Method for loading poorly water-soluble compounds into extracellular vesicles

Abstract

To provide a method for loading poorly water-soluble drugs into extracellular vesicles without using an organic solvent.SOLUTION: A method for loading a poorly water-soluble compound into extracellular vesicles without using an organic solvent, comprises the steps of: forming a water-soluble complex of a poorly water-soluble compound and a polymer by mixing the poorly water-soluble compound and the polymer, and then preparing the water-soluble complex to obtain a complex dispersion; and mixing the complex dispersion with an extracellular vesicle dispersion containing extracellular vesicles, and loading the poorly water-soluble compound into the extracellular vesicles by exchanging the poorly water-soluble compound from the polymer to the extracellular vesicles, wherein the polymer is at least one or more selected from the group consisting of polyethylene glycol, polyethyleneimine, cyclodextrin, polysaccharide, and polypeptide.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present disclosure relates to a method for loading poorly water-soluble compounds into extracellular vesicles. [Background technology]

[0002] Extracellular vesicles such as exosomes are vesicles with a lipid bilayer structure secreted by living organisms (animals, microorganisms, and plants) into their microenvironments, and are being applied to regenerative medicine and functional materials in cosmetics. Since extracellular vesicles are components derived from living organisms, they have excellent biocompatibility. Furthermore, since high selectivity can be achieved for target cell types depending on the cell and species from which they are derived, they are also expected to be used as a DDS base material.

[0003] The development of a technology for loading drugs into extracellular vesicles is essential to expand the range of their application. Conventionally, loading of water-soluble drugs (small molecules, siRNA, mRNA, proteins) has been achieved by electroporation. On the other hand, loading of poorly water-soluble drugs has been achieved by injecting poorly water-soluble drugs dissolved in organic solvents such as DMSO or ethanol, which are freely miscible with water, into the extracellular vesicle dispersion.

[0004] For example, Non-Patent Documents 1 and 2 disclose a treatment method using exosomes loaded with curcumin. In addition, Non-Patent Documents 2 and 3 disclose loading various drugs into extracellular vesicles and methods for loading the drugs. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] A.Kalani et al., The International Journal of Biochemistry & Cell Biology 79 (2016) 360-369 [Non-Patent Document 2] D.Li et al., Molecular Pharmaceutics 2023 20 (9), 4453-4467 [Non-Patent Document 3] Sarwareddy Kartik Kumar and Manda Venkata Sasidhar, Molecular Pharmaceutics 2023 20 (8), 3829-3842 Summary of the Invention [Problem to be solved by the invention]

[0006] When an organic solvent is used to load a poorly water-soluble drug into an extracellular vesicle as in the above-mentioned conventional method, the organic solvent induces denaturation of the membrane proteins constituting the extracellular vesicle and weakens the membrane structure. Furthermore, the biological reactions induced by these organic solvent components in cells, tissues, individuals, etc. cannot be ignored.

[0007] In addition, as in Non-Patent Document 2, when a drug added in excess to cells is loaded onto passively secreted extracellular vesicles, there are problems with the amount that can be loaded into the extracellular vesicles, uniformity, selectivity of host cells, etc., and the method lacks versatility. There is also a problem that it is extremely difficult to scale up.

[0008] The technology disclosed herein has been made in consideration of these points, and its purpose is to provide a method for loading poorly water-soluble compounds into extracellular vesicles without using organic solvents. [Means for solving the problem]

[0009] The inventors of the present application have developed a method for loading polymers and poorly water-soluble compounds into extracellular vesicles by host exchange.

[0010] Specifically, the method of the present disclosure comprises: A method for loading a poorly water-soluble compound into extracellular vesicles without using an organic solvent, comprising the steps of: A step of mixing a poorly water-soluble compound and a polymer to form a water-soluble complex of the poorly water-soluble compound and the polymer, and then preparing the water-soluble complex to obtain a complex dispersion; mixing the complex dispersion with an extracellular vesicle dispersion containing extracellular vesicles to exchange the poorly water-soluble compound from the polymer with the extracellular vesicles, thereby loading the poorly water-soluble compound into the extracellular vesicles; The polymer is at least one selected from the group consisting of polyethylene glycol, polyethyleneimine, cyclodextrin, polysaccharides, and polypeptides.

[0011] The poorly water-soluble compound is preferably at least one selected from the group consisting of fullerene derivatives, porphyrin derivatives, carborane derivatives, polyphenol analogues, polycyclic aromatic hydrocarbons, aggregation-induced luminescence compounds, pharmacologically active substances, and cell-labeling compounds.

[0012] As a result of intensive research by the present inventors, the findings obtained have shown that the method of loading a poorly water-soluble compound into extracellular vesicles of the present disclosure makes it possible to load a poorly water-soluble compound into extracellular vesicles without using an organic solvent. This method makes it possible to load a poorly water-soluble compound at a high concentration, prevents destabilization of the structure of the extracellular vesicles and aggregation of the extracellular vesicles, and can solubilize the poorly water-soluble compound in a stable state. In addition, it is extremely easy to scale up regardless of the type of extracellular vesicles to be used, making it highly versatile. Effect of the Invention

[0013] As described above, according to the present disclosure, it is possible to load a poorly water-soluble compound into an extracellular reticulum without using an organic solvent. [Brief description of the drawings]

[0014] [Figure 1] 2 shows UV-vis absorption spectra of the complexes of poorly water-soluble compounds and natural polymers according to Examples 2 to 4. [Diagram 2]1 shows a UV-vis absorption spectrum of a complex of a poorly water-soluble compound and a natural polymer according to Example 5. [Diagram 3] 4 is a graph showing the stability of complexes between poorly water-soluble compounds and natural polymers according to Examples 2 to 4. [Figure 4] 1 is a 1H NMR spectrum according to Example 1. [Diagram 5] 1 is a 1H NMR spectrum according to Example 2. [Figure 6] 1H NMR spectrum according to Example 3. [Figure 7] 1H NMR spectrum according to Example 4. [Figure 8] 1H NMR spectrum according to Example 5. [Figure 9] 1H NMR spectrum of Example 6. [Figure 10] 1H NMR spectrum of Example 7. [Figure 11] 1H NMR spectrum of Example 8. [Figure 12] 1H NMR spectrum of Example 9. [Figure 13] 13 is a fluorescence spectrum according to Example 6. [Figure 14] 13 is a fluorescence spectrum according to Example 7. [Figure 15] 1 shows UV-vis absorption spectra according to Examples 2 to 4. [Figure 16] 1 is a UV-vis absorption spectrum according to Example 5. [Figure 17] 1 is a UV-vis absorption spectrum according to Example 6. [Figure 18] 1 is a UV-vis absorption spectrum according to Example 7. [Figure 19] 1 is a UV-vis absorption spectrum according to Example 9. [Figure 20] 1 is a graph showing the stability of extracellular vesicles loaded with poorly water-soluble compounds according to Examples 2 to 4. [Figure 21] This is an image of extracellular vesicles taken by a transmission electron microscope. [Figure 22] 1 is an image of extracellular vesicles loaded with a poorly water-soluble compound of Example 1 taken by a transmission electron microscope. [Diagram 23] This is an image of extracellular vesicles loaded with a poorly water-soluble compound of Example 6 taken by a transmission electron microscope. [Figure 24] This is an image of extracellular vesicles loaded with a poorly water-soluble compound of Example 7 taken by a transmission electron microscope. [Diagram 25] This is an image of extracellular vesicles loaded with a poorly water-soluble compound of Example 8 taken by a transmission electron microscope. [Figure 26] This is an image of extracellular vesicles loaded with a poorly water-soluble compound of Example 9 taken by a transmission electron microscope. [Figure 27] 1 is a UV-vis absorption spectrum comparing the disclosed method with a conventional method. [Figure 28] 1 is a UV-vis absorption spectrum comparing the disclosed method with a conventional method. [Figure 29] Fluorescence spectra showing the reaction behavior between a synthetic polymer / porphyrin complex and exosomes. [Diagram 30] Fluorescence spectra showing the reaction behavior between polysaccharide / porphyrin complexes and exosomes. [Diagram 31] Fluorescence spectra showing the reaction behavior between a polypeptide / porphyrin complex and exosomes. [Diagram 32] 1 is a graph showing the anti-allergic activity of resveratrol-loaded extracellular vesicles obtained in Example 2. [Diagram 33] 1 is a graph showing the antitumor activity of PTX-loaded extracellular vesicles obtained in Example 4. [Diagram 34] 1 is a graph showing the photodynamic activity of fullerene-loaded extracellular vesicles obtained in Example 5. [Diagram 35] 1 is a graph comparing the BNCT activity of the carborane-loaded extracellular vesicles obtained in Example 1 against Colon 26 cells with that of a clinical drug. [Diagram 36] 1 is a graph showing the BNCT activity of carborane-loaded extracellular vesicles obtained in Example 1 against cancer model mice. [Figure 37] Photographs comparing the tumor delivery properties of liposomes and carborane-loaded extracellular vesicles. [Figure 38] 1 is a graph comparing the blood retention of liposomes and carborane-loaded extracellular vesicles. [Figure 39] 13 is a photograph comparing the delivery properties of liposomes and extracellular vesicles to intestinal tissue. [Diagram 40] 1 is a graph comparing the delivery properties of liposomes and extracellular vesicles to intestinal tissue. [Diagram 41] 1 is a graph showing the change in rectal temperature indicating the effect of suppressing food allergies. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The method of loading poorly water-soluble compounds into extracellular vesicles according to the present disclosure is described in detail below.

[0016] The method of loading a poorly water-soluble compound into extracellular vesicles according to the present disclosure is as follows: 1) A poorly water-soluble compound and a polymer are mixed to form a water-soluble complex of the poorly water-soluble compound and the polymer, and then this water-soluble complex is prepared to obtain a complex dispersion. 2) The complex dispersion is mixed with an extracellular vesicle dispersion containing extracellular vesicles, and the poorly water-soluble compound is exchanged from the polymer to the extracellular vesicles, thereby loading the poorly water-soluble compound into the extracellular vesicles. The above reactions 1) and 2) are carried out without using an organic solvent.

[0017] In the present disclosure, "poorly water-soluble" means that the solubility in water at 25°C is 0.1 g / L or less, and includes being completely insoluble in water at 25°C.

[0018] [Poorly water-soluble compounds] Poorly water-soluble compounds applicable to the extracellular vesicle loading method of the present disclosure are not particularly limited, and examples thereof include poorly water-soluble pharmacologically active substances, poorly water-soluble functional substances for use in the food industry such as beverages and supplements, and poorly water-soluble compounds in the chemical material industry that traditionally required dissolution in an organic solvent.

[0019] The poorly water-soluble pharmacologically active substance is, for example, classified into class 2 (low solubility, good membrane permeability) or class 4 (low solubility, poor membrane permeability) in the Biopharmaceutical Classification System defined by the FDA. Specific examples include, but are not limited to, taxane anticancer drugs such as paclitaxel (taxol) and docetaxel, antitumor drugs such as bortezomib, and antiulcer drugs such as omeprazole. Bortezomib and omeprazole are poorly water-soluble pharmacologically active substances having a nitrogen-containing heterocycle.

[0020] Poorly water-soluble functional substances in the food and chemical materials fields include, but are not limited to, porphyrin derivatives, fullerene derivatives, carborane derivatives, polyphenol analogues such as curcumin, resveratrol, and astaxanthin, polycyclic aromatic hydrocarbons such as anthracene, phenanthrene, chrysene, pyrene, and fluoranthene, and aggregation-induced luminescence compounds such as tetraphenylethylene and hexaphenylethylene. Cell labeling compounds for labeling specific sites of cells include Nile Red, Nile Blue, Janus Green, Oil Red, and Bismarck Brown. Application of these functional substances also makes it possible to construct an imaging system.

[0021] These poorly water-soluble compounds may be used alone or in combination of two or more.

[0022] [Polymer] Polymers applicable to the method of loading extracellular vesicles of the present disclosure are synthetic polymers and natural polymers that can form a complex with the above-mentioned poorly water-soluble compound to dissolve or disperse the poorly water-soluble compound in water. Preferred examples of synthetic polymers include polyethylene glycol and polyethyleneimine. Preferred examples of natural polymers include cyclodextrin, polysaccharides, and polypeptides. These polymers may be used whose structure has been changed by chemical modification reactions or the like within the scope of the effects of the present invention.

[0023] The polyethylene glycol is not particularly limited, and can be appropriately selected from commercially available products. The terminal of the polyethylene glycol is not limited to a hydroxyl group, and may be other functional groups. Polyethyleneimine can also be used as the synthetic polymer. A cationic polymer such as polyethyleneimine strongly interacts with anionic extracellular vesicles, and is therefore considered to be able to form a water-soluble complex with the extracellular vesicles in the same way as polyethylene glycol.

[0024] Examples of polysaccharides include, but are not limited to, glycogen, dextran, pectin, amylopectin, amylose, chitin, chitosan, heparin, hyaluronic acid, chondroitin sulfate, alginic acid, pullulan, mannan, levan, inulin, xyloglucan, hydroxyethyl cellulose, hydroxypropyl cellulose, and carboxymethyl cellulose.

[0025] The polypeptide is not particularly limited, but examples thereof include gelatin, collagen, polyglutamic acid, polylysine, and the like.

[0026] These polymers may be used alone or in combination of two or more.

[0027] [Preparation of composite dispersion] By mixing the poorly water-soluble compound and the polymer, a water-soluble complex of the poorly water-soluble compound and the polymer is formed. Then, this water-soluble complex is prepared to obtain a complex dispersion. The poorly water-soluble compound is preferably mixed with the polymer in a pulverized state. As a more specific preparation method, for example, the poorly water-soluble compound and the polymer are pulverized and mixed using a ball mill or a mortar and pestle to obtain a pulverized mixture. A pulverization and mixing method using a high-speed vibration pulverizer may be used as a type of ball mill. In the high-speed vibration pulverizer, hard balls are reciprocated at high speed in the hollow part of a container containing the poorly water-soluble compound and the polymer, thereby pulverizing and mixing the poorly water-soluble compound and the polymer. Specifically, for example, it is a ball mill that can make the particle size from several nm to several hundred μm by a vibration rotation speed of 180 to 2100 rpm (3 to 35 Hz). Water is added to the pulverized mixture, and a complex dispersion of the poorly water-soluble compound and the polymer is obtained as an aqueous solution of the pulverized mixture. The solvent of the complex dispersion is water, and does not contain an organic solvent.

[0028] [Extracellular vesicles] In the present disclosure, extracellular vesicles refer to vesicles having a secreted lipid bilayer structure. The size is a few nm to a few μm in diameter. Examples of extracellular vesicles include exosomes, microvesicles, ectosomes, membrane particles, exosome-like vesicles, and apoptotic vesicles. There is no particular limit to the origin of the extracellular vesicles. For example, they may be derived from animals or plants. For example, they may be derived from animal body fluids such as blood components and breast milk, or from plants such as fruits and fruit juices.

[0029] [Preparation of extracellular vesicles loaded with poorly water-soluble compounds] A complex dispersion in which a water-soluble complex is prepared to a predetermined concentration is mixed with an extracellular vesicle dispersion, which is an aqueous solution containing extracellular vesicles, and the poorly water-soluble compound is exchanged from the polymer to the extracellular vesicles, thereby loading the poorly water-soluble compound onto the extracellular vesicles. Specifically, the concentration of the water-soluble complex is determined by various methods, and the complex dispersion is adjusted to a predetermined mol% relative to the lipids of the extracellular vesicles to prepare a complex dispersion. The adjusted complex dispersion is then added to the extracellular vesicle dispersion. The amount of the complex dispersion added relative to the lipids of the extracellular vesicles is a general value as the ratio of the compound to the lipids in the drug delivery system, and is preferably 20 mol% or less, and more preferably 10 mol% or less. After the complex dispersion is added to the extracellular vesicle dispersion, the mixture is stirred at a temperature that does not affect the extracellular vesicles, for example, at room temperature to about 40°C. The poorly water-soluble compound that formed a complex with the polymer leaves the polymer and is loaded onto the extracellular vesicles. In this process, the solvent is water and does not contain an organic solvent. EXAMPLES

[0030] Examples of the present invention will be described below. Note that the present invention is not limited to these examples, and these examples can be modified or changed based on the spirit of the present invention, and are not excluded from the scope of the present invention.

[0031] [Separation and purification of extracellular vesicles] -Collection of extracellular vesicles from milk Acetic acid was added to 1 L of milk, and the resulting precipitate was separated by centrifugation (10,000 g, 60 min, 4°C) and passed through a 0.22 μm filter. It was then concentrated by tangential flow filtration. After sufficient concentration, it was recovered by ultracentrifugation (120,000 g, 70 min, 4°C). It was suspended in an optimal amount of phosphate buffered saline (PBS). Collection of extracellular vesicles from lemon Fresh lemons were squeezed, and the juice was centrifuged (10,000g, 60 min, 4°C) to remove large insoluble matter. It was then passed through a 0.22 μm filter. It was then concentrated by tangential flow filtration. After sufficient concentration, it was recovered by ultracentrifugation (120,000g, 70 min, 4°C). It was suspended in an appropriate amount of PBS. In the following examples, the above-mentioned milk-derived exosomes or lemon-derived exosomes were used.

[0032] [Preparation of composite dispersion] In Examples 1 to 5, 2-hydroxypropyl-β-cyclodextrin (HP-β-CDx) or 2-hydroxypropyl-γ-cyclodextrin (HP-γ-CDx) was used as the polymer, and each of the poorly water-soluble compounds shown in Table 1 was used. In Examples 6 to 10, HP-β-CDx was used as the polymer, and each of the poorly water-soluble compounds shown in Table 2 was used.

[0033] [Table 1]

[0034] [Table 2]

[0035] In Example 1, the molar amounts of β-cyclodextrin and o-carborane (CB) were equal, in Example 2, 5 equivalents of β-cyclodextrin relative to resveratrol (Res), in Example 3, 3 equivalents of β-cyclodextrin relative to curcumin (Cur), in Example 4, 2 equivalents of β-cyclodextrin relative to paclitaxel (PTX), and in Example 5, cationic fullerene (cC 60In Example 6, β-cyclodextrin was weighed out so that it was 2 equivalents to pyrene (Py). In Example 7, β-cyclodextrin was weighed out so that it was 2.5 equivalents to tetrakis (4-hydroxyphenyl) ethylene (TPE-OH). In Example 8, β-cyclodextrin was weighed out so that it was 4.0 equivalents to bortezomib (BTZ). In Example 9, β-cyclodextrin was weighed out so that it was 3.0 equivalents to omeprazole (Omp). In Example 10, β-cyclodextrin was weighed out so that it was 4.0 equivalents to Nile Red (NR).

[0036] The weighed poorly water-soluble compounds and cyclodextrin were placed in an agate container together with two agate balls and mixed using a high-speed vibration grinder (25 Hz, 30 min). 2 mL of pure water was added to this mixture to suspend it, and the supernatant was collected after centrifugation (4500 rpm, 20 min). This supernatant is the water-soluble complex in which the poorly water-soluble compound is dissolved or dispersed.

[0037] The concentration of the resulting water-soluble complex was determined by ICP emission spectrometry or UV-vis absorption spectrometry.

[0038] 100 μL of the water-soluble complex of Example 1 was taken and treated with aqua regia, then diluted to a total volume of 5 mL and subjected to ICP measurement. As a result, the concentration of boron derived from CB solubilized by HP-β-CDx was 12.5 ppm.

[0039] For the water-soluble complexes of Examples 2 to 7, the UV-vis absorption spectra were measured (25°C, 1 mm cell) using a spectrophotometer (Shimadzu, UV-3600). Figure 1 shows the UV-vis absorption spectra of the complexes of poorly water-soluble compounds and natural polymers of Examples 2 to 4, and Figure 2 shows the UV-vis absorption spectrum of the complex of poorly water-soluble compound and natural polymer of Example 5. The molar absorption coefficient Cur:ε 430 =44000(mol -1 dm 3 cm -1),PTX:ε 235 =31000(mol -1 dm 3 cm -1 ),Res:ε 308 =25000(mol -1 dm 3 cm -1 ),cC 60 :ε 322 =43100(mol -1 dm 3 cm -1 ) and the concentrations were calculated from the absorbance of each spectrum to be 170 μM, 64 μM, 2600 μM, and 5500 μM, respectively.

[0040] For Examples 2 to 4, the stability of each compound during complex formation was examined by measuring the change in absorbance at the wavelength showing the peak top of the UV-vis absorption spectrum, as shown in FIG. 3. The peak top wavelengths of each complex (poorly water-soluble compound / polymer) were: Example 2: Res / HP-β-CDx, peak top wavelength 308 nm; Example 3: Cur / HP-β-CDx, peak top wavelength 431.5 nm; Example 4: PTX / HP-β-CDx, peak top wavelength 235.5 nm. The most stable was Res / HP-β-CDx in Example 2, with an absorbance of about 49% after 7 days. On the other hand, the absorbance of Cur / HP-β-CDx in Example 3 and PTX / HP-β-CDx in Example 4 decreased to 16% and 12%, respectively, after 1 day, suggesting rapid release from the guest molecules.

[0041] [Preparation of extracellular vesicles loaded with poorly water-soluble compounds] 500μL of the complex dispersion obtained by preparing the water-soluble complex to be 10mol% relative to the lipid of the exosome was added to 500μL of the exosome dispersion (final protein concentration 3.0mg / mL) that had been heated at 40℃ for 30 minutes. The complex dispersion was added and stirred for 2 hours to carry out an exchange reaction of the poorly water-soluble compound from the CDx complex to the exosome membrane. Then, centrifugation (4500rpm, 5min, 25℃) was performed to remove the poorly water-soluble compound that had not been associated and transferred to the exosome membrane. The supernatant was separated to obtain exosomes loaded with the poorly water-soluble compound. The fact that the poorly water-soluble compound was loaded on the exosome was confirmed by nuclear magnetic resonance spectroscopy, UV-Vis spectroscopy, fluorescence spectroscopy and IPC luminescence measurement. In all of the compounds obtained in Examples 1 to 10, a change in spectrum was observed before and after mixing with the exosome, and it was determined that the poorly water-soluble compound was loaded on the exosome.

[0042] [Confirmation of extracellular vesicles loaded with poorly water-soluble compounds by nuclear magnetic resonance spectroscopy] Nuclear magnetic resonance spectroscopy (Varian, 400 MHz) was performed to confirm that each poorly water-soluble compound was loaded into exosomes.

[0043] FIG. 4 shows the nuclear magnetic resonance spectra of Example 1, including the spectrum of HP-β-CDx, the spectrum of the complex of carborane and cyclodextrin (CB / HP-β-CDx), and the spectrum of carborane-loaded exosomes (CB / Lexo). In FIG. 4, the black circle indicates the anomeric proton of free HP-β-CDx, and the white circle indicates the peak derived from the CB / HP-β-CDx complex. The peak derived from the CB / HP-β-CDx complex disappeared and became broad, suggesting the loading of carborane into the exosome membrane.

[0044] Figure 5 shows the nuclear magnetic resonance spectra of Example 2, including the spectrum of HP-β-CDx, the spectrum of the complex of resveratrol and cyclodextrin (Res / HP-β-CDx), and the spectrum of resveratrol-loaded exosomes (Res / Lexo). In Figure 5, the black circle indicates the anomeric proton of free HP-β-CDx. The disappearance and broadening of the peak derived from the complex suggested the loading of resveratrol into the exosome membrane.

[0045] Figure 6 shows the nuclear magnetic resonance spectra of Example 3, including the spectrum of HP-β-CDx, the spectrum of the complex of curcumin and cyclodextrin (Cur / HP-β-CDx), and the spectrum of curcumin-loaded exosomes (Cur / Lexo). In Figure 6, the black circle indicates the anomeric proton of free HP-β-CDx, and the enlarged part indicates the peak derived from the Cur / HP-β-CDx complex. The disappearance and broadening of the peak derived from the complex suggested the loading of curcumin into the exosome membrane.

[0046] FIG. 7 shows the nuclear magnetic resonance spectrum of Example 4, which includes the spectrum of HP-β-CDx, the spectrum of the complex of paclitaxel and cyclodextrin (PTX / HP-β-CDx), and the spectrum of paclitaxel-loaded exosomes (PTX / Lexo). In FIG. 7, the black circle indicates the anomeric proton of free HP-β-CDx, and the enlarged portion indicates the peak derived from the PTX / HP-β-CDx complex. After mixing and stirring with exosomes, strong singlet peaks (2.20, 7.31, 7.66 ppm) were observed. This is thought to be a paclitaxel peak, and it was suggested that paclitaxel may be stuck on the membrane surface of exosomes or may be supported on exosomes by some kind of interaction.

[0047] FIG. 8 shows the nuclear magnetic resonance spectrum of Example 5, including the spectrum of HP-γ-CDx, the spectrum of the cationic fullerene-cyclodextrin complex (cC 60 / HP-β-CDx) and cationic fullerene-loaded exosomes (cC 60In FIG. 8, the black circles indicate the anomeric protons of free HP-γ-CDx, and the white circles indicate the cC 60 The peak derived from the / HP-β-CDx complex is shown. The disappearance and broadening of the peak derived from the complex suggested that cationic fullerene was loaded onto the exosome membrane.

[0048] Figure 9 shows the nuclear magnetic resonance spectra of Example 6, including the spectrum of HP-γ-CDx, the spectrum of the complex of pyrene and cyclodextrin (Py / HP-β-CDx), and the spectrum of pyrene-loaded exosomes (Py / Lexo). In Figure 9, black circles indicate peaks derived from the Py / HP-β-CDx complex. The disappearance and broadening of the peaks derived from the complex suggested the loading of pyrene into the exosome membrane.

[0049] FIG. 10 shows the nuclear magnetic resonance spectra of Example 7, including the spectrum of HP-γ-CDx, the spectrum of a complex of tetrakis(4-hydroxyphenyl)ethylene and cyclodextrin (TPE-OH / HP-β-CDx), and the spectrum of tetrakis(4-hydroxyphenyl)ethylene-loaded exosomes (TPE-OH / Lexo). In FIG. 10, the black circles indicate peaks derived from the TPE-OH / HP-β-CDx complex. The disappearance and broadening of the peaks derived from the complex suggested the loading of tetrakis(4-hydroxyphenyl)ethylene into the exosome membrane.

[0050] FIG. 11 shows the nuclear magnetic resonance spectra of Example 8, including the spectrum of HP-γ-CDx, the spectrum of a complex of bortezomib and cyclodextrin (BTZ / HP-β-CDx), and the spectrum of bortezomib-loaded exosomes (BTZ / Lexo). In FIG. 11, black circles indicate peaks derived from the BTZ / HP-β-CDx complex, and white circles indicate peaks derived from BTZ / Lexo. The appearance of a peak derived from BTZ / Lexo suggested the loading of pyrene into the exosome membrane.

[0051] Figure 12 shows the nuclear magnetic resonance spectra of Example 9, including the spectrum of HP-γ-CDx, the spectrum of a complex of omeprazole and cyclodextrin (Omp / HP-β-CDx), and the spectrum of omeprazole-loaded exosomes (Omp / Lexo). In Figure 12, the black circles indicate peaks derived from the Omp / HP-β-CDx complex. In the nuclear magnetic resonance spectrum of omeprazole-loaded exosomes after 2 days, the peaks derived from the Omp / HP-β-CDx complex had partially disappeared, suggesting that omeprazole had been loaded onto the exosome membrane.

[0052] [Confirmation of extracellular vesicles loaded with poorly water-soluble compounds by fluorescence spectrum measurement] In Examples 6 and 7, the fluorescence spectra of the water-soluble complex before mixing with exosomes and the poorly water-soluble compound-loaded exosomes after mixing were measured using a fluorometer, and it was confirmed whether the poorly water-soluble compound was loaded onto the extracellular vesicles by the fluorescence of the poorly water-soluble compound. As shown in Figures 13 and 14, the fluorescence spectrum of the water-soluble complex (Py / CDx, TPE-OH / CDx) before mixing with exosomes was different from the fluorescence spectrum of the water-soluble compound-loaded exosomes (Py / Lexo, TPE-OH / Lexo) after mixing with exosomes, suggesting that the poorly water-soluble compound was loaded onto the extracellular vesicles.

[0053] [Concentration determination of extracellular vesicles loaded with poorly water-soluble compounds] The concentration of the obtained extracellular vesicles loaded with poorly water-soluble compounds (poorly water-soluble compounds / Lexo) was determined by ICP emission spectroscopy or UV-vis absorption spectroscopy.

[0054] 100 μL of the CB / Lexo prepared in Example 1 was taken, treated with aqua regia, and diluted to a total volume of 5 mL, and then ICP measurement was performed. The ICP measurement was performed using an ICP emission analyzer. As a result, the boron concentration in the exosome membrane was 2.04 ppm. This indicates that the boron derived from the CB solubilized by HP-β-CDx was exchanged into the exosome membrane. In addition, ICP measurement was performed on the poorly water-soluble compound-loaded extracellular vesicles prepared in Examples 6 to 10 in the same manner as in Example 1, and the loading of the poorly water-soluble compound into the extracellular vesicles was confirmed.

[0055] The results of measuring the UV-vis absorption spectrum of the poorly water-soluble compound-loaded extracellular vesicles Res / Lexo, Cur / Lexo, and PTX / Lexo prepared in Examples 2 to 4 are shown in FIG. 15. 60 The results of measuring the UV-vis absorption spectrum of Res / Lexo are shown in Figure 16. Using the same molar absorption coefficient as that of the poorly water-soluble compound and polymer complex, the concentrations were calculated from the spectrum: Res / Lexo: 210 μM, Cur / Lexo: 53 μM, cC 60 / Lexo: 46 μM. 60 For cC, the peak broadening observed upon complex formation with cyclodextrin was observed, and 60 The decrease in the motility of the β-amyloides, i.e., the progress of the exchange reaction, was shown. Moreover, the concentration (1 mM) of the β-amyloides was quantitative with respect to the amount of the β-amyloides added.

[0056] In addition, the results of measuring the UV-vis absorption spectra of the water-soluble complex (Py / CDx) and extracellular vesicles loaded with a poorly water-soluble compound (Py / Lexo) prepared in Example 6 are shown in Figure 17, and the results of measuring the UV-vis absorption spectra of the water-soluble complex (TPE-OH / CDx) and extracellular vesicles loaded with a poorly water-soluble compound (TPE-OH / Lexo) prepared in Example 7 are shown in Figure 18.

[0057] FIG. 19 shows the results of measuring the UV-vis absorption spectrum of the water-soluble complex (Omp / CDx) and the extracellular vesicles loaded with a poorly water-soluble compound (Omp / Lexo) prepared in Example 9. The loading of each compound into the exosomes was confirmed from the change in the UV-vis absorption spectrum. For Example 9, the UV-vis absorption spectrum was also measured two days after the formation of the extracellular vesicles loaded with a poorly water-soluble compound (Omp / Lexo) (Omp / Lexo 2day in FIG. 19). The spectrum after two days was almost the same as the spectrum immediately after the formation of Omp / Lexo, confirming that the extracellular vesicles loaded with a poorly water-soluble compound were stable.

[0058] The long-term stability, which is important when using the poorly water-soluble compound-loaded extracellular vesicles prepared in Examples 2 to 4 as drugs, was examined by measuring the change in absorbance at the wavelength showing the peak top of the UV-vis absorption spectrum of the exosome dispersion loaded with each compound. As shown in FIG. 20, PTX / Lexo maintained the highest absorbance over 7 days, and no attenuation was observed. It is believed that secondary aggregation of exosomes due to the influence of membrane proteins and sugar chains present on the exosome membrane surface also caused aggregation of PTX, resulting in these results. Cur / Lexo aggregated by about 80% one day after preparation. Res / Lexo and PTX / Lexo remained stable for 7 days after preparation. This indicates high stability as a formulation.

[0059] [Characterization of exosomes loaded with poorly water-soluble compounds by dynamic light scattering] The average particle size, polydispersity, and zeta potential of the exosomes and the carborane-loaded exosomes (CB / Lexo) obtained in Example 1 were measured by dynamic light scattering (DLS). The results are shown in Table 3.

[0060] [Table 3]

[0061] As shown in Table 3, there was no significant change in the average particle size before and after the exchange reaction, suggesting that carborane was incorporated into exosomes. In addition, the PDI value was about 0.2, indicating that no aggregation of exosomes was observed, which may be induced by membrane proteins or glycans present on the exosome membrane surface, strongly suggesting that the particle size distribution was monodisperse.

[0062] The average particle size polydispersity and zeta potential of the exosomes and the poorly water-soluble compound-loaded exosomes (Res / Lexo, Cur / Lexo, PTX / Lexo) obtained in Examples 2 to 4 were measured by dynamic light scattering (DLS, Zeta Sizer Nano, Malvern). The results are shown in Table 4.

[0063] [Table 4]

[0064] From the results shown in Table 4, the concentration of the compound that could be loaded onto exosomes was the highest for resveratrol, and the particle size was about 130 nm with a PDI value of 0.22, suggesting that it was a single particle. In addition, the surface charge of Res / Lexo was significantly more negative than that of exosomes alone.

[0065] [Morphological observation of exosomes loaded with poorly water-soluble compounds using a transmission electron microscope] Exosomes and the carborane-loaded exosomes of Example 1 were added to the grid, and after absorbing with filter paper, the mixture was stained with 1% phosphotungstic acid and observed with a transmission electron microscope (JEOL, JEM-1400). Figure 21 shows the observation results of the exosomes and Figure 22 shows the observation results of the carborane-loaded exosomes. Spherical structures with a particle size similar to that measured using dynamic light scattering (DLS) were observed. In addition, the poorly water-soluble compound-loaded exosomes of Examples 6 to 9 were also observed with a transmission electron microscope. Figure 23 shows the pyrene-loaded exosomes of Example 6, Figure 24 shows the tetrakis(4-hydroxyphenyl)ethylene-loaded exosomes of Example 7, Figure 25 shows the bortezomib-loaded exosomes of Example 8, and Figure 26 shows the observation results of the omeprazole-loaded exosomes of Example 9. In all cases, the exosomes maintained a spherical shape even after loading with a water-soluble compound.

[0066] [Comparison with the thin film method] The conventional thin film method using an organic solvent was used to load poorly water-soluble compounds into extracellular vesicles, and the results were compared with the method disclosed herein. In this comparative study, o-carborane (CB), curcumin (Cur), and cationic fullerene (cC 60 ) was used. o-Carborane and curcumin were dissolved in chloroform, and cationic fullerene was dissolved in toluene. The poorly water-soluble compound was weighed out in a vial so that it was 10 mol% relative to the lipids in the exosomes, and dissolved in an appropriate organic solvent. The solvent was then removed with nitrogen gas to form a thin film of the poorly water-soluble compound. The exosome dispersion was added to this vial and stirred in the same manner as in the method of the present disclosure (40°C, 30 min). ICP measurement of the extracellular vesicles loaded with carborane revealed that the boron concentration derived from CB was 119 ppm (method of the present disclosure: 2.04 ppm). Figure 27 shows the UV-vis absorption spectra of curcumin-loaded exosomes by the method of the present disclosure (solid line) and curcumin-loaded exosomes by the thin film method (dotted line). Figure 28 shows the UV-vis absorption spectra of cC 60 Loaded exosomes (solid line) and cC by thin film method 60The UV-vis absorption spectrum of the loaded exosomes (dotted line) is shown. As shown in FIG. 27, in the extracellular vesicles loaded with curcumin, no peaks derived from Cur were confirmed (concentration of poorly water-soluble compound by the loading method of the present disclosure: 53 μM). In the extracellular vesicles loaded with fullerene, the concentration of the poorly water-soluble compound calculated from the UV-vis absorption spectrum in FIG. 28 was 18 μM (concentration of poorly water-soluble compound by the loading method of the present disclosure: 1 mM). With the extracellular vesicle loading method of the present disclosure, it was possible to load curcumin and cationic fullerene at higher concentrations than with conventional techniques.

[0067] [Extracellular vesicle loading method using synthetic polymers, polysaccharides, and polypeptides] In Examples 1 to 5, cyclodextrin was used as the polymer, but other polymers, such as synthetic polymers, polysaccharides, and polypeptides, can be applied to the method of the present disclosure. The following shows the results of a preparation method using a synthetic polymer (polyethylene glycol (PEG)), a polysaccharide (pullulan (Pul)), and a polypeptide (poly-γ-glutamic acid (PGA)) as a host for the complex. A porphyrin derivative (5,10,15,20-Tetrakis(4-aminophenyl)porphyrin, TAPP; 5 μM) was used as a poorly water-soluble compound. The PEG / TAPP complex, Pul / TAPP complex, or PGA / TAPP complex prepared by high-speed vibration grinding was allowed to interact with extracellular vesicles. The loading ability was evaluated from the recovery of the fluorescence of the porphyrin derivative present in the complex. The fluorescence is quenched when complexed with polysaccharides or polypeptides, but is restored by migration to extracellular vesicles. In both systems, the recovery of fluorescence was confirmed, and the loading of the porphyrin derivative into the extracellular vesicles was confirmed. Figure 29 is a fluorescence spectrum showing the behavior of a complex of a synthetic polymer and porphyrin when it reacts with exosomes, Figure 30 is a fluorescence spectrum showing the behavior of a complex of a polysaccharide and porphyrin when it reacts with exosomes, and Figure 31 is a fluorescence spectrum showing the behavior of a complex of a polypeptide and porphyrin when it reacts with exosomes. In all of Figures 29 to 31, the dotted line is the spectrum immediately after mixing the complex with exosomes, the gray line is the spectrum 1 hour after mixing the complex with exosomes, and the solid line is the spectrum 3 hours after mixing the complex with exosomes.

[0068] [Biological activity of extracellular vesicles loaded with poorly water-soluble compounds] Anti-allergic activity of polyphenol-loaded extracellular vesicles The anti-allergic activity of extracellular vesicles loaded with resveratrol as a polyphenol was examined. Anti-DNP IgE was added to rat-derived basophilic leukemia cells (RBL-2H3) and sensitized (16 hours). Resveratrol-loaded extracellular vesicles prepared by the loading method of the present disclosure were added thereto, and after acting for 30 minutes, an antigen (DNP-BNA) was added. A substrate for β-hexosaminidase was added thereto, and the allergy suppression effect was evaluated from the activity value (Figure 32). As a result, the activity was not reduced by the use of extracellular vesicles alone, but the suppression of allergic reactions was confirmed by loading resveratrol.

[0069] [Antitumor activity of PTX-loaded extracellular vesicles] The antitumor activity of PTX, an anticancer drug, was evaluated by adding it to tumor cells. The extracellular vesicles loaded with PTX prepared by the method of the present disclosure were added to mouse squamous cell carcinoma cells (SCC-VII cells) and incubated for 24 hours. The cell viability was then colored and quantified using a Cell Counting Kit to determine the antitumor activity. As a control, PTX dissolved in DMSO was compared with PTX, and the activity was equal to or greater than that of DMSO, and the effect of improving bioavailability by imparting water solubility was obtained (Figure 33).

[0070] [Photodynamic activity of extracellular vesicles loaded with fullerene derivatives] The activity of the fullerene derivative as a phototherapeutic agent for photodynamic therapy, which is expected to be a minimally invasive phototherapy, was examined. The fullerene derivative-loaded extracellular vesicles prepared by the method of the present disclosure were added to mouse colon cancer cells (Colon26), and after 24 hours, the cells were irradiated with red light (>620 nm), which has excellent biopermeability, for 30 minutes. The antitumor activity after another 24 hours was evaluated by a colorimetric method using a Cell Counting Kit. As shown in FIG. 34, while no toxicity was observed in the dark, it was revealed that cell death could be induced in a concentration-dependent manner under light irradiation. In other words, it was revealed that the present system has high activity as a phototherapeutic agent for photodynamic therapy.

[0071] [BNCT activity of carborane-loaded extracellular vesicles] We investigated the activity of the boron compound as a boron drug for boron neutron capture therapy (BNCT), which is expected to be a minimally invasive radiation therapy. The carborane-loaded extracellular vesicles prepared by the method disclosed herein were added to mouse colon cancer cells (Colon26), and after 24 hours, the cells were irradiated with thermal neutrons. The antitumor activity after another 24 hours was considered by a colorimetric method using a Cell Counting Kit. Here, the therapeutic performance was compared with that of the clinical drug L-BPA / fructose complex as a control, and therapeutic performance surpassed that of the clinical drug (Figure 35). In Figure 35, ◯: L-BPA / fructose complex, ●: carborane-loaded extracellular vesicles (CB / Lexo). This efficacy was also shown in tumor-bearing model mice in which Colon26 cells were transplanted, and the therapeutic results here also surpassed those of the L-BPA / fructose complex prepared by the conventional method. Moreover, no sudden weight loss was observed during the course of this treatment, proving the safety of this system (Figure 36). In Figure 36, ◯: PBS, ◇: L-BPA / fructose, □: carborane-loaded extracellular vesicles (CB / Lexo) are shown, with the white markers representing the non-neutron beam irradiation group and the black markers representing the neutron beam irradiation group.

[0072] [Comparison of tumor delivery with conventional technology] The delivery to tumor tissue was compared between the conventional technology (liposomes) and the extracellular vesicles prepared by the method of the present disclosure. Carborane-loaded extracellular vesicles were stained with a fluorescent dye DiD that can be excited in the near infrared region and administered to the tail vein of a cancer-bearing mouse. As a comparison, liposomes were stained in the same manner and administered to the tail vein of another cancer-bearing mouse. Accumulation was visualized and compared using an imaging system immediately after administration (0 hours), 1 hour, 3 hours, 6 hours, and 24 hours later. As a result, as shown in FIG. 37, the carborane-loaded extracellular vesicles (EVs) prepared by the method of the present disclosure achieved higher tumor accumulation than liposomes. In addition, blood was collected at the same time to compare blood retention as shown in FIG. 38, and the carborane-loaded extracellular vesicles were also superior.

[0073] [Comparison of delivery to intestinal tissue with conventional technology] When aiming at food allergy, it is desirable that polyphenols are delivered to immune cells in the intestinal tissue. Therefore, we compared the difference in delivery ability between extracellular vesicles and liposomes by oral administration. Exosomes were stained with a fluorescent dye DiD that can be excited in the near infrared region and orally administered to mice using a probe. As a comparison, liposomes were stained in the same way and orally administered to another mouse using a probe. After administration, the intestines were collected 24 hours later and quantified using an imaging system. Figure 39 shows a photograph taken with the imaging system, and Figure 40 shows a graph of the quantification using the imaging system. It was revealed that exosomes (DiD-Lexosomes) showed higher delivery ability than liposomes (DiD-Liposomes), and that even 24 hours later, a large amount of exosomes was delivered to the intestinal tract.

[0074] [Study on food allergy treatment efficacy] We investigated whether there is an inhibitory effect on food allergy induction in a food allergy model induced by excessive administration of ovalbumin (OVA) and adjuvant (Alum). From 3 weeks after the start of induction, phosphate-buffered saline (PBS), exosomes (Lexo), resveratrol-loaded exosomes (Res / Lexo), DMSO, and DMSO solution of resveratrol (Res / DMSO) were orally administered daily by a sonde. After one week of treatment, the above-mentioned inhibitors were administered, and OVA or PBS was administered 30 minutes later, and the change in rectal temperature was measured to evaluate the inhibitory effect on allergy induction. The results are shown in Figure 41. In Figure 41, ●: OVA-nonimmunized mice, ○: OVA-immunized mice, △: PBS, ■: Res / Lexo, □: Lexo, ◆: Res / DMSO, ◇: DMSO, respectively. As shown in Figure 41, the groups containing resveratrol (Res / DMSO, Res / Lexo) suppressed the decrease in rectal temperature compared to the vehicle (DMSO only, exosomes only). In addition, a higher inhibitory effect was achieved by delivering resveratrol via exosomes (Res / Lexo) than by dissolving resveratrol in DMSO (Res / DMSO). As described above, it has become clear that exosomes are more advantageous in activating the intestinal immune system.

Claims

1. A method for loading a poorly water-soluble compound into extracellular vesicles without using an organic solvent, comprising the steps of: A step of mixing a poorly water-soluble compound and a polymer to form a water-soluble complex of the poorly water-soluble compound and the polymer, and then preparing the water-soluble complex to obtain a complex dispersion; mixing the complex dispersion with an extracellular vesicle dispersion containing extracellular vesicles to exchange the poorly water-soluble compound from the polymer with the extracellular vesicles, thereby loading the poorly water-soluble compound into the extracellular vesicles; The method for loading a poorly water-soluble compound into extracellular vesicles, wherein the polymer is at least one selected from the group consisting of polyethylene glycol, polyethyleneimine, cyclodextrin, polysaccharides, and polypeptides.

2. The method for loading a poorly water-soluble compound into extracellular vesicles according to claim 1 , wherein in the step of preparing the complex dispersion, the poorly water-soluble compound is mixed in a pulverized state with the polymer.

3. The method for loading a poorly water-soluble compound into extracellular vesicles described in claim 2, wherein the poorly water-soluble compound is at least one selected from the group consisting of fullerene derivatives, porphyrin derivatives, carborane derivatives, polyphenol analogues, polycyclic aromatic hydrocarbons, aggregation-induced luminescence compounds, pharmacologically active substances, and cell labeling compounds.

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