KR20190023450A - Drug delivery system for targeting lymph node using exosome from serum - Google Patents

Abstract

The present invention relates to uses of exosomes derived from mice, cattle, and humans while having a diameter of 100 nm or less, and more specifically, to a lymph node targeting a drug delivery agent having the exosomes with biomolecules encapsulated therein and an immunity enhancing composition thereof. The lymph node targeting drug delivery agent can effectively deliver drugs to lymph nodes. Yields of the serum-derived exosomes can also be increased.

Description

[0001] DRUG DELIVERY SYSTEM FOR TARGETING LYMPH NODE USING EXOSOME FROM SERUM [0002]

The present invention relates to a lymph node targeted drug delivery system and a composition for enhancing immunity using serum-derived exosomes.

The exosome is a kind of small vesicle with a membrane structure of 50 - 200 nm that is secreted out of the cell with protein, DNA, and RNA for intercellular signaling, and can be separated from blood, urine, serum and the like. Exosomes are used for various purposes such as drug delivery systems and vaccines. In recent years, the use of exosomes secreted from tumor cells in the bloodstream to diagnose cancer has been studied.

Currently, studies have been reported to deliver antigens or nucleotides using dendritic cells or exocomes isolated from tumors. However, because exonsome isolated from cells or tumors has a low yield and has a size of 100 nm or more, Is used as a drug delivery vehicle.

Korean Patent Publication No. 10-2016-0055687

In the present invention, the present inventors have completed the present invention by confirming that exosome of 100 nm or less can be isolated from serum to be used as a drug delivery vehicle targeting lymph nodes.

To achieve the above object, the present invention provides a lymph node target-oriented drug delivery system in which a biomolecule is encapsulated in exosomes having a diameter of 100 nm or less derived from a mouse, a bovine, or a human serum.

The present invention also provides a composition for immunomodulating an immune enhancer encapsulated in an exosome having a diameter of 100 nm or less derived from a mouse, a bovine or a human serum.

The serum-derived exosomes according to the present invention can be separated from the serum and obtained at a high yield, and have a size of 100 nm or less, so that the drug can be effectively delivered to the lymph node.

FIG. 1 is a diagram illustrating a process of extracting exosomes by extracting serum from a blood sample of a mouse, a cow, and a human, centrifuging after mixing with an ExoQuick solution, and b) Schematic diagram showing that the drug is delivered to the lymph node when the drug is injected into the extracted exosome.
Figure 2 is a TEM image of exosomes isolated from bovine and rat serum in one embodiment of the present invention.
FIG. 3 is a graph showing the emission intensity of a fluorescent dye encapsulated in exosomes isolated from human and bovine serum in RAW 264.7 cells, according to an embodiment of the present invention.
Figure 4 is an image showing the degree of lymph node targeting of exosomes isolated from human and bovine serum in RAW 264.7 cells, in one embodiment of the present invention.
FIG. 5 is a graph showing the amount of cytokine secreted by a drug delivery system using exosomes isolated from bovine serum in RAW 264.7 cells according to an embodiment of the present invention. FIG.
Figure 6 shows the amount of cytokine secreted by the drug delivery system using exosomes isolated from bovine serum in RAW 264.7 cells (a and b) and dendritic cells (BMDC) (c and d) in one embodiment of the present invention Graph.
FIG. 7 is a graph showing cell viability by free MPLA encapsulated in the free MPLA and MPLA according to an embodiment of the present invention. FIG.

Hereinafter, the present invention will be described in detail with reference to embodiments of the present invention. It should be understood, however, that the invention is not limited thereto and that various changes and modifications may be made therein without departing from the spirit and scope of the invention as defined by the appended claims and their equivalents. .

The present invention provides a lymph node targeted drug delivery system in which biomolecules are encapsulated in exosomes having a diameter of 100 nm or less derived from a mouse, a bovine, or a human serum.

The present invention also provides a composition for immunomodulating an immune enhancer encapsulated in an exosome having a diameter of 100 nm or less derived from a mouse, a bovine or a human serum.

The exosomes can be isolated from rats, small or human sera by the process shown in Fig. Specifically, serum can be extracted from blood of rats, cows, and humans by centrifugation, and the mixture can be centrifuged by mixing with ExoQuick solution to extract exosomes.

The extracted exosome may be injected into the individual after the biomolecule is encapsulated to transfer the biomolecule to the lymph node (FIG. 1B). The subject may be a mammal, for example a human.

The exosome derived from the serum of the bovine or the mouse may have a diameter of 50 nm or less, and the exosome derived from the human serum may have a diameter of 100 nm or less.

Exosomes derived from the serum of cows or rats may have a zeta potential of -30 mV or less, for example, -25 mV or less, and the exosomes derived from the human serum may be -20 mV or less, for example, And may have a zeta potential of less than -15 mV.

The biomolecule can be used without limitation as long as it is a drug commonly used in the art, and can be, for example, an anticancer agent or an immunostimulant.

The anticancer agent can be used without limitation as long as it is usable in the art and includes, for example, cisplatin, carboplatin, procarbazine, mechlorethamine, cyclophosphamide but are not limited to, cyclophosphamide, ifosfamide, melphalan, chlorambucil, bisulfan, nitrosourea, dactinomycin, daunorubicin, , Doxorubicin, bleomycin, plicomycin, mitomycin, etoposide, tamoxifen, paclitaxel, transplatinum, and the like. 5-fluorouracil, adriamycin, vincristin, vinblastin, methotrexate, and the like.

The immunostimulant may be used without limitation as long as it is usable in the art, and may include a toll-like receptor agonist, saponin, aluminum salt, ovalbumin, and the like. The immunostimulants include, for example, QS21, Quil A, QS7, QS17, CpG, monophosphoryl lipid A (MPLA), imiquimod, recipe mod, flagellin, poly (I: ], Ovalbumin, and the like.

The drug delivery or immunoconjugate composition targets the lymph node, and the drug or the immunostimulating agent can be transported to the lymph node for immunological activity.

The present invention will be described in more detail with reference to the following examples. However, the following examples are only for the purpose of illustrating the present invention, and thus the present invention is not limited thereto.

Comparative Example 1. Extraction and isolation of exosome from cells

RAW 264.7 cells (1.5 x 10 ⁷ cells / T75 flask), one of the macrophage cells, were cultured in serum-containing medium for 2 days and then cultured in an exosome depleted 10% FBS medium for 24 hours. , And the supernatant was obtained and mixed with ExoQuick-TC at a volume ratio of 1: 0.2. The mixed solution was incubated at 4 ° C for 12 hours or more, and then the supernatant was removed by centrifugation at 1500 xg for 30 minutes, and the supernatant was completely removed by centrifugation for an additional 5 minutes. The obtained pellet was used by dissolving in 1 x PBS.

Example 1. Isolation of exosomes from rats, human and bovine serum

Rat and human serum were diluted by mixing 550 μl and 1 μl PBS, respectively, and then filtered through a 0.22 μm filter which was sufficiently wet with 5 ml of 1 × PBS. The filtered serum was centrifuged at 10000 x g for 30 minutes, and then the supernatant was obtained and mixed with exo-quick at a volume ratio of 1: 0.2. The mixed solution was sufficiently inverted and then incubated overnight at 4 ° C. The next day, the supernatant was removed by centrifugation at 1500 x g for 30 minutes, centrifugation was further carried out for 5 minutes, the supernatant was completely discarded, and the pellet was dissolved in 100 ul of 1 x PBS.

One milliliter of bovine serum was centrifuged at 3000 x g for 15 minutes, and the supernatant was obtained. The supernatant was mixed with 1: 0.2 volume ratio of exo-quick. The mixture was sufficiently inverted and then incubated overnight at 4 < 0 > C. The next day, the supernatant was removed by centrifugation at 1500 x g for 30 minutes, centrifuged at the same rcf for 5 minutes, the supernatant was completely discarded, and the pellet was dissolved in 100 μl of 1 x PBS.

Experimental Example 1. Characterization of isolated exosomes

The exosomes extracted as in Example 1 were assayed using DLS and Zeta potential for characterization. To compare the yields, three types of exosomes were quantified using the BCA assay used for protein determination. The results are shown in Table 1 below. The exosomes isolated from the serum of bovine and mouse have a diameter of 50 nm or less and a surface charge of about 20 vmV. The exosomes isolated from human serum have a diameter of about 100 nm Diameter and a surface charge of about 12 vmV. Protein quantification values in serum were 1.52 ± 0.25, 13.12 ± 4.55, and 15.49 ± 2.36 mg / ml in the order of cattle, mouse and human, respectively, and were found to be about 10 times smaller in bovine serum than in mouse and human serum.

In addition, the exosomes isolated from bovine serum and mouse serum were transferred to a 300 mesh copper grid at a protein concentration of 300 μg or more, followed by washing three times with DW. The TEM was then measured by coating a grid of exosomes with uranyl acetate. As a result, a vesicle having a diameter of 50 nm or less was observed, as shown in FIG. 2, and a larger number of vesicles were confirmed in bovine serum despite the similar amount of protein.

Example 2: Preparation of a drug delivery vehicle encapsulated with an immunostimulant

Immunostimulators OVA, CpG and MPLA (monophosphoryl lipid A) were encapsulated using exosomes extracted from bovine serum. The FITC labeled OVA and CpG were inserted into the exosome using electroporation and MPLA was sutured by the hydrophobic nature of the plasma membrane of lipid A and exosomes.

 For OVA-FITC, 160 ug OVA-FITC and 160 ug exosomes were mixed with 160 mM of 50 mM trehalose in 1 x PBS, followed by electroporation at 0.2 kV pulse. Then, 1 x PBS was added so that the sample volume became 500 μl using 300 k nanosep, and centrifugation was performed at 5000 x g for 10 minutes. This procedure was repeated three times, and the supernatant was obtained to prepare the exosome in which OVA-FITC was encapsulated. Loading efficiency was quantified by FITC fluorescence.

 For CpG, 40 ug CpG and 160 ug exosomes were mixed with 80 mM of 50 mM trehalose in 1 x PBS, followed by electroporation with a 0.2 kV pulse. To completely remove the unloaded CpG, DNase I was added and reacted at 37 ° C for 1 hour. Then, 1 × PBS was added to the sample volume of 500 μl using 300 k nanosep and centrifuged at 5000 × g for 10 minutes . This procedure was repeated three times, and the supernatant was obtained to prepare CpG-encapsulated exosomes. Loading efficiency was confirmed by the picogreen assay known as DNA quantitation kit for CpG.

 For MPLA, 400 ug exosomes and 4 ug MPLA were reacted in 10% DMSO in 400 μl of DW for 2 hours at 37 ° C., DW was added so that the sample volume was 500 μl using 300 k nanosep, And centrifuged for 10 minutes. This procedure was repeated three times, and the supernatant was obtained. MPLA was quantitated using LAL kit to prepare the exosome encapsulated with MPLA. The amount of CpG and MPLA encapsulated in the exosomes was then checked by BCA assay (Table 2).

Experimental Example 2. In vivo experiment of exosomes isolated from cells and plasma

In order to demonstrate the transfer to the lymph node, which is one of the lymphatic systems using exosomes, fluorescent dyes were encapsulated in the exosomes prepared as in Comparative Example 1 and Example 1 (Fig. 3 Table 3). For fluorescent dyes, ICG-TBAI (Indocyanine green Tetrabutylammonium iodide), an ionic complex that was used in previous studies to confirm lymph node targeting, was used. 200 ug and 20 ug of ICG-TBAI, respectively, in RAW 264.7 cells, human serum and bovine serum were mixed in 130 μl of 7.7% DMSO in 1 x PBS and reacted for 2 hours at 37 ° C. Thereafter, the glass dyes were removed using a 40k desalting column to prepare exosome encapsulated with ICG-TBAI.

As can be seen in FIG. 3 and Table 3, exosomes isolated from cells have a diameter of 200 nm or more, whereas exosomes isolated from human and bovine serum were found to be about 100 nm or 50 nm Of the diameter.

The ICR-TBAI in Bovine EXO, ICG-TBAI in Bovine EXO, and ICG-TBAI in RAW EXO, ICG-TBAI in RAW EXO and ICG- ul. As shown in Fig. 4, ICG-TBAI in Bovine EXO was found to be 2.2, 2.19, and 2.9 in the order of free ICG-TBAI, ICG-TBAI in RAW EXO, and ICG-TBAI in Human in popliteal LN, Which is 1.87 times higher than that of the other samples. As a result, effective lymph node targeting of ICG-TBAI in Bovine EXO was confirmed.

Experimental Example 3. Confirmation of secretion amount of cytokine

In order to confirm the activity and the cellular uptake of CpG encapsulated CpG encapsulated CpG prepared in Example 2, 0, 0.5 and 1.5 ug / ml of CpG were treated in RAW 264.7 cells to obtain 24 After culturing for a period of time, medium was obtained and the secretion amount of TNF-a was quantified by ELISA. As a result, as shown in FIG. 5, when CpG in EXO was treated with CpG at a concentration of 0.5 and 1.5 ug / ml, the amount of TNF-a was 12.5 and 8.5 times higher than that of Free CpG, respectively.

Free MPLA and MPLA in EXO were treated with macrophage RAW 264.7 cells and dendritic cells (BMDC) in order to confirm that the immunosuppressive agent encapsulated in the exosomes isolated from bovine serum was effectively active. For RAW 264.7 cells, cells were inoculated at 12 × 10 ⁵ cells / well in a 12-well plate and cultured for 24 hours. Free MPLA and MPLA in EXO were incubated with 24, Time. Transfection of MPLA (adjuvant) through the obtained medium was then confirmed as a cytokine (TNF-a, IL-6) expressed in the cells. The cytokines of TNF-a and IL-6 were quantified using an ELISA kit. Primary dendritic cells (BMDC) were inoculated in 96-well plates at 5 × 10 ⁴ cells / well. After 24 hours, the cells were treated with the same MPLA concentration for 24 hours, and cytokines were quantitated in the medium . As a result, as shown in FIG. 6, it was confirmed that MPLA in EXO encapsulated with immunosuppressant in the exosomes isolated from bovine serum showed high cytokine secretion amount.

Experimental Example 4. Confirmation of cell viability

The effects of free MPLA and MPLA in EXO were confirmed by macrophage RAW 264.7 cells, fibroblast NIH3T3 cells and dendritic cells BMDC using cell counting kit (CCK). Concentrations of MPLA were 0, 0.45, 1.5 and 4.5 ug / ml in RAW 264.7 and NIH3T3 cells and 0, 0.1, 0.3, 1 and 3 ug / ml in BMDC, respectively. As can be seen in FIG. 7, in both macrophages, the proliferation phenomenon can be observed by increasing the number of cells, but it has been confirmed that there is no change in fibroblasts and dendritic cells. That is, MPLA in EXO was found to be non-toxic at a concentration of 0-4.5 ug / ml.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (6)

A lymph node target-oriented drug delivery system in which biomolecules are encapsulated in exosomes having a diameter of 100 nm or less derived from a mouse, bovine or human serum. 2. The drug delivery system according to claim 1, wherein the biomolecule is an anti-cancer agent or an immunomodulator. 2. The medicament according to claim 1, wherein the exosome derived from the rat or cattle has a diameter of 50 nm or less. 2. The medicament according to claim 1, wherein the exosome has a zeta potential of less than -30 mV. Wherein the immunosuppressive agent is encapsulated in an exosome having a diameter of 100 nm or less derived from a mouse, a bovine, or a human serum. 6. The composition according to claim 5, wherein the composition targets lymph nodes.

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