Composition for improving intestinal health function containing milk-derived exosomes as an active ingredient and method for producing the same

Abstract

The present invention provides a composition for improving intestinal health function, comprising milk-derived exosomes and a therapeutic agent, the therapeutic agent being DNA, RNA or a small molecule, and the therapeutic agent being located and encapsulated in the inner space of the exosomes.The present invention also provides a method for producing the composition for improving intestinal health function, the method comprising the steps of: (a) isolating exosomes from milk; (b) electroporating the isolated exosomes to form micropores on the surface of the exosomes; (c) mixing the electroporated exosomes of step (b) with the therapeutic agent; and (d) removing the therapeutic agent that is not encapsulated in the exosomes.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority based on Korean Patent Application No. 10-2022-0009019 filed on January 21, 2022, and Korean Patent Application No. 10-2022-0099821 filed on August 10, 2022, the entire contents of which are incorporated by reference into this application.

[0002] State-sponsored research and development explanation This research was conducted under the supervision of the Korea Institute of Science and Technology and was supported by the Ministry of Science and ICT-Korea Institute of Science and Technology's Research Operating Expenses Support (R&D) (Major Project Costs) Project (Hyper-connected Medical Device Reverse Medication Research Project, Project ID: 1711152094), the Ministry of Science and ICT-Korea Institute of Science and Technology's Research Operating Expenses Support (Major Project Costs) Project (Custom-made diagnosis, treatment, regeneration, rehabilitation, and development of new drugs, Project ID: 1711173292), and the Ministry of Science and ICT-Korea Institute of Science and Technology's Basic Platform Technology Development Project (Development of innovative siRNA oral therapeutic drug for the treatment of inflammatory bowel disease, Project ID: 1711160075).

[0003] The present disclosure relates to a composition for improving intestinal health function, comprising milk-derived exosomes as an active ingredient. [Background technology]

[0004] Exosomes are a type of extracellular endoplasmic reticulum with a diameter of 30 to 150 nm, and are secreted from various types of cells. It is known that such exosomes play an important role in transmitting intercellular information, and they have begun to be used in next-generation drug delivery technology. However, the amount of exosomes secreted by cells is too small, making it difficult to ensure high purity, and their relatively unstable physical properties limit mass production. Recently, food-derived exosomes have been in the spotlight. Such food-derived exosomes are easily available and generally safe. Among them, milk-derived exosomes in particular have anti-inflammatory properties and contain microRNAs in the exosomes themselves that are important for mediating pathological and physiological activities. Milk-derived exosomes can be isolated and purified from milk with a high yield, and maintain high stability even under sudden environmental changes, such as pH and temperature. In addition, milk-derived exosomes have a high bioavailability, making it possible to administer drugs orally, and are particularly useful when there is a lesion in the gastrointestinal tract, such as inflammatory bowel disease. Summary of the Invention [Problem to be solved by the invention]

[0005] In one aspect, an object of the present invention is to provide a composition for improving intestinal health function.

[0006] In one aspect, the present invention aims to provide a composition for improving intestinal health function, characterized in that a therapeutic agent is encapsulated by being located in the internal space of an exosome.

[0007] In one aspect, an object of the present invention is to provide a composition which can be orally administered and is for improving intestinal-related diseases or functions.

[0008] In one aspect, an object of the present invention is to provide a method for producing a composition for improving intestinal health function. [Means for solving the problem]

[0009] In one aspect, a composition for improving intestinal health function is provided, comprising: a milk-derived exosome; and a therapeutic agent, the therapeutic agent being DNA or RNA, and the therapeutic agent being located and encapsulated in the internal space of the exosome.

[0010] In another aspect, there is provided a method for producing a composition for improving intestinal health function, comprising the steps of: (a) isolating exosomes from milk; (b) electroporating the isolated exosomes to form micropores on the surface of the exosomes; (c) mixing the electroporated exosomes of step (b) with a therapeutic agent; and (d) removing the therapeutic agent that is not encapsulated inside the exosomes. Effect of the Invention

[0011] In order to overcome the low yield and high maintenance cost of extracellular vesicles derived from cells, exosomes can be easily isolated and purified in high yield from milk, which has a smooth supply and is easily accessible. In addition, instead of extracellular vesicles derived from cells, which have relatively unstable physical properties, therapeutic agents can be encapsulated in milk exosomes, which have the property of maintaining high stability even under sudden environmental changes such as pH and temperature, by electroporation. Such milk-derived exosomes have the advantage of being highly bioavailable, allowing oral administration of drugs, and are particularly useful when lesions exist in the gastrointestinal tract, such as inflammatory bowel disease. [Brief description of the drawings]

[0012] [Figure 1a] Expression levels of exosomal protein markers (Alix, Tsg-101, and CD9) and non-exosomal protein marker (GM130) are shown. [Figure 1b] 1 is a size distribution graph of milk-derived exosomes. [Figure 1c] Representative TEM images of milk-derived exosomes are shown. [Figure 2a]Confocal microscopy images of RAW264.7 and NCM460 cells cultured with 0.05 or 0.1 mg / ml Cy5.5-labeled Col-exo for 0, 1, 4, 8, 12 or 24 hours. In the figure, the dark small circles represent DAPI and the light circles represent Col-exo. [Figure 2b] Figure 1 shows the cell proliferation rate of NCM460 and RAW264.7 cells cultured with 0, 0.1, 0.2, 0.5 or 1 mg / ml milk-derived exosomes for 24 hours (n=6; ns=not significant, **p<0.01 and ****p<0.0001). [Figure 2c] 1 is a graph showing the cell viability of NCM460. [Figure 3a] The images confirm the reduction of intracellular ROS. [Figure 3b] This is a graph showing the expression of mRNA encoding inflammatory cytokines in M1 macrophages (n=4; *p<0.05, **p<0.01, ****p<0.0001). [Figure 4a-4b] 4 shows biodistribution images of Cy5.5-labeled milk-derived exosomes. Figure 4a shows a whole organ image and Figure 4b shows a cross-sectional image of the organ. [Figure 4c] FIG. 1 is a schematic diagram showing the schedule of DSS colitis induction treatment and oral administration of milk-derived exosomes. [Figure 4d] From the left, graphs show weight loss and changes in DAI index in DSS-induced colitis-induced mice (n≧10; ns=not significant, *p<0.05, **p<0.01, and ****p<0.0001), and graphs show the colon length measured after sacrificing mice on day 27 (n≧10; ns=not significant, ***p<0.001, and ****p<0.0001). [Figure 5a-5c]Figure 5 shows the effect of Example 1 in attenuating the severity of DSS-induced colitis. Figure 5a shows histopathological images of colonic tissue analyzed by hematoxylin and eosin (H&E) staining on day 27 (○ surface epithelial loss, △ ulceration, ↑ inflammatory cell infiltration). Figure 5b shows representative immunohistochemistry (IHC) images for COX-2, with the arrow indicating the location where COX-2 protein is highly expressed. Figure 5c shows representative images of colonic necrosis detected by TUNEL staining. [Figure 6a] Colonic ROS measured by chemiluminescence analysis is shown (n≧7; ns>0.05, ***p<0.001 and ****p<0.0001). [Figure 6b] Representative images of immunofluorescence (IF) staining for iNOS (blue, dark gray in the figure: DAPI; green, light gray in the figure: iNOS). [Figure 6c] Expression of mRNA encoding inflammatory cytokines in colonic tissue is shown (n=4; *p<0.05 and ***p<0.001). [Figure 7] 1 is a graph showing the stability of milk exosomes against electroporation. [Figure 8] 1 shows an image of siRNA carried by milk-derived exosomes confirmed on an agarose gel. [Figure 9] 13 is a graph comparing milk-derived exosome encapsulation efficiency of siRNA according to electroporation parameters. [Figure 10a] and [Figure 10b] 1 shows a visualization photograph of milk-derived exosomes encapsulating TNF-α siRNA and a graph showing the relative size of the fluorescent area. [Figure 11] 13 is an intracellular uptake image of milk exosomes encapsulating TNF-α siRNA (Cy3 labeled). [Figure 12] 1 shows confocal images and fluorescence intensity graphs illustrating the intracellular delivery rate of milk exosomes encapsulating TNF-α siRNA. [Figure 13] 1 is a graph showing the gene silencing effect (in vitro) of milk exosomes encapsulated with TNF-α siRNA. [Figure 14a] FIG. 1 is a schematic diagram showing IBD induction and treatment schedule. [Figure 14b] and [Figure 14c] 1 is a graph showing the observation of weight loss and changes in DAI index in IBD-induced mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Definition of Terms The term "intracellular delivery rate" used herein means the amount of encapsulated TNF-α siRNA delivered into the cells, expressed as a percentage by measuring the relative fluorescence intensity of TNF-α siRNA in the cells, based on the relative fluorescence intensity (100%) of cells stained after transfection with TNF-α siRNA using Lipofectamine.

[0014] The present invention will be described in detail below.

[0015] In one aspect, the present invention provides a composition for improving intestinal health function, comprising milk-derived exosomes; and a therapeutic agent, the therapeutic agent being DNA, RNA or a small molecule, and the therapeutic agent being located and encapsulated in the internal space of the exosome. As used herein, "located and encapsulated in the internal space of the exosome" means that a substance such as a therapeutic agent completely enters the internal space of the exosome and is encapsulated, and does not mean that the substance is present only on the surface of the exosome by a method such as attachment, adsorption or adhesion.

[0016] In another aspect of the present invention, there is provided a method for improving intestinal health function in a subject, the method comprising the step of administering to a subject in need thereof milk derived exosomes; and an effective amount of a therapeutic agent, wherein the therapeutic agent is DNA, RNA or a small molecule, and the therapeutic agent is located and encapsulated in the internal space of the exosome.

[0017] In another aspect of the present invention, there is provided a use of milk-derived exosomes; and a therapeutic agent in the manufacture of a composition for improving intestinal health function.

[0018] In other aspects of the present invention, there are provided milk-derived exosomes and therapeutic agents for improving intestinal health function.

[0019] In an exemplary embodiment, the milk may be, but is not limited to, milk from a human, cow, sheep, goat, goat, camel, buffalo, or yak, or may be colostrum.

[0020] In an exemplary embodiment, the RNA may be one or more RNAs selected from the group consisting of siRNA, miRNA, mRNA, shRNA, ncRNA, and antisense RNA, but is not limited thereto.

[0021] In an exemplary embodiment, the siRNA may be one or more siRNAs selected from the group consisting of TNF-α siRNA, TGF-β siRNA, JAK3 siRNA, and IL-6R siRNA.

[0022] In an exemplary embodiment, the miRNA may be one or more miRNAs selected from the group consisting of Let-7b-5p, miR-320a-3p, and miR-21.

[0023] In an exemplary embodiment, the composition may be in a liquid dosage form and may contain the therapeutic agent-encapsulated milk-derived exosomes at a concentration of 100 to 1000 μg / ml. Specifically, the milk-derived exosomes may have a concentration of 100 μg / ml or more, 150 μg / ml or more, 200 μg / ml or more, 250 μg / ml or more, 300 μg / ml or more, 350 μg / ml or more, 400 μg / ml or more, 450 μg / ml or more, or 500 μg / ml or more, and 1000 μg / ml or less, 950 μg / ml or less, 900 μg / ml or less, 850 μg / ml or less, 800 μg / ml or less, 750 μg / ml or less, 700 μg / ml or less, 650 μg / ml or less, 600 μg / ml or less, or 550 μg / ml or less.

[0024] In an exemplary embodiment, the composition may be in a lyophilized form and may contain the milk-derived exosomes encapsulating the therapeutic agent in an amount ranging from 0.001 to 100% by weight based on the total weight of the composition. Specifically, the content may be adjusted by those skilled in the art depending on the concentration of the milk-derived exosomes encapsulating the therapeutic agent or the type of solvent. For example, the content may be 0.001% by weight or more, 0.01% by weight or more, 0.1% by weight or more, 1% by weight or more, 5% by weight or more, 10% by weight or more, 15% by weight or more, 20% by weight or more, 25% by weight or more, 30% by weight or more, 35% by weight or more, 40% by weight or more, 45% by weight or more, or 50% by weight or more, and 100% by weight or less, 95% by weight or less, 90% by weight or less, 85% by weight or less, 80% by weight or less, 75% by weight or less, 70% by weight or less, 65% by weight or less, 60% by weight or less, or 55% by weight or less.

[0025] In an exemplary embodiment, the composition may be administered orally.

[0026] In another aspect, the present invention provides a method for producing a composition for improving intestinal health function, the method comprising the steps of: (a) isolating exosomes from milk; (b) electroporating the isolated exosomes to form micropores on the surface of the exosomes; (c) mixing the electroporated exosomes of step (b) with a therapeutic agent; and (d) removing the therapeutic agent that is not encapsulated inside the exosomes.

[0027] In an exemplary embodiment, the electroporation in step (b) may be performed by applying a voltage of 250 to 350 V. Specifically, the voltage may be 250 V or more, 255 V or more, 260 V or more, 265 V or more, 270 V or more, 275 V or more, 280 V or more, 285 V or more, 290 V or more, 295 V or more, or 300 V or more, and 350 V or less, 345 V or less, 340 V or less, 335 V or less, 330 V or less, 325 V or less, 320 V or less, 315 V or less, 310 V or less, or 305 V or less.

[0028] In an exemplary embodiment, the electroporation in step (b) may be performed by applying 3 to 7 pulses, specifically, the number of pulses may be 3 or more, 4 or more, 5 or more, and 7 or less, or 6 or less.

[0029] In an exemplary embodiment, the electroporation in step (b) may be performed by applying a pulse for a time period of 20 to 40 ms, and the pulse may be 20 ms or more, 21 ms or more, 22 ms or more, 23 ms or more, 24 ms or more, 25 ms or more, 26 ms or more, 27 ms or more, 28 ms or more, 29 ms or more, or 30 ms or more, and 40 ms or less, 39 ms or less, 38 ms or less, 37 ms or less, 36 ms or less, 35 ms or less, 34 ms or less, 33 ms or less, 32 ms or less, or 31 ms or less.

[0030] In an exemplary embodiment, the milk in step (a) may be milk from a human, cow, sheep, goat, goat, camel, buffalo, or yak, or may be colostrum.

[0031] In an exemplary embodiment, the therapeutic agent in step (c) may be DNA, and step (d) may involve adding dnase to remove any unencapsulated therapeutic agent.

[0032] In an exemplary embodiment, the therapeutic agent in step (c) may be RNA, and step (d) may involve adding RNase to remove any unencapsulated therapeutic agent.

[0033] In an exemplary embodiment, the RNA in step (c) may be one or more RNAs selected from the group consisting of siRNA, miRNA, mRNA, iRNA, ncRNA, and antisense RNA.

[0034] In an exemplary embodiment, the siRNA in step (c) may be one or more siRNAs selected from the group consisting of TNF-α siRNA, TGF-β siRNA, JAK3 siRNA, and IL-6R siRNA.

[0035] In an exemplary embodiment, the miRNA in step (c) may be one or more miRNAs selected from the group consisting of Let-7b-5p, miR-320a-3p, and miR-21.

[0036] The present invention will be described in more detail below through examples. Note that these examples are merely for the purpose of illustrating the present invention, and the scope of the present invention is not limited by these examples. Anything that has substantially the same configuration and exhibits the same effects as the technical idea described in the claims of the present invention is included in the technical scope of the present invention. EXAMPLES

[0037] 1. Confirmation of the preventive and therapeutic effects of bovine colostrum-derived exosomes on inflammatory bowel disease (1)Cell culture NCM460 and RAW264.7 cells (40071, Korea Cell Line Bank) were grown in high glucose (SH30243.01, Hyclone, USA) Dulbecco's modified eagle medium (DMEM) containing 10% fetal bovine serum (FBS, FP-0500-A, Atlas Biologicals, USA) and 1% antibiotic-antimycotic (15240096, ThermoFisher Scientific, USA). The cells were incubated in a 37°C incubator with 5% carbon dioxide (CO2). To induce M1-like macrophages, RAW264.7 cells were stimulated with 100 ng / ml LPS (L4524, sigma, USA) and 20 ng / ml IFN-γ (315-05, PeproTech, UK) for 24 h.

[0038] (2) Isolation of exosomes from colostrum Exosomes were produced by sequential centrifugation at 4°C. Bovine colostrum (obtained from Lee Seong-yeon Changbuk Farm, 20, Bongsan Gwan-dong 1-ro, Gyeseong-myeon, Changnyeong-gun, Gyeongsangnam-do) was centrifuged at 5,000xg for 30 min and 12,000xg for 1 h using a high-speed centrifuge with a JA-14 rotor (Avanti JE, Beckman Coulter, USA). The supernatant was passed through a 40 μm strainer (93040, SPL, Korea) and stored at -80°C until use. The filtered milk was centrifuged at 35,000xg for 1 h and 70,000xg for 3 h at maximum acceleration / decal using an ultracentrifuge with a 45 Ti rotor (Optima XE-100, Beckman Coulter, USA). The solution was separated into three layers, and the middle layer was filtered sequentially through syringe filters with pore sizes of 0.8, 0.45, and 0.2 μm (16592-K, 16555-K, and 16534-K, Sartorius Stedim Biotech, Germany).

[0039] The pellet, which was considered to be exosomes, was obtained by centrifugation at 100,000xg for 1 hour. The exosomes were resuspended in Dulbecco's phosphate buffered saline (DPBS, LB001, Welgene, Korea) and rotated overnight at 4°C. The exosomes thus obtained are hereinafter referred to as bovine colostrum-derived exosomes (Col-exo, Example 1).

[0040] For the preparation of exosome-free milk (Comparative Example 1), the clear solution, excluding the exosome pellet, was centrifuged at 200,000×g for 3 hours to remove remaining exosomes, and the supernatant was considered as exosome-free milk.

[0041] (3) Western blot The concentration of exosome proteins was quantified using a BCA assay kit (23225, Thermofisher Scientific, USA), and proteins were denatured with SDS-PAGE loading buffer. Samples were electrophoresed on sodium dodecyl sulfate (SDS) polyacrylamide gels. Separated proteins were transferred to nitrocellulose membranes and blocked with 5% nonfat milk. Primary antibodies were bound to target proteins for 24 h. The membranes were rinsed with tris-buffered saline, 0.1% Tween 20 detergent (TBST), and proteins were labeled by incubation with HRP-conjugated secondary antibodies for 40 min at room temperature. Labeled proteins were visualized with Clarity Western ECL substrate (1705061, Bio-Rad, USA).

[0042] The antibodies used were Alix (1:500, NB100-65678, Novus, USA), Tsg-101 (1:1000, ab83, Abcam, USA), CD9 (1:1000, NB500-494, Novus, USA), GM130 (1:500, PA1077, Thermofisher Scientific, USA), goat anti-mouse IgG antibody (HRP) (1:1000, GTX21311-01, GeneTex, USA), and goat anti-rabbit IgG antibody (HRP) (1:1000, GTX21310-01, GeneTex, USA).

[0043] As shown in Figure 1a, the results for Example 1 and Comparative Example 1 show that exosomal protein markers Alix, Tsg-101, and CD9 were confirmed in Example 1, whereas non-exosomal protein GM130 was found in Comparative Example 1. These results indicate that Comparative Example 1 (Exo-free milk) and Example 1 (Col-exo) can be distinguished by the presence or absence of exosomes.

[0044] (4) Dynamic lighting scattering (DLS) and transmission electron microscopy (TEM) For DLS, exosomes were diluted to 0.1 μg / μl with distilled water and transferred to a disposable cuvette (art.01960-00, Kartell LABWARE, Italy). The size distribution of exosomes was measured by DLS (Zetasizer Nano ZS, Malvern Instruments, UK) at a fixed angle of 173°. Three independent measurements were performed for each sample and averaged for analysis by the software.

[0045] For TEM image confirmation, 10 μg of exosomes were deposited on carbon-coated copper grids (CF200-Cu, Emgrid Australia, Australia) for 30 min at room temperature. For negative staining of exosomes, the copper grids were contrasted with uranyl acetate for 1 min. Exosome morphology was visualized by TEM (FEI Tecnai F20 G2, Philips, Netherlands).

[0046] As shown in FIG. 1b and FIG. 1c, it was confirmed that the exosomes of Example 1 had an average diameter of 85.2 nm and were spherical.

[0047] (5) Cellular uptake of exosomes Exosomes were labeled with 650 nM Cyanine 5.5 NHS ester overnight at 4°C. Unbound fragments were washed twice using an Airfuge. Cells were 2 x 10 for RAW264.7 cells. 5 Cells / dish, 3 × 10 for NCM460 cells 5 The cells were plated on confocal dishes at a density of 100 x 100 cells / dish. Exosomes were treated at various concentrations (0.05 and 0.1 mg / ml) and washed with DPBS at each time point. To capture images, cells were fixed with formaldehyde and then 4',6-diamidino-2-phenylindole (DAPI) was added to stain nucleic acids for 15 min. Images were captured with a confocal microscope (Leica TCS SP6, Leica, Germany).

[0048] (6) Cell proliferation and viability assay Cell viability analysis was performed according to the Cell counting Kit-8 (CCK-8) (CK04, Dojindo, Japan) guide. NCM460 and RAW264.7 cells were seeded in 96-well plates at a density of 6,000 and 8,000 cells / well, respectively, and allowed to attach for 24 hours. Cells were treated with various concentrations of Comparative Example 1 (milk without exosomes) or exosomes of Example 1 (0.5 mg / ml for viability analysis) for 24 hours. As an additional step for viability analysis, cells were incubated with 2.5% DSS (0216011080, MP Biomedicals, USA) solution (w / v) for 24 hours. The medium was replaced with serum-free medium containing 10% CCK-8 solution. Absorbance at 450 nm wavelength was measured with a microplate reader (SpectraMAX 340, Molecular Devices, USA). RAW264.7 cells were cultured with 100 ng / ml LPS and 20 ng / ml INF-γ for 24 hours before treatment with exosomes.

[0049] The intracellular delivery efficiency of Example 1 (Col-exo) was investigated. RAW264.7 cells were cultured with Example 1 labeled with Cy5.5 at a concentration of 0.05 or 0.1 mg / ml for 0, 1, 4, 8, 12 or 24 hours (Figure 2a). In Figure 2a, the dark small circles represent DAPI and the light circles represent Col-exo. Confocal images show that significant intracellular absorption of Example 1 (Col-exo) occurred within 8 hours, and the images show that the absorption was saturated within 24 hours. A similar pattern was also observed in NCM460 cells. Based on these data, the absorption time of Example 1 (Col-exo) was set to 24 hours in subsequent in vitro and in vivo experiments.

[0050] Next, to investigate the relationship between Example 1 (Col-exo) and cell growth, cell proliferation analysis was performed on NCM460 cells and RAW264.7 cells (Figure 2b). It can be seen that when NCM460 cells were treated with Example 1 (Col-exo) at a concentration of 0.1 mg / ml, cell proliferation significantly increased to 113.1 ± 3.4%. This cell proliferation was similarly maintained up to the concentration of 1 mg / ml of Example 1. Another cell line, RAW264.7, showed a significant increase in cell growth at a concentration of 0.5 mg / ml, especially at a concentration of 1 mg / ml, which increased to 121.8 ± 4.7%.

[0051] In addition, colon epithelial cells were damaged by dextran sulfate sodium (DSS, 36,000 - 50,000 Da), a chemical that induces inflammatory bowel disease (IBD), and the ability of Example 1 (Col-exo) to repair the external damage was observed (Figure 2c). Cell damage was caused by treatment with 2.5% DSS for 24 hours, and as a result, the cell growth rate decreased to 82.7 ± 3.7%. The group treated with Comparative Example 1 (Exo-free milk) showed a similar level of cell viability to that treated with DSS, while Example 1 (Col-exo) was able to restore cell viability to 91.8 ± 2.6%. As a result, it was found that Example 1 (Col-exo) increased the proliferation of NCM460 and RAW264.7 cells.

[0052] (7) Confirmation of anti-inflammatory effect We then used the DCF-DA assay to confirm whether bovine colostrum-derived exosomes could reduce the concentration of intracellular reactive oxygen species (ROS). 5 The cells were seeded at a density of 3 × 10 and treated with 100 ng / ml LPS and 20 ng / ml IFN-γ for 24 hours to induce M1 macrophages. 5The cells were cultured at a density of 10 cells / well for 24 hours. Exosome-free milk or exosomes were treated at a concentration of 0.1 mg / ml for 24 hours. Oxidative stress was induced by providing 100 μM H2O2 for 30 minutes and incubating 50 μM 2',7'-dichlorofluorescin diacetate (DCFDA) with the cells for 30 minutes. After washing the cells with DPBS, fluorescent images were obtained using a confocal microscope.

[0053] As shown in FIG. 3a, in both NCM460 cells and RAW264.7 cells, the intracellular ROS values ​​(light circles) in the Example 1 (Col-exo) treatment group were significantly reduced compared to the hydrogen peroxide treatment group or Comparative Example 1 (Exo-free milk) treatment group.

[0054] (8) Quantification of cytokine levels in vitro In addition, the expression levels of mRNAs encoding intracellular cytokines involved in inflammatory responses were analyzed. 5 M1-induced cells (1000 cells / ml) were plated in culture dishes and stimulated with 100 ng / ml LPS and 20 ng / ml IFN-γ for 24 h. M1-induced cells were treated with exosome-free milk or exosomes for 24 h and then washed twice with DPBS. Total RNA was extracted with Trizol™ reagent according to the manufacturer's guidelines, and cDNA was generated with oligo(dT) primer after reverse transcription. Cytokine quantification was performed by quantitative real-time PCR (qRT-PCR) using SYBR Green qPCR premix (RT501S, Enzynomics, Korea) and each primer (Table 1), and the PCR cycle was set according to the premix protocol.

[0055] [Table 1] First, RAW264.7 cells were differentiated into M1 macrophages and treated with Example 1 (Col-exo) and Comparative Example 1 (Exo-free milk), respectively. As shown in FIG. 3b, quantitative real-time PCR (qRT-PCR) data showed that the mRNA expression levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were reduced by 0.69-fold and 0.61-fold in Example 1 compared to the control group. In contrast, the mRNA expression level of interleukin-10 (IL-10), an anti-inflammatory cytokine, was increased by 1.7-fold in Example 1 (Col-exo) compared to the control group. Therefore, it was found that Example 1 has an anti-inflammatory effect at the cellular level.

[0056] (9) Ex vivo distribution of colostrum-derived exosomes Before confirming the effect of Example 1 (Col-exo) in a mouse model of colitis, the relative biodistribution in organs was measured after oral administration of Cy5.5-labeled Example 1 (Col-exo). BALB / c mice were orally administered Cy5.5-labeled exosomes. After oral gavage of the mice, the gastrointestinal tract and other organs including heart, lung, spleen, kidney, and liver were collected at 5 and 24 hours. The fluorescence intensity of the isolated organs was estimated using an in vivo imaging system (IVIS) (Lumina SeriesIII PerkinElmer, USA). The absorption of exosomes was observed from formalin-fixed, paraffin-embedded gastrointestinal organs. The paraffin-embedded tissues were sectioned to a thickness of 5 μm. Representative images were captured using a confocal microscope.

[0057] As a result, as shown in Figure 4a, when the fluorescence intensity was measured 5 hours after feeding Cy5.5-labeled Col-exo, a strong signal was observed in the gastrointestinal tract, especially in the colon. It was also confirmed that the fluorescence signal was significantly maintained in the stomach and colon at 24 hours. Meanwhile, as can be seen from Figure 4b, the stomach, small intestine, and colon were sliced, respectively, and the absorption of Cy5.5-labeled Example 1 (Col-exo) into the organs was observed. The majority of the absorption of Cy5.5-labeled Example 1 (Col-exo) was found in the stomach and colon. It was confirmed that the absorbed exosomes were significantly reduced after 24 hours. For this reason, Example 1 was orally administered to the mice every day.

[0058] (10) Estimation of inflammatory responses in a mouse model of colitis Eight-week-old BALB / c female mice (Orient Bio, Korea) were used for the ulcerative colitis model. The mice were randomly divided into three groups: healthy control (saline), DSS-induced colitis (DSS), and Col-exo pretreatment (Example 1), and were allowed to adapt for 10 days. The Example 1 (Col-exo) group was pretreated with 1 mg of colostrum-derived exosomes by oral gavage every day for one week, while the other groups were given saline only. Inflammatory bowel disease (IBD) was chemically induced by administering 2.5% DSS solution ad libitum in drinking water for one week. On day 27, the mice were sacrificed and colon length was estimated (Figure 4c).

[0059] 1) Disease Activity Index (DAI) Disease activity indices were assessed by three independent observers for weight loss, fecal viscosity, and fecal bleeding characteristics for each mouse daily from days 18 to 27 (Table 2). Total scores range from 0 to 12, with higher scores indicating greater disease severity.

[0060] [Table 2] As a result of carrying out the animal experiment according to the set schedule, the DSS group showed rapid weight loss, whereas the Example 1 (Col-exo) group effectively suppressed weight loss in mice with colitis induced by DSS (Figure 4d).

[0061] 2) Ex vivo ROS imaging On day 27, L-012 (120-04891, Fujifilm Wako Chemicals, Japan) was dissolved in DPBS and stored in a dark room at room temperature. L-012 solution was intraperitoneally injected at a dose of 20 mg / kg, and the mouse colon was isolated 15 min later. ROS in the isolated colon was imaged by an in vivo imaging system (IVIS) (Lumina Series III PerkinElmer, USA). Data were measured for 5 min (p / s / cm 2 / sr)

[0062] As a result of carrying out the animal experiment according to the set schedule, the DSS group showed rapid weight loss, while the Example 1 (Col-exo) group effectively suppressed weight loss in mice with DSS-induced colitis. In addition, the DAI (disease activity index) score of the Example 1 (Col-exo) group was significantly lower than that of the DSS group. The length of the colon of the Example 1 (Col-exo) group was significantly longer than that of the DSS group, and recovered to a similar level to that of the saline group (Figure 4d). Through the above experiment, it was confirmed that Example 1 alleviates DSS-induced colitis.

[0063] (11)Histological analysis Isolated mouse colon tissue was prepared by the Swiss-roll technique. Colon samples were fixed overnight in formalin, dehydrated, and embedded in paraffin. All tissue specimens were cut at a thickness of 5 μm.

[0064] 1) H&E (Hmatoxylin and eosin) Sections were dewaxed with xylene and stained with hematoxylin. After rinsing with tap water, nuclear staining was completed with a bluing agent. Proteins were nonspecifically stained with eosin (ab245880, Abcam, USA). Slides were mounted with toluene and observed using a light microscope (BX51, Olympus Corporation, Japan).

[0065] As a result, severe superficial epithelial damage, ulcers, and infiltration of immune cells were confirmed in the colon of the DSS group ( FIG. 5 a), whereas the group of Example 1 showed slight epithelial proliferation.

[0066] 2) Immunohistochemistry (IHC) Because COX-2 is a core molecule that induces the initiation of inflammation, we used immunohistochemistry (IHC) to examine the expression level of COX-2 protein in each group. The expression of COX-2 from colonic tissues was detected using a Mouse and Rabbit Specific HRP / DAB (ABC) Detection IHC kit (ab64264, Abcam, USA) according to the manufacturer's protocol. The target protein was tagged with COX-2 antibody (1:500, ab15191, Abcam, USA) in PBS containing 1% BSA and 0.5% Triton X-100. Tissue sections were stained with DAB substrate and counterstained with hematoxylin. Images were captured by light microscope.

[0067] As can be seen from FIG. 5b, the expression of COX-2 protein was decreased in the large intestine of the Example 1 (Col-exo) group compared to the DSS group.

[0068] 3) Immunofluorescence (IF) To visualize the extent of tissue cell death, TUNEL analysis was performed according to the manufacturer's guidelines for the fluorometric TUNEL System (G3250, Promega, USA). Rehydrated tissue sections were fixed with formaldehyde and proteinase K solution was added for permeabilization. Formaldehyde fixation was repeated and incubated with equilibration buffer. Apoptotic areas were displayed by terminal deoxynucleotidyl transferase (TdT) reaction and counterstained with DAPI.

[0069] As shown in Figure 5c, colon cell death was severely observed in the DSS group and significantly improved in the Example 1 (Col-exo) group, as shown by the TUNEL staining data. As a result, Example 1 was effective in reducing the severity of colitis and cell death.

[0070] (12) Quantification of in vivo cytokine levels To confirm the effect of Col-exo on alleviating colonic inflammation, colonic ROS levels were measured using a luminol-based chemiluminescence probe. Mouse colon tissues were homogenized using a homogenizer (HG-15D, Wise Tis, Korea) with TrizolTM reagent. After homogenization, total RNA of colonic tissue was obtained according to the procedure manual of TrizolTM reagent. After cDNA synthesis, qRT-PCR was performed using SYBR Green (RT501M, Enzynomics, Korea) according to the manufacturer's protocol. All qRT-PCR was performed using a QuantStudioTM1 Real-Time PCR instrument (Applied Biosystems, USA). Each primer is summarized in (Table 1).

[0071] As a result, the ROS level was significantly increased in the DSS group and decreased to less than half in the Example 1 (Col-exo) group (FIG. 6a).

[0072] (13) Confirmation of the effect of improving intestinal inflammation Next, to confirm the effect of Col-exo in improving intestinal inflammation, we used immunofluorescence (IF) to confirm the expression of iNOS protein, a marker of infectious M1 macrophages. Infiltration of macrophages in mouse colon tissue was detected by staining for iNOS. Deparaffinized tissues were rehydrated and boiled in antigen retriever buffer (Citrate buffer pH 6.0) (ab93678, Abcam, USA). Slides were washed with PBS, 0.05% Tween 20 detergent (PBST), and blocked with PBST containing 5% BSA (w / v) for 10 minutes at room temperature. Sections were stained overnight at 4°C with Alexa fluor 488-conjugated iNOS monoclonal antibody (53-5920-82, eBiosience, USA) prepared in PBS containing 1% BSA (w / v) and 0.5% Triton-100 (v / v). Nucleic acids were stained with DAPI for 15 minutes at room temperature. The slides were mounted with water-soluble mounting solution (0100-01, Southern Biotech, USA) and imaged with a confocal microscope. As a result, as shown in Figure 6b, iNOS protein was overexpressed in the DSS group, whereas it was hardly observed in the Example 1 (Col-exo) group.

[0073] In addition, qRT-PCR was used to examine the mRNA expression of inflammatory cytokines (Figure 6C). The results of qRT-PCR showed that the mRNA levels of TNF-α and IL-6 were increased 3.8-fold and 3.6-fold, respectively, in the DSS group compared to the saline group. In contrast, these mRNA expression levels were found to be similar to those in the saline group. However, the results of qRT-PCR for anti-inflammatory cytokines showed the opposite trend, with the IL-10 mRNA expression level in the DSS group decreasing by about 20-fold compared to the saline group. In particular, the IL-10 expression level in the Example 1 (Col-exo) treatment group was slightly restored to about 5-fold that of the DSS treatment group. These results indicate that Example 1 reduces excessive intestinal inflammatory responses.

[0074] (14) Statistical analysis Statistical analysis of data was performed using parametric one-way ANOVA and two-way ANOVA with Prism 8.0. All results were expressed as mean ± standard deviation. All graphs were constructed with Prism 8.0 (GraphPad Software Inc, USA).

[0075] 2. Efficacy of RNA-encapsulated milk-derived exosomes in treating inflammatory bowel disease (1) Extraction of exosomes The milk used was commercially available pasteurized milk, and exosomes were extracted using the same method as described in Example 1.(2) above.

[0076] (2) Confirmation of exosome stability against electroporation 0.1 mg / ml cell-derived exosomes (HEK exo) and milk-derived exosomes (milk exo) were subjected to electric shock of 300 V, 30 ms, 5 pulses, and 1 s interval, respectively, and incubated at 4° C. for 3 hours. The size change of the exosomes before and after the electric shock was observed using the same method as the dynamic light scattering (DLS) experimental method in Example 1.(4).

[0077] As a result, as shown in FIG. 7, it was confirmed that the size of cell-derived exosomes decreased after electroporation, whereas the size distribution of milk-derived exosomes remained almost unchanged before and after electroporation.

[0078] (3) Encapsulation of siRNA After creating micropores in the exosomes by electroporation, TNF-α siRNA was mixed into the exosome solution to encapsulate TNF-α siRNA inside the exosomes, and RNase was used to remove siRNA that was not encapsulated inside the exosomes. Non-electroporated exosomes, electroporated exosomes, the result of mixing non-electroporated exosomes with TNF-α siRNA, and electroporated exosomes mixed with TNF-α siRNA and encapsulated exosomes were loaded onto a 1% agarose gel and electrophoresed at 100V for 10 minutes in an electrophoresis apparatus. Then, the gel image analyzer was used to visually confirm that TNF-α siRNA was efficiently encapsulated inside the exosomes when the exosomes were electroporated, as shown in FIG. 8. Hereinafter, the exosomes in which TNF-α siRNA is encapsulated are referred to as Example 2, and the exosomes in which TNF-α siRNA is not encapsulated are referred to as Comparative Example 2.

[0079] (4) Comparison of siRNA entrapment efficiency by electroporation parameters To confirm the voltage conditions, the number of electroporations was fixed at 5 times and the time was fixed at 30 ms, and the encapsulation efficiency of TNF-α siRNA was compared when voltages of 200, 250, 300, 350, and 400 V were applied to 0.1 mg / ml milk-derived exosomes. To confirm the electroporation number conditions, the voltage was fixed at 300 V and the time was fixed at 30 ms, and the encapsulation efficiency of TNF-α siRNA was compared when 1, 2, 3, 4, and 5 electroporations were applied. To confirm the electroporation time conditions, the voltage was fixed at 300 V, the number of electroporations was fixed at 5 times, and the encapsulation efficiency of TNF-α siRNA was compared when electric shocks were applied for 10, 20, 30, 40, 50, and 60 ms. It was confirmed that TNF-α siRNA was most efficiently encapsulated in exosomes when electroporation was performed at 300 V, 5 times, and for 30 ms (FIG. 9).

[0080] (5) Visualization of milk-derived exosomes encapsulating TNF-α siRNA Milk-derived exosomes were subjected to an electric shock of 300V, 30ms, 5pulses, and 1s interval, and then TNF-α siRNA (FAM labeled) was encapsulated and incubated for 3 hours, and milk-derived exosomes were labeled with Cy5.5. Milk-derived exosomes encapsulating TNF-α siRNA (Example 2) were mixed with Fluoromount-G solution and fixed on a slide glass. Milk-derived exosomes encapsulating TNF-α siRNA were then measured and visualized in the x, y, and z directions by super resolution microscopy (SRM) (FIG. 10a), and the relative fluorescent area was plotted (FIG. 10b). Looking at the data after encapsulating TNF-α siRNA, in the case of Example 2, it was confirmed that TNF-α siRNA labeled with green (dark gray in the figure) FAM was located in the center of the exosome labeled with red (white in the figure) Cy5.5. In addition, in the case of the relative fluorescence area, Cy5.5, which showed a fluorescence area of ​​over 100 in Comparative Example 2 containing only exosomes, decreased to below 100 when TNF-α siRNA was encapsulated, and it was confirmed that the fluorescence area labeled with FAM relatively increased. This means that TNF-α siRNA was not carried on the surface of the exosome by adhesion or other means, but rather TNF-α siRNA was accurately inserted into the center of the internal region of the exosome through minute gaps generated by electroporation and encapsulated.

[0081] (6) Confirmation of cellular uptake of milk-derived exosomes encapsulating TNF-α siRNA Cavities were formed in milk-derived exosomes by the same electroporation method as in Example 2, Cy3-labeled TNF-α siRNA was encapsulated, and the mixture was incubated for 3 hours. The exosomes were then treated with NCM460 cells, and the degree of cellular absorption was visualized by confocal microscopy after 24 hours. As shown in FIG. 11 (in the figure, dark gray indicates DPAI, and bright small dots indicate siRNA), it can be confirmed that in Example 2, in which TNF-α siRNA was encapsulated in exosomes, the inflow into cells was more efficient than in the case where cells were treated with only TNF-α siRNA.

[0082] (7) Confirmation of intracellular delivery rate of milk-derived exosomes encapsulating TNF-α siRNA by electroporation The same electroporation method as in Example 2 was used to form voids in milk-derived exosomes, and Cy3-labeled TNF-α siRNA was encapsulated and stabilized for 3 hours at 4°C. In the case of RAW264.7 cells, 3 × 10 5 The cells were plated on a confocal dish at a density of 1000 x 1000 cells / dish. As a control, TNF-α siRNA was transfected using Lipofectamin. RAW264.7 cells were treated with the control and Example 2, respectively, and after 24 hours, the cells were washed with DPBS, fixed with formaldehyde, and then stained with Hoechst33342 for 10 minutes. The stained cells were imaged for relative fluorescence intensity using a confocal microscope (n=4). The fluorescence intensity of the captured images was measured by the Image J program.

[0083] As shown in FIG. 12 (in the figure, dark gray indicates Hoechst and light gray indicates TNF-si-RNA), the relative TNF-α siRNA fluorescence intensity in cells treated with the control group was set to 100 (Lipofectamin: 100±17.19), which was set as an intracellular delivery rate of 100%. In comparison, the relative TNF-α siRNA fluorescence intensity in cells treated with Example 2 was about 75% (Example 2: 75.04±11.25). In other words, it can be seen that the intracellular delivery rate of Example 2 is about 75% (t-test: ns).

[0084] (8) Confirmation of gene silencing effect of milk-derived exosomes encapsulating TNF-α siRNA (in vitro) Cavities were formed in milk-derived exosomes under the same conditions as the electroporation method, and milk-derived exosomes and TNF-α siRNA were mixed at a ratio of 1 mg:14 μg and incubated for 3 hours. Milk-derived exosomes (Example 2) encapsulating TNF-α siRNA were treated at different concentrations in RAW264.7 cells pre-activated with LPS (1 μg / ml, 24 hours). After 24 hours, the level of TNF-α mRNA was measured by qRT-PCR. As a result, it was confirmed that the expression of TNF-α mRNA was reduced in a concentration-dependent manner (FIG. 13).

[0085] (9) Anti-inflammatory effect (in vivo) Inflammatory bowel disease (IBD) animal model (BALB / c, female, 8 weeks) was induced with DSS 2.5%. Milk-derived exosomes (Comparative Example 2) and milk-derived exosomes encapsulating TNF-α siRNA (Example 2) were intraperitoneally injected at 3-day intervals, and the body weight change and disease activity index (DAI) were measured. The scores are shown in Table 2. The length of the intestine and the colonic tissue were observed by H&E, and the H&E experimental method was the same as that in Example 1.(12).

[0086] As a result of intraperitoneal injection of Example 2 at 3-day intervals, it was confirmed that the weight loss typically observed in the IBD model was alleviated (FIG. 14b), and the DAI score was reduced (FIG. 14c), indicating that milk exosomes encapsulating TNF-α siRNA have a therapeutic effect on IBD inflammation.

[0087] Examples - Example 1: A composition for improving intestinal health function, comprising: milk-derived exosomes; and a therapeutic agent, the therapeutic agent being DNA, RNA or a small molecule, and the therapeutic agent being located and encapsulated in the inner space of the exosome.

[0088] - Specific Example 2: A composition for improving intestinal health function as described in Specific Example 1, characterized in that the composition has an intracellular delivery rate of 60 to 90%.

[0089] - Example 3: A composition for improving intestinal health function according to Examples 1-2, wherein the milk is cow's milk from a human, cow, sheep, goat, goat, camel, buffalo or yak, or is colostrum.

[0090] - Specific Example 4: A composition for improving intestinal health function according to Specific Examples 1 to 3, wherein the RNA is one or more RNAs selected from the group consisting of siRNA, miRNA, mRNA, shRNA, ncRNA, and antisense RNA.

[0091] - Specific Example 5: A composition for improving intestinal health function described in Specific Example 4, wherein the siRNA is one or more siRNAs selected from the group consisting of TNF-α siRNA, TGF-β siRNA, JAK3 siRNA, and IL-6R siRNA.

[0092] - Example 6: A composition for improving intestinal health function described in Example 4, wherein the miRNA is one or more miRNAs selected from the group consisting of Let-7b-5p, miR-320a-3p, and miR-21.

[0093] - Example 7: A composition for improving intestinal health function according to Examples 1 to 6, wherein the composition is in a liquid dosage form and contains milk-derived exosomes encapsulating the therapeutic agent at a concentration of 100 to 1000 μg / ml.

[0094] - Example 8: A composition for improving intestinal health function according to Examples 1 to 7, wherein the composition is in a lyophilized dosage form and contains milk-derived exosomes encapsulating the therapeutic agent in an amount ranging from 0.001 to 100% by weight based on the total weight of the composition.

[0095] - Example 9: A composition for improving intestinal health function according to any one of Examples 1 to 8, wherein the composition is administered orally.

[0096] - Example 10: A method for producing a composition of Examples 1 to 9, comprising the steps of: (a) isolating exosomes from milk; (b) electroporating the isolated exosomes to form micropores on the surface of the exosomes; (c) mixing the electroporated exosomes of step (b) with a therapeutic agent; and (d) removing the therapeutic agent that is not encapsulated inside the exosomes.

[0097] - Example 11: The method described in Example 10, wherein the electroporation in step (b) is performed by applying a voltage of 250 to 350 V.

[0098] - Example 12: The method described in Examples 10 to 11, wherein the electroporation in step (b) is characterized by applying 3 to 7 pulses.

[0099] - Example 13: The method described in Examples 10 to 12, wherein the electroporation in step (b) is characterized by applying a pulse for a time period of 20 to 40 ms.

[0100] - Example 14: The method described in Examples 10 to 13, characterized in that the milk in step (a) is milk from a human, cow, sheep, goat, goat, camel, buffalo or yak, or is colostrum.

[0101] - Example 15: The method described in Examples 10 to 14, wherein the therapeutic agent in step (c) is DNA, and step (d) is a step of adding dnase to remove any unencapsulated therapeutic agent.

[0102] - Example 16: The method described in Examples 10 to 15, wherein the therapeutic agent in step (c) is RNA, and step (d) is a step of adding RNase to remove any unencapsulated therapeutic agent.

[0103] - Example 17: The method described in Example 16, wherein the RNA is one or more RNAs selected from the group consisting of siRNA, miRNA, mRNA, shRNA, ncRNA, and antisense RNA.

[0104] - Example 18: The method described in Example 17, wherein the siRNA is one or more siRNAs selected from the group consisting of TNF-α siRNA, TGF-β siRNA, JAK3 siRNA, and IL-6R siRNA.

[0105] Example 19: The method described in Example 17, wherein the miRNA is one or more miRNAs selected from the group consisting of Let-7b-5p, miR-320a-3p, and miR-21.

Claims

1. milk-derived exosomes; and a therapeutic agent, the therapeutic agent is DNA, RNA or a small molecule; A composition for improving intestinal health function, wherein the therapeutic agent is encapsulated by being located in the internal space of the exosome.

2. The composition for improving intestinal health function according to claim 1, characterized in that the composition has an intracellular delivery rate of 60-90%.

3. 2. The composition for improving intestinal health function according to claim 1, wherein the milk is milk from a human, cow, sheep, goat, goat, camel, buffalo or yak, or is colostrum.

4. The composition for improving intestinal health function according to claim 1 , wherein the RNA is one or more RNAs selected from the group consisting of siRNA, miRNA, mRNA, shRNA, ncRNA, and antisense RNA.

5. The composition for improving intestinal health function according to claim 4 , wherein the siRNA is one or more siRNAs selected from the group consisting of TNF-α siRNA, TGF-β siRNA, JAK3 siRNA, and IL-6R siRNA.

6. The composition for improving intestinal health function according to claim 4 , wherein the miRNA is one or more miRNAs selected from the group consisting of Let-7b-5p, miR-320a-3p, and miR-21.

7. The composition is in liquid form, 2. The composition for improving intestinal health function according to claim 1, comprising the milk-derived exosomes encapsulating the therapeutic agent at a concentration of 100 to 1000 μg / ml.

8. The composition is in a lyophilized form; 2. The composition for improving intestinal health function according to claim 1, comprising the milk-derived exosomes encapsulating the therapeutic agent in an amount ranging from 0.001 to 100% by weight based on the total weight of the composition.

9. The composition for improving intestinal health function according to claim 1, wherein the composition is administered orally.

10. A method for producing a composition according to any one of claims 1 to 9, comprising the steps of: (a) isolating exosomes from milk; (b) electroporating the isolated exosomes to form micropores on the surface of the exosomes; (c) mixing the electroporated exosomes of step (b) with a therapeutic agent; and (d) removing the therapeutic agent not encapsulated inside the exosomes.

11. The method according to claim 10, wherein the electroporation in step (b) is performed by applying a voltage of 250 to 350V.

12. The method according to claim 10, wherein the electroporation in step (b) is performed by applying 3 to 7 pulses.

13. The method according to claim 10, wherein the electroporation in step (b) is performed by applying a pulse for a time period of 20 to 40 ms.

14. 11. The method according to claim 10, wherein the milk in step (a) is milk from a human, cow, sheep, goat, goat, camel, buffalo or yak, or is colostrum.

15. The therapeutic agent in step (c) is DNA; The method of claim 10, wherein step (d) is the addition of dnase to remove unencapsulated therapeutic agent.

16. The therapeutic agent in step (c) is RNA; The method of claim 10, wherein step (d) is a step of adding rnase to remove unencapsulated therapeutic agent.

17. The method of claim 16 , wherein the RNA is one or more RNAs selected from the group consisting of siRNA, miRNA, mRNA, shRNA, ncRNA, and antisense RNA.

18. The method of claim 17, wherein the siRNA is one or more siRNAs selected from the group consisting of TNF-α siRNA, TGF-β siRNA, JAK3 siRNA, and IL-6R siRNA.

19. The method of claim 17, wherein the miRNA is one or more miRNAs selected from the group consisting of Let-7b-5p, miR-320a-3p, and miR-21.

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