Remington's Pharmaceutical Sciences (19 th It is described in detail in Ed., 1995.

[0057] The inventors have previously identified through prior research that stem cells trained with culture media of cancer cells or inflammatory cells exhibit enhanced targeting ability against tumor tissues or inflammatory tissues. Based on this, they hypothesized that ectosomes secreted from such trained stem cells would also possess targeting ability against lesions such as tumor tissues or inflammatory tissues. To verify this hypothesis, ectosomes were isolated from trained stem cells and mixed with PLGA nanoparticles, a biodegradable polymer, to prepare an ectosome-biodegradable nanoparticle complex coated with an ectosome membrane (see Fig. 1), and the inventors investigated whether this nanoparticle complex effectively targets tumor tissues and inflammatory tissues. As a result, it was confirmed that the ectosome-biodegradable nanoparticle complex isolated from trained stem cells according to one embodiment of the present invention exhibited significantly higher tumor cell targeting ability compared to an ectosome-biodegradable polymer nanoparticle complex derived from untrained stem cells (see Figs. 5, 6, and 8). Furthermore, when an anticancer drug such as doxorubicin was loaded onto the above ectosome-biodegradable polymer nanoparticle composite and administered to a tumor model mouse, it was confirmed that tumor growth was significantly inhibited compared to the administration of the anticancer drug alone or the administration of biodegradable polymer nanoparticles loaded with the anticancer drug (see Fig. 9).

[0058] Furthermore, the inventors investigated whether the enhancement of the tumor cell targeting ability described above is similarly observed in other lesions, such as inflammatory tissues. Specifically, the inventors prepared an ectosome-PLGA nanoparticle complex by isolating ectosomes from adipose-derived stem cells trained with fibroblast-like synovial cells (FLSs) isolated from inflammatory tissues and coating them onto PLGA nanoparticles as described above. When this was administered to a collagen-induced arthritis model animal, it was confirmed that the ectosome-PLGA nanoparticle complex aggregated specifically in inflammatory tissues (see Fig. 10b).

[0059] These results suggest that the ectosome-biodegradable polymer nanoparticle composite derived from trained stem cells according to one embodiment of the present invention can be usefully used as a drug delivery vehicle for an effective drug against a lesion by increasing the targeting ability against the same type of lesion as the lesion-derived cells used for training.

[0060] The present invention will be described in more detail below 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.

[0061] Example 1: Isolation of ectosomes (nanovesicles) from adipose-derived mesenchymal stem cells (AD-MSCs) trained with cancer cells to enhance targeting ability

[0062] According to one embodiment of the present invention, ectosomes (nanovesicles) to be used in the production of a polymer (PLGA) coated with ectosomes (nanovesicles) derived from adipose-derived mesenchymal stem cells (ADMSC) that have enhanced targeting ability by being educated in a cancer cell medium were isolated.

[0063] 1-1: Teaching Methods for Stem Cells

[0064] To enhance the ability to target cancer cells, the inventors placed adipose-derived mesenchymal stem cells (AD-MSC), pancreatic cancer cells (PANC-1), and brain tumor cells (U87MG) in 100π culture plates at 5x10 wells per well. 4 After dispensing a few times, the cells were cultured at 37°C for 48 hours. Culture medium was collected from a plate in which pancreatic cancer cells or brain tumor cells were being cultured, and 5 ml was dispensed into the stem cells being cultured and cultured for an additional 24 hours.

[0065] 1-2: Isolation of ectosomes from trained stem cells

[0066] 5 μl of cytochalasin B 10 μg / μl was added to a plate containing adipose-derived mesenchymal stem cells (AD-MSCs) cultured in cancer cell culture medium. After detaching the cells, the plate was vortexed for 3 minutes. Centrifugation was performed at 1000 rpm for 10 minutes using a centrifuge to collect the supernatant, and centrifugation was performed at 4000 rpm for 15 minutes to remove the supernatant to obtain an ectosome pellet. This pellet was resuspended in 10 μl of DW and then quantified using a nanodrop.

[0067] Example 2: Ectosome-PLGA Coating

[0068] 2-1: Preparation of PLGA Nanoparticles

[0069] Polylactate-co-glycolate (PLGA) is a representative biodegradable polymer. To proceed with PLGA synthesis, a tip sonicator was operated at 30 Hz, and 1 ml of a PLGA solution prepared by dissolving the PLGA copolymer ([CAS#: 26780-50-7], 50:50 Carboxylated End Group (nominal), Lactel, Part #B6013-2P) in acetone at a concentration of 5 mg / ml was added dropwise to 3 ml of 1% PVA. The solvent, acetone, was removed by concentrating under vacuum for 10 minutes using a rotary concentrator. Subsequently, residual PLGA that did not form nanoparticles was removed by centrifuging at 4°C and 17,000 xg for 10 minutes to remove the supernatant. Then, the amount of generated PLGA nanoparticles was quantified using a spectrophotometer (UV-VIS).

[0070] 2-2: Fabrication of Ectosome-Coated PLGA Nanoparticles

[0071] The ectosomes extracted in Example 1 above and the PLGA nanoparticles prepared in Example 2-1 above were mixed in a 1:10 (mass ratio) ratio and sonicated in a bath sonicator for 5 minutes. Transmission electron microscopy (TEM) was used to confirm that the ectosomes were properly attached to the PLGA (Fig. 2).

[0072] Example 3: Preparation of DID,DIO-labeled ectosome-PLGA

[0073] To prepare a composite in which the fluorescent diene DiD or DIO is encapsulated in an ectosome-PLGA composite, the inventors operated a tip sonicator at 30 Hz, mixed DID (1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt, D7757, Thermo Fisher Scientific, USA) or DIO (3,3'-Dioctadecyloxacarbocyanine Perchlorate, D275, Thermo Fisher Scientific, USA) with 3 ml of 1% PVA, and then added 1 ml of PLGA with a concentration of 5 mg / ml dissolved in acetone dropwise. Subsequently, the solvent, acetone, was removed under vacuum for 10 minutes using a rotary concentrator. Then, centrifugation was performed using a centrifuge at 4°C and 17,000 xg for 10 minutes to remove the supernatant, thereby removing the remaining PLGA that was not nano-sized. After removal, the generated PLGA nanoparticles were quantified using a spectroscopic analyzer (UV-VIS). Then, the ectosomes separated in Example 1 were mixed with the DID or DIO encapsulated above in a 1:10 (mass ratio) ratio, and sonication was performed in a bath sonicator for 5 minutes. Then, the presence of fluorescence was checked using a Victor 1420 Multilabel Counter (PerkinElmer, Waltham, MA, USA) at an excitation wavelength of 646 nm and an emission wavelength of 661 nm. As a result, as confirmed in Figure 4, it was found that the PLGA encapsulated with DID or DIO had fluorescence properly attached.

[0074] Experimental Example 1: Physical and Chemical Properties

[0075] The size distribution and potential of the ectosome-PLGA were analyzed using a nanoparticle size analyzer (Anton Paar LiteSizer 500). As a result, as confirmed in Figure 3, the ectosome-PLGA nanoparticle composite of one embodiment of the present invention exhibited particle size and potential similar to simple PLGA nanoparticles. Interestingly, both the PLGA nanoparticles and the ectosome-coated PLGA nanoparticles are very similar in particle size to the ectosome itself.

[0076] Experimental Example 2: Analysis of the absorption capacity of the ectosome-PLGA nanoparticle complex into tumor cells

[0077] The inventors investigated the uptake ability of an ectosome-PLGA nanoparticle complex according to one embodiment of the present invention into tumor cells. Specifically, the inventors seeded brain tumor cells U87MG and pancreatic cancer cells PANC-1 onto a poly-d-lysine-coated coverslip (5 x 10 4 Cells were cultured overnight. Subsequently, 0.5 μg / mL of PLGA nanoparticles labeled with DIO, a fluorescent substance in the GFP wavelength range (PLGA), an ectosome-PLGA nanoparticle complex isolated from uneducated adipose-derived stem cells labeled with DIO (ADMSC-PLGA), and an ectosome-PLGA nanoparticle complex isolated from educated adipose-derived stem cells according to one embodiment of the present invention labeled with DIO were added to the cultured cells, and the cells were cultured at 37°C for 6 hours. After 6 hours, all samples were fixed overnight in 4% paraformaldehyde in an intermediate medium at 4°C, and DIO fluorescence within the tumor cells was captured using an LSM700 confocal microscope (Carl Zeiss). As a result, as confirmed in Figure 5, no fluorescence was detected inside tumor cells treated with simple PLGA nanoparticles, and fluorescence was observed inside tumor cells treated with a non-educated stem cell-derived ectosome-PLGA nanoparticle complex, but the intensity of the fluorescence was low compared to when treated with a different educated stem cell-derived ectosome-PLGA nanoparticle complex in one embodiment of the present invention.

[0078] Experimental Example 3: Analysis of Ectosome-PLGA Tumor Tissue Targeting Ability in a Pancreatic Cancer Model

[0079] According to one embodiment of the present invention in vivo The targeting ability of ectosome-PLGA was analyzed in animal models.

[0080] Specifically, pancreatic cancer cells (PANC-1-luciferase-GFP) genetically engineered to express fluorescent protein and bioluminescent enzyme (luciferase) in BALB / c nude mice were 1 x 10⁶ 6 A pancreatic cancer model was prepared by injecting individual units via the tail vein. Subsequently, the size of the pancreatic cancer was assessed using an in vivo fluorescence imaging system (IVIS) for 4 weeks to confirm cancer formation. Afterward, ectosome-PLGA (1x10⁶), isolated from AD-MSCs trained using pancreatic cancer cells, was used. 6 cells, 5 mg / kg), free AD-MSC (1x10 6 cells), trained AD-MSC (1x10 6 cells), PLGA (5 mg / kg) were each intravenously injected via the tail vein of the above-mentioned pancreatic cancer model mice once a day for a total of 3 days. On the 4th day, whole-body in vivo imaging was performed on the experimental animals, and after sacrificing the animals, the tumor-formed pancreas was excised and ex vivo ( ex vivo By performing imaging, the targeting ability of ectosome-PLGA to tumor tissue was analyzed (Figs. 6a and 6b).

[0081] As a result, as confirmed in Figures 6a and 6b, it was found that the targeting ability to tumor tissue was enhanced in the case of ectosome-PLGA trained by treating with a cancer cell culture medium, which suggests that stem cells trained according to one embodiment of the present invention can be used very efficiently for cancer cell-specific drug delivery, particularly for drug delivery to pancreatic cancer, a representative intractable malignant tumor.

[0082] Example 4: Preparation of Doxorubicin-Loaded Ectosome-PLGA

[0083] To prepare doxorubicin-loaded ectosome-PLGA, after operating a tip sonicator at 30 Hz, 1 ml of a PLGA solution prepared by dissolving a PLGA copolymer ([CAS#: 26780-50-7], 50:50 Carboxylated End Group (nominal), Lactel, Part #B6013-2P) in acetone at 5 mg / ml was added dropwise to 3 ml of a 1% PVA solution mixed with 600 μl of 1 mg / ml doxorubicin. Then, the solvent acetone was removed by concentrating under vacuum for 10 minutes using a rotary concentrator. Subsequently, residual PLGA that did not form nanoparticles was removed by centrifuging at 4°C and 17,000 xg for 10 minutes using a centrifuge to remove the supernatant. Then, the amount of PLGA nanoparticles produced was quantified using a spectrophotometer (UV-VIS), while the particle size and potential of the composite produced in the same manner as in Experimental Example 1 were analyzed. As a result, as confirmed in Figure 7, the doxorubicin-loaded trained stem cell-derived ectosome-PLGA nanoparticle complex also exhibited a particle size distribution and potential similar to PLGA nanoparticles or doxorubicin-loaded PLGA nanoparticles.

[0084] Experimental Example 4: Analysis of Ectosome-PLGA Tumor Tissue Targeting Ability in a Brain Tumor Model

[0085] According to one embodiment of the present invention in vivo The targeting ability of ectosome-PLGA was analyzed in animal models.

[0086] Specifically, brain tumor cells (U87MG-luciferase-GFP) genetically engineered to express fluorescent protein and bioluminescent enzyme (luciferase) were used in BALB / c nude mice at a rate of 3x10 5It was formed by directly injecting into the brain one by one via surgery. After confirming the formation of the brain tumor by verifying the size using an in vivo fluorescence imaging system (IVIS imaging system), ectosome-PLGA (1x10⁶) extracted from the trained AD-MSCs was used. 6 cells, 5 mg / kg), free AD-MSC (1x10 6 cells), trained AD-MSC (1x10 6 cells) and PLGA (5 mg / kg) were each intravenously injected via the tail vein of the above brain tumor model mice once a day for a total of 3 days. On the 4th day, whole-body in vivo imaging was performed on the experimental animals, and after sacrificing the animals, the brain cancer with formed tumors was excised and ex vivo ( ex vivo By performing imaging, the targeting ability of ectosome-PLGA to tumor tissue was analyzed.

[0087] As a result, as confirmed in Figures 8a and 8b, it was found that the targeting ability to brain tumor tissue was enhanced in the case of ectosome-PLGA trained by treating with cancer cell culture medium. This experimental result was achieved by injecting a biodegradable polymer nanoparticle complex containing stem cell-derived ectosome components, rather than stem cells, into experimental animals via intravenous injection. It demonstrates that the ectosome-polymer nanoparticle complex according to one embodiment of the present invention accurately moved to the brain tumor by passing through the blood-brain barrier, thereby providing a clue to solving a significant technical challenge in the treatment of brain tumors, where anticancer drug treatment has been limited due to the presence of the blood-brain barrier.

[0088] Experimental Example 5: Analysis of the Pancreatic Cancer Inhibitory Efficacy of Ectosome-PLGA

[0089] According to one embodiment of the present invention in vivo The antitumor efficacy of ectosome-PLGA nanoparticles was analyzed in an animal model.

[0090] Specifically, in vivoFluorescence imaging was used to confirm the cytotoxic efficacy of ectosome-PLGA against cancer cells in a fluorescent / luminescent PANC-1-luciferase-GFP tumor mouse model using the IVIS animal fluorescence imaging system. First, luciferase-GFP fluorescent pancreatic cancer cells (PANC-1) were injected into nude mice, and 4 weeks later, ectosome-PLGA (5 mg / kg) and PLGA-DOX (5 mg / kg) extracted from trained AD-MSCs were intravenously injected twice a week for 3 weeks, for a total of 6 times. After activating luciferase by injecting 150 mg / kg of luciferin IVly 1 hour prior to measurement, changes in the fluorescence of pancreatic cancer cells (PANC-1) were analyzed over time using a fluorescence imaging device.

[0091] As a result, as confirmed in Figure 9, the administration of ectosome-PLGA was found to effectively inhibit the production of pancreatic cancer cells expressing luciferase-GFP fluorescence.

[0092] Example 5: Isolation of ectosomes (nanovesicles) from adipose-derived mesenchymal stem cells (AD-MSCs) trained with inflammatory cells to enhance targeting ability

[0093] According to one embodiment of the present invention, ectosomes (nanovesicles) to be used in the production of a polymer (PLGA) coated with ectosomes (nanovesicles) derived from adipose-derived mesenchymal stem cells (ADMSC) that have been educated in an inflammatory cell medium to enhance their targeting ability for inflammatory sites were isolated.

[0094] 5-1: Methods of Teaching About Stem Cells

[0095] To enhance the ability to target inflammatory cells, the inventors placed adipose-derived mesenchymal stem cells (AD-MSC), inflammatory synovial cells (FLS), and inflammatory macrophages (J774) in 100π culture plates at 5 x 10 wells per well. 4 After dispensing a few times, they were cultured at 37°C for 48 hours. Culture medium was collected from the plate in which the inflammatory cells were being cultured, and 5 ml was dispensed into the stem cells being cultured and cultured for an additional 24 hours.

[0096] 5-2: Isolation of ectosomes from trained stem cells

[0097] 5 μl of cytochalasin B 10 μg / μl was added to a plate cultured with adipose-derived mesenchymal stem cells (AD-MSCs) cultured in an inflammatory cell culture medium. After detaching the cells, the plate was vortexed for 3 minutes. Centrifugation was performed at 1000 rpm for 10 minutes using a centrifuge to collect the supernatant, and centrifugation was performed at 4000 rpm for 15 minutes to remove the supernatant to obtain an ectosome pellet. This pellet was resuspended in 10 μl of DW and then quantified using a nanodrop.

[0098] Example 6: Ectosome-PLGA Coating

[0099] The coating of PLGA nanoparticles and DIO or DID-labeled PLGA nanoparticles on ectosomes isolated from inflammatory cell education adipose-derived stem cells isolated in Example 5 above was performed in the same manner as described in Example 2.

[0100] Experimental Example 6: Analysis of Ectosome-PLGA Uptake in FLSs and J774 Cells

[0101] The inventors investigated the uptake ability of an ectosome-PLGA nanoparticle complex according to one embodiment of the present invention into inflammatory cells. Specifically, the inventors seeded fibroblast-like synoviocytes (FLSs) and 774 cells onto a poly-d-lysine-coated coverslip (5 x 10⁶ 4Cells were cultured overnight. Subsequently, 0.5 μg / mL of PLGA nanoparticles (PLGA) encapsulated with DIO, a fluorescent substance in the GFP wavelength range, and an ectosome-PLGA nanoparticle complex isolated from trained adipose-derived stem cells according to one embodiment of the present invention, encapsulated with DIO, were added to the cells and cultured at 37°C for 6 hours. After 6 hours, all samples were fixed overnight in 4% paraformaldehyde in an intermediate medium at 4°C, and DIO fluorescence within the inflammatory cells was captured using an LSM700 confocal microscope (Carl Zeiss). As a result, as confirmed in the upper right corner of Fig. 10b, no fluorescence was detected inside the inflammatory cells treated with simple PLGA nanoparticles, while fluorescence was detected when treated with the ectosome-PLGA nanoparticle complex derived from trained stem cells according to one embodiment of the present invention.

[0102] Experimental Example 7: Formation of a CIA (collagen-induced arthritis) model

[0103] DBA / 1 mice (male, 4–6 weeks old, body weight 20–25 g) were purchased from Orient Bio (Seoul, Korea) and acclimatized prior to the experiment by housing them in a temperature and humidity-controlled, pathogen-free environment with a 12-hour light / dark cycle (lighting at 6:30 AM) for 7–14 days. All animal experiments were performed in accordance with the Gachon University Guide for the Care and Use of Laboratory Animals. Additionally, arthritis was induced by tail-based intradermal injection of 2 mg / mL CFA (Chondrex). After the first signs of inflammation were observed on day 27, animals with AIA-induced arthritis were randomly assigned to six groups (n = 6).

[0104] Experimental Example 7: Analysis of the Arthritis Targeting Capability of Ectosome-PLGA in an Arthritis Model

[0105] Specifically, to evaluate the arthritic targeting ability of ectosome-PLGA in DBA / 1 mice with an arthritis model, CIA-induced mice were established via intradermal injection of the complete Freund's adjuvant (CFA) (Fig. 10a) (L. Bevaart, et al ., Arthritis Rheum . 66-3362(8): 2192-2205, 2010). The targeting ability of ectosome-PLGA to arthritic cells was analyzed using an in vivo fluorescence imaging system (IVIS imaging system) and histological images. Ectosome-PLGA-DID (1x10⁶) extracted from trained AD-MSCs 6 ectosome-PLGA (5 mg / kg) and PLGA-DID (5 mg / kg) were each intravenously injected once a day for a total of 3 days via the tail vein of arthritis model mice. On the 4th day, whole-body in vivo imaging was performed on the experimental animals to analyze the targeting ability of ectosome-PLGA to arthritis cells. As a result, as confirmed in Fig. 10b, it was confirmed that the stem cell-derived ectosome-PLGA nanoparticle complex trained with inflammatory cells according to one embodiment of the present invention selectively accumulated in inflammatory tissue. Therefore, it was confirmed that the biodegradable nanoparticle complex coated with a stem cell-derived ectosome membrane trained with cells derived from lesions such as tumors or inflammation according to one embodiment of the present invention possesses lesion-specific targeting ability from which the cells used for training originated. This lesion-specific education can be used to manufacture lesion-specific drug delivery systems by using ectosomes derived from the stem cells when stem cells are educated using cells that are highly relevant to the pathological symptoms present in lesions where neurodegenerative diseases or various metabolic-related diseases occur, in addition to tumors or inflammation.

[0106] Although the present invention has been described with reference to the embodiments described above, this is merely illustrative, and those skilled in the art will understand that various modifications and equivalent alternative embodiments are possible therefrom. Accordingly, the true technical scope of protection of the present invention should be determined by the technical spirit of the appended claims.

Claims

Claim 1 A method for preparing an ectosome-PLGA nanoparticle complex with enhanced targeting ability against the lesion, comprising: a step of producing trained adipose-derived mesenchymal stem cells by contacting and culturing adipose-derived mesenchymal stem cells in a culture medium in which lesion-derived cells selected from a group consisting of brain tumor tissue, pancreatic cancer tissue, inflammatory synovial cells, and inflammatory macrophages are being cultured; a step of isolating ectosomes from the trained adipose-derived mesenchymal stem cells; and a step of mixing the ectosomes with PLGA nanoparticles to produce PLGA nanoparticles coated with the ectosomes. Claim 2 delete Claim 3 delete Claim 4 delete Claim 5 delete Claim 6 delete Claim 7 delete Claim 8 A method of manufacturing, wherein the nanoparticles of claim 1 have a diameter of 100 to 350 nm.

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