KR102088555B1 - Drug delivery system for targeting lymph node using exosome from serum - Google Patents

Abstract

The present invention relates to the use of exosomes having a diameter of 100 nm or less derived from serum of rats, cows, or humans, and specifically, as a composition for targeting drug delivery agents and immune enhancing lymph nodes targeted with biomolecules enclosed in exosomes It is about.

Description

DRUG DELIVERY SYSTEM FOR TARGETING LYMPH NODE USING EXOSOME FROM SERUM}

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

Exosomes (exosomes) are small vesicles of a membrane structure of 50-200 nm in size that are secreted out of cells with proteins, DNA, RNA, etc. for intercellular signaling, and can be separated from blood, urine, serum, etc. Exosomes are used for various purposes, such as drug delivery systems and vaccines. Recently, exosomes are analyzed for cancer by analyzing exosomes secreted from tumor cells present in the bloodstream.

Currently, studies have been reported to deliver antigens or nucleotides using dendritic cells or exosomes isolated from tumors. It is difficult to use it as a drug delivery agent that targets.

Korean Patent Publication No. 10-2016-0055687

In the present invention, the present invention was completed by confirming that it can be used as a drug delivery agent targeting a lymph node by separating exosomes of 100 nm or less from serum.

In order to achieve the above object, the present invention provides a targeted drug delivery system in which a biomolecule is encapsulated in a exosome having a diameter of 100 nm or less derived from serum of rat, bovine or human.

In addition, the present invention provides a composition for enhancing immunity in which an immunopotentiator is encapsulated in exosomes having a diameter of 100 nm or less derived from serum of rat, cow or human.

The serum-derived exosome according to the present invention can be obtained from a serum in high yield, and has a size of 100 nm or less to effectively deliver drugs to the lymph nodes.

1 is in one embodiment of the present invention, (a) extracting serum through centrifugation from rat, bovine and human blood, mixing it with ExoQuick solution and centrifuging to extract exosomes and ( b) It is a schematic diagram showing that the drug is encapsulated in the extracted exosomes and delivered to the lymph nodes.
FIG. 2 is a TEM image of exosomes isolated from bovine and rat serum in one embodiment of the present invention.
3 is a graph showing the luminescence intensity of fluorescent dye encapsulated in exosomes isolated from human and bovine serum in RAW 264.7 cells in one embodiment of the present invention.
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.
5 is a graph showing cytokine secretion by a drug delivery system using exosomes isolated from bovine serum in RAW 264.7 cells in one embodiment of the present invention.
Figure 6, in one embodiment of the present invention, RAW 264.7 cells (a and b) and dendritic cells (BMDC) (c and d) showing cytokine secretion by drug delivery using exosomes isolated from bovine serum It is a graph.
7 is a graph showing cell viability by free MPLA and MPLA encapsulated in exosomes according to the present invention.

Hereinafter, the present invention will be described in detail with embodiments of the present invention. However, the following embodiments are presented as examples of the present invention, whereby the present invention is not limited, and the present invention is capable of various modifications and applications within the scope of the claims and the equivalents interpreted therefrom. .

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

In addition, the present invention provides a composition for enhancing immunity in which an immunopotentiator is encapsulated in exosomes having a diameter of 100 nm or less derived from serum of rat, cow or human.

The exosomes can be isolated from rat, bovine or human serum by the process shown in Figure 1A. Specifically, serum may be extracted from the blood of rats, cows, and humans by centrifugation, and mixed with an ExoQuick solution to centrifuge to extract exosomes.

The extracted exosomes can be injected into an individual after biomolecules have been encapsulated to deliver the biomolecules to the lymph nodes (FIG. 1B). The individual may be a mammal, for example, a human.

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

Exosomes derived from bovine or rat serum may have a zeta potential of -30 mV or less, for example -25 mV or less, and exosomes derived from human serum may be -20 mV or less, for example, It may have a zeta potential of -15 mV or less.

The biomolecule may be used without limitation as long as it is a drug generally used in the field, and may be, for example, an anti-cancer agent or an adjuvant.

The anticancer agent may be used without limitation as long as it can be used in the art, for example, cisplatin, carboplatin, procarbazine, mechlorethamine, cyclophosphamide (cyclophosphamide), ifosfamide, melphalan, chlorambucil, bisulfan, nitrosourea, dactinomycin, daunorubicin , Doxorubicin, bleomycin, plicomycin, mitomycin, etoposide, tamoxifen, paclitaxel, transplatinum , 5-fluorouracil, adriamycin, vincristin, vinblastin, methotrexate, and the like.

The adjuvant may be used without limitation as long as it can be used in the field, and may include toll-like receptor agonist, saponin, aluminum salt, ovalbumin, and the like. The adjuvants are, for example, QS21, Quil A, QS7, QS17, CpG, MPLA (monophosphoryl lipid A), imiquimod, leciquimod, flagellin, polyacyl (poly (I: C) ], Ovalbumin, and the like.

The drug delivery system or the composition for enhancing immunity targets the lymph nodes, and can transport the drug or the adjuvant to the lymph nodes to activate the immune activity.

The present invention will be described in more detail with reference to the following examples. However, the following examples are only intended to embody the contents of the present invention, and the present invention is not limited thereto.

Comparative Example 1. Extraction and separation of exosomes from cells

One of the macrophage cells, RAW 264.7 cells 1.5 x 10 ⁷ cells / T75 flask was cultured for 2 days in a medium containing serum, and then cultured for 24 hours in a medium of exosome depleted 10% FBS to obtain 15 minutes at 3000 xg. During centrifugation, supernatant was obtained and mixed with ExoQuick-TC in a volume ratio of 1: 0.2. After incubating the mixed solution at 4 ° C. for at least 12 hours, the supernatant was removed by centrifugation at 1500 xg for 30 minutes, and centrifugation for an additional 5 minutes to completely remove the supernatant. The obtained pellet was dissolved in 1 x PBS and used.

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

Serum from rats and humans was diluted by mixing 550 ul and 500 ul of 1 x PBS, respectively, and filtered through a 0.22 um filter soaked with 5 ml of 1 x PBS. The filtered serum was centrifuged at 10000 x g for 30 minutes, and then only the supernatant was obtained and mixed with exo-quick in a volume ratio of 1: 0.2. After sufficiently inverting the mixed solution, it was incubated at 4 ° C overnight. The next day, the supernatant was removed by centrifugation at 1500 x g for 30 minutes, centrifugation was further performed for 5 minutes, the supernatant was completely discarded, and the pellet was dissolved with 100 ul of 1 x PBS.

After 1 ml of bovine serum was centrifuged at 3000 x g for 15 minutes, a supernatant was obtained and mixed with exo-quick to a volume ratio of 1: 0.2. After the mixture was sufficiently inverted, it was incubated at 4 ° C overnight. The next day, the supernatant was removed by centrifugation at 1500 x g for 30 minutes, centrifugation was performed at the same rcf for 5 minutes, the supernatant was completely discarded, and pellets were dissolved with 100 ul of 1 x PBS.

Experimental Example 1. Characterization of isolated exosomes

The exosomes extracted as in Example 1 were measured using DLS and zeta potential to analyze the properties. To compare the yield, three types of exosomes were quantified using the BCA assay used for protein quantification. As a result, as shown in Table 1 below, exosomes isolated from bovine and rat serums have a diameter of 50 nm or less and a surface charge of about 20 vmV, and exosomes separated from human serum are around 100 nm. It was confirmed to have a diameter and a surface charge of about 12 vmV. Protein values in serum were 1.52 ± 0.25, 13.12 ± 4.55, and 15.49 ± 2.36 mg / ml in the order of cattle, rats and humans.

Additionally, exosomes separated from bovine serum and rat serum were transferred to a 300 mesh copper grid to be 300 ug or more based on protein quantification, and then washed three times with DW. Then, a grid on which exosomes were raised with uranyl acetate was coated to measure TEM. As a result, as shown in FIG. 2, vesicles having a diameter of 50 nm or less were observed, and despite a similar amount of protein, a larger number of vesicles was confirmed in bovine serum.

Example 2. Preparation of drug delivery system encapsulated with an adjuvant

Using exosomes extracted from bovine serum, adjuvants OVA, CpG and MPLA (monophosphoryl lipid A) were respectively sealed. FITC-labeled OVA and CpG were inserted into exosomes using an electroporation method, and MPLA was sealed by the hydrophobicity of lipid A and the plasma membrane of exosomes.

 In the case of OVA-FITC, 160 ug OVA-FITC and 160 ug exosomes were mixed with 50 mM trehalose in 160 ul of 1 x PBS, and then electroporation was performed at a 0.2 kV pulse. Then, 1 x PBS was added to a sample volume of 500 ul using 300 k nanosep and centrifuged at 5000 x g for 10 minutes. This process was repeated 3 times, and a supernatant was obtained to prepare exosomes encapsulated with OVA-FITC. Loading efficiency was quantified by FITC fluorescence value.

 For CpG, 40 ug CpG and 160 ug exosomes were mixed with 50 mM trehalose in 80 ul of 1 x PBS, and electroporation was performed with a 0.2 kV pulse. To completely remove the unloaded CpG, DNase I was mixed and reacted at 37 ° C for 1 hour. Then, 1 x PBS was added to a sample volume of 500 ul using 300k nanosep and centrifuged at 5000 xg for 10 minutes. . This process was repeated 3 times, and a supernatant was obtained to prepare exosomes encapsulated with CpG. Loading efficiency of CpG was confirmed by a picogreen assay known as a DNA quantification kit.

 In the case of MPLA, 400 ug exosomes and 4 ug MPLA were reacted in 10% DMSO in 400 ul of DW for 2 hours at 37 ° C., and then DW was added using a 300 k nanosep so that the sample volume was 500 ul and at 5000 xg. Centrifuge for 10 minutes. After repeating this process 3 times, supernatant was obtained to quantify the degree of encapsulation of MPLA using a LAL kit, and exosomes encapsulated with MPLA were prepared. Then, the loading amount was confirmed by BCA assay for CpG and MPLA encapsulated in exosomes (Table 2).

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

Fluorescent dye was encapsulated in the exosomes prepared as in Comparative Example 1 and Example 1 to verify the delivery to lymph nodes, one of the lymphatic systems, using exosomes and confirmed this (FIG. 3 and Table 3). For the fluorescent dye, ICG-TBAI (Indocyanine green Tetrabutylammonium iodide), an ionic complex used as a prior study, was used to confirm lymph node targeting. 200 ug and 20 ug of ICG-TBAI isolated from RAW 264.7 cells, human serum and bovine serum, respectively, were mixed in 130 ul of 1 × PBS in 7.7% DMSO and reacted at 37 ° C. for 2 hours. Thereafter, the glass dye was removed using a 40k desalting column, and ICG-TBAI-encapsulated exosomes were prepared.

As can be seen in Figure 3 and Table 3, exosomes isolated from cells have a diameter of 200 nm or more, whereas exosomes isolated from human and bovine serum are about 100 nm or 50 nm even when fluorescent dye is encapsulated. It was confirmed that the diameter of the degree was shown.

Then, each sample 30 was subcutaneously subcutaneously in the hind paw of a 6-week-old female ICR rat using a free ICG-TBAI and ICG-TBAI in RAW EXO, ICG-TBAI in Human EXO, and ICG-TBAI in Bovine EXO to reach 1 g of ICG-TBAI standard. ul was injected. As a result, as shown in FIG. 4, ICG-TBAI in Bovine EXO in popliteal LN, a short-distance lymph node in the footpad, was 2.2, 2.19 in order of Free ICG-TBAI, ICG-TBAI in RAW EXO, and ICG-TBAI in Human. It was confirmed that it showed a high strength of 1.87 times. As a result, effective lymph node targeting of ICG-TBAI in Bovine EXO was confirmed.

Experimental Example 3. Confirmation of cytokine secretion

CpG criteria 0, 0.5 and 1.5 ug / ml were treated in RAW 264.7 cells to confirm the activity and cellular uptake of CpG encapsulated exosomes prepared as in Example 2 and cellular uptake. After incubation for an hour, a medium was obtained to quantify the secretion of TNF-a by ELISA. As a result, as shown in FIG. 5, when CpG in EXO was treated than Free CpG, it was confirmed that the amount of TNF-a was 12.5 and 8.5 times higher at 0.5 and 1.5 ug / ml concentration, respectively.

Free MPLA and MPLA in EXO were treated with macrophages RAW 264.7 cells and dendritic cells (BMDC) to confirm that the adjuvant encapsulated in exosomes isolated from bovine serum effectively showed activity. For RAW 264.7 cells, inoculate cells with 5 x 10 ⁵ cells / well in a 12-well plate and incubate for 24 hours, followed by free MPLA and MPLA in EXO based on MPLA concentrations of 0.1, 0.3, 1, 3 ug / ml. Time treated. Thereafter, the transferability of MPLA (adjuvant) through the obtained medium was confirmed by cytokines (TNF-a, IL-6) expressed in cells. Cytokines of TNF-a and IL-6 were quantified using an ELISA kit. Primary dendritic cells (BMDC), primary dendritic cells, were inoculated into 96-well plates at 5 x 10 ⁴ cells / well, and after 24 hours, treated with the above MPLA concentration and cultured for 24 hours, and cytokines were quantified in the medium. . As a result, as can be seen in FIG. 6, it was confirmed that MPLA in EXO in which an immunoadjuvant was encapsulated in exosomes isolated from bovine serum showed high cytokine secretion.

Experimental Example 4. Confirmation of cell viability

Free MPLA and MPLA in EXO were used to determine the effect on macrophage RAW 264.7 cells, fibroblasts NIH3T3 cells, and dendritic cells BMDC with a cell counting kit (CCK). The concentrations treated by MPLA were 0, 0.45, 1.5, and 4.5 ug / ml in RAW 264.7 cells and NIH3T3 cells, and 0, 0.1, 0.3, 1, and 3 ug / ml in BMDC. As can be seen in FIG. 7, in the macrophages, proliferation phenomenon can be observed in both samples by increasing the number of cells, but it was confirmed that there was no change in fibroblasts and dendritic cells. That is, MPLA in EXO was confirmed to be non-toxic at a concentration of 0-4.5 ug / ml.

So far, the present invention has been focused on the preferred embodiments. Those skilled in the art to which the present invention pertains will understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered in terms of explanation, not limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the equivalent range should be construed as being included in the present invention.

Claims (6)

A method for increasing secretion of TNF-a or IL-6, comprising administering a drug delivery system in which MPLA is encapsulated in exosomes having a diameter of 50 nm or less derived from bovine serum to a subject other than humans. delete delete The method of claim 1,
The exo-some having a zeta potential of -30 mV or less. delete The method of claim 1,
The drug delivery method is to target the lymph nodes.

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