Composition for material delivery, including exosome mimetics derived from red blood cells, and use thereof

Abstract

The present invention relates to a composition for material delivery, including exosome mimetics derived from red blood cells, and a use thereof and the composition for material delivery according to an exemplary embodiment of the present invention includes exosome mimetics derived from red blood cells, which are capable of being loaded with a drug, a radioactive material, or a fluorescent material, and thus may be usefully utilized for a drug delivery use, a cell labeling use, a contrast medium, or the like, and when the composition for material delivery according to an exemplary embodiment of the present invention is used, treatment and diagnosis may be simultaneously performed.

Claims

1. A composition for material delivery, comprising exosome mimetics derived from red blood cells loaded with a target material, wherein the exosome mimetics are vesicles artificially prepared by extruding red blood cells by a filter, wherein the target material comprises technetium-99m (.sup.99mTc), wherein the exosome mimetics comprises hemoglobin derived from red blood cells and the technetium-99m (.sup.99mTc) is bound to hemoglobin inside the exosome mimetics derived from red blood cells, wherein the exosome mimetics derived from red blood cells have a diameter of 100 nm to 300 nm.

2. The composition of claim 1, wherein the target material further comprises one or more selected from the group consisting of a drug, a radioactive material other than technetium-99m, and a fluorescent material.

3. The composition of claim 2, wherein the drug is one or more selected from the group consisting of a compound, a peptide, a protein, and a nucleic acid.

4. The composition of claim 2, wherein the radioactive material is a diagnostic radionuclide or a therapeutic radionuclide.

5. The composition of claim 4, wherein the therapeutic radionuclide is one or more selected from the group consisting of .sup.131I, .sup.186Re, .sup.188Re, .sup.153Sm, and .sup.32P.

6. The composition of claim 2, wherein the fluorescent material is one or more selected from the group consisting of a fluorescent protein, a photoprotein, a luciferase, and a fluorescent dye.

7. The composition of claim 1, wherein the composition for material delivery is used to treat a liver disease.

8. The composition of claim 1, wherein the composition for material delivery is used to treat arthritis.

9. The composition of claim 1, wherein the composition for material delivery is used to treat tumors.

10. The composition for claim 1, wherein the composition for material delivery is used to label cells.

11. A method for preparing the composition for material delivery of claim 1, the method comprising: (a) obtaining exosome mimetics derived from red blood cells, wherein the exosome mimetics are vesicles artificially prepared by extruding red blood cells by a filter; (b) incubating a mixture in which the exosome mimetics derived from red blood cells obtained in step (a) and a target material are mixed; (c) obtaining a pellet by ultracentrifuging the mixture incubated in step (b); (d) separating exosome mimetics derived from red blood cells loaded with a target material by washing the pellet obtained in step (c) and using a density gradient; (e) incubating a mixture in which the exosome mimetics derived from red blood cells separated in step (d) and tin (II) chloride are mixed; and (f) adding technetium-99m (.sup.99mTc) to the mixture incubated in step (e) and incubating the resulting mixture.

12. The method of claim 11, wherein the target material is one or more selected from the group consisting of a drug, a radioactive material other than technetium-99m, and a fluorescent material.

13. A contrast medium comprising the composition for material delivery of claim 1.

14. The contrast medium of claim 13, wherein the contrast medium is applied to nuclear medical imaging.

15. The contrast medium of claim 14, wherein the nuclear medical imaging is positron emission tomography (PET) or single-photon emission computed tomography (SPECT), or gamma camera imaging.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 illustrates a visual image of exosome derived from red blood cells (RBC-Exo) and exosome mimetics derived from red blood cells (RBC-EM), which are prepared from the same number of red blood cells.

(2) FIG. 2A illustrates the concentrations (number prepared/ml) of RBC-Exo and RBC-EM, which are prepared from the same number of red blood cells, and FIG. 2B illustrates the total protein contents of RBC-Exo and RBC-EM, which are prepared from the same number of red blood cells.

(3) FIG. 3 is a schematic view schematically illustrating a process of labeling RBC-EM with .sup.99mTc.

(4) FIG. 4 illustrates a radiochemical purity of exosome mimetics derived from red blood cells labeled with .sup.99mTc (.sup.99mTc-RBC-EM) over time.

(5) FIG. 5 illustrates the stability of .sup.99mTc-RBC-EM over time.

(6) FIG. 6A illustrates the size distribution of RBC-Exo, RBC-EM, and .sup.99mTc-RBC-EM, and FIG. 6B illustrates the average sizes of RBC-Exo, RBC-EM, and .sup.99mTc-RBC-EM.

(7) FIG. 7 illustrates an image in which RBC-EM and .sup.99mTc-RBC-EM are observed by field emission transmission electron microscopy (FE-TEM).

(8) FIG. 8 illustrates an image in which RBC-EM and .sup.99mTc-RBC-EM are observed by high-angle annular dark field scanning transmission electron microscopy (HAADF-STEM).

(9) FIG. 9A illustrates an in vivo gamma camera image of free .sup.99mTc and .sup.99mTc-RBC-EM, and FIG. 9B illustrates quantitative values of the measured radiation.

(10) FIG. 10 illustrates results of measuring the biodistribution of .sup.99mTc and .sup.99mTc-RBC-EM after free .sup.99mTc and .sup.99mTc-RBC-EM are administered to mice via intravenous injection. In FIG. 10, the upper graph illustrates the radiation dose measured from different organs, specifically illustrating the radiation dose measured in division to the lower left panel and right panel.

(11) FIG. 11A and FIG. 11B illustrate the results in which fluorescent imaging is performed on RBC-EM.sup.DiD. Specifically, FIG. 11A illustrates a fluorescent image of RBC-EM.sup.DiD in vivo. FIG. 11B illustrates a fluorescent image (left image) of RBC-EM.sup.DiD in the liver and spleen collected from mice and a result (right graph) of quantifying the same.

(12) FIG. 12A and FIG. 12B illustrate the results of performing an immunofluorescence assay on RBC-EM.sup.DiD in the section of a liver tissue. Specifically, FIG. 12A illustrates an immunofluorescence assay image on RBC-EM.sup.DiD in the section of a liver tissue, and FIG. 12B illustrates fluorescence values observed in the immunofluorescence assay of CD68+(left graph) and DiD (right graph).

(13) FIG. 13 illustrates the results in which exosome mimetics derived from red blood cells (RBC-EM) are successfully loaded with dexamethasone and curcumin.

(14) FIG. 14A illustrates amounts of radiation measured in 8505C cells according to the culture time and the dose of .sup.99mTc-RBC-EM. FIG. 14B illustrates amounts of .sup.99mTc-RBC-EM and free .sup.99mTc with the same dose (4 μCi) absorbed in cells.

(15) FIG. 15 illustrates a .sup.18F-FDG PET/CT image of acute inflammatory mice.

(16) FIGS. 16A to 16D illustrate that in an acute inflammation mouse model, white blood cells labeled with .sup.99mTc may be effectively used to track inflammation. Specifically, FIG. 16A illustrates a gamma camera image in an acute inflammation mouse model into which .sup.99mTc-WBC is infused via the tail vein. FIG. 16B illustrates amounts of radiation measured from the left and right feet of the acute inflammation mouse model to which .sup.99mTc-WBC is injected. FIG. 16C illustrates the biodistribution of .sup.99mTc-WBC. FIG. 16D illustrates the amounts of .sup.99mTc-WBC taken up by the left and right feet.

(17) FIGS. 17A and 17B illustrate the results of performing gamma camera imaging after infusing .sup.99mTc-DiD-RBC-EM into the articular cavity of an animal model with rheumatoid arthritis. Specifically, FIG. 17A illustrates a gamma camera image of mice with rheumatoid arthritis, and FIG. 17B quantitatively illustrates amounts of measured radiation. The left graph of FIG. 17B illustrates absolute values of amounts of radiation measured (background corrected counts) in a portion injected into the arthritic lesion, and the right graph illustrates the amounts of radiation (retention rate) remaining in the injected lesion after radiation decay correction in the same data.

(18) As a result of performing fluorescent imaging after .sup.99mTc-DiD-RBC-EM is infused into the articular cavity of the animal model with rheumatoid arthritis, FIG. 18A illustrates a fluorescent image over time, and FIG. 18B quantitatively illustrates amounts of fluorescent material measured.

(19) FIG. 19 illustrates a fluorescent image over time when RBC-EM-porphyrin is infused into the tumor.

(20) FIG. 20A illustrates amounts of porphyrin measured subcutaneously when RBC-EM-porphyrin is infused subcutaneously, and

(21) FIG. 20B illustrates amounts of porphyrin measured in tumor when RBC-EM-porphyrin is infused into the tumor.

(22) FIG. 21A illustrates an ex-vivo fluorescent image of various organs and FIG. 21B illustrates quantitative values of porphyrin measured in various organs.

DETAILED DESCRIPTION

(23) Hereinafter, the present invention will be described in more detail through the Examples. However, these Examples are provided only for exemplarily explaining the present invention, and the scope of the present invention is not limited by these Examples.

Example 1. Preparation of Exosome Mimetics Derived from Red Blood Cells

(24) 1-1. Preparation of Exosomes Derived From Red Blood Cells (RBC-Exo) and Exosome Mimetics (RBC-EM) Derived From Red Blood Cells (RBC-EM)

(25) Blood samples were collected from Sprague Dawley rats (150 g; 6 weeks old; Hana Corp., Korea) in the presence of a citrate-dextrose solution (Sigma-Aldrich, USA) as an anticoagulant.

(26) The collected blood was centrifuged at 200 g at 18° C. to 22° C. for 15 min to 20 min. A buffy coat of a blood supernatant was obtained from the top of concentrated red blood cells (packed RBCs), and the same was used to separate red blood cells. Subsequently, the concentrated red blood cells were collected. The red blood cells (RBCs) were obtained by a one-time centrifugation step under the conditions of 1,500 g at 4° C. for 20 min.

(27) Exosomes were obtained from red blood cells by a previously publicly known method (Varga et al., Cancer Biother. Radiopharm. 31, 168-173, 2016), and exosomes derived from red blood cells were named as RBC-Exo.

(28) Red blood cells (RBC) were diluted by adding PBS to red blood cells (RBC:PBS=1:9 volume ratio), and the diluted red blood cells were extruded by a 1-μm pore size polycarbonate membrane filter (Nuclepore, Whatman, Inc., Clifton, N.J., USA) once to four times using a mini-extruder (Vanti Polar Lipids, Birmingham, Ala., USA). The extruded sample was diluted by adding 20×PBS to the sample, and centrifuged at 3,000 g for 10 min in order to remove RBC, larger vesicles, and debris. The centrifuged sample was filtered by a 0.22 μm syringe filter, and ultra-centrifuged (Beckman Coulter, CA, USA) at 4° C. and 100,000 g for 1 hour. After the ultracentrifugation, a two-step density gradient ultracentrifugation was performed at 4° C. using iodixanol (OptiPrep™ Density Gradient Medium, Sigma-Aldrich, USA). The exosome mimetics derived from red blood cells were obtained at the intersection point of a 60% iodixanol layer and a 20% iodixanol layer, and were immediately used without an additional treatment. The obtained exosome mimetics derived from red blood cells were named as RBC-exosome mimetics (RBC-EM).

(29) FIG. 1 illustrates a visual image of RBC-Exo and RBC-EM, which are prepared from the same number of red blood cells. The dotted line of FIG. 1 indicates the positions of RBC-Exo and RBC-EM. As illustrated in FIG. 1, it can be confirmed that the number of RBC-EM is larger than that of naturally-derived RBC-Exo.

(30) 1-2. Analysis of Numbers of RBC-Exo and RBC-EM and Protein Amounts

(31) By ultracentrifugation and extraction methods, the numbers of RBC-Exo and RBC-EM produced from 5×10.sup.6 red blood cells (RBCs) were determined. FIG. 2A illustrates the concentrations (number prepared/ml) of RBC-Exo and RBC-EM, which are prepared from the same number of red blood cells. As illustrated in FIG. 2A, the number of RBC-EM prepared from the same number of red blood cells was about 130-times larger than the number of RBC-Exo prepared from the same number of red blood cells (P<0.001).

(32) The protein amounts of RBC-Exo and RBC-EM were measured by a previously publicly known method (Gangadaran et al., Oncotarget 8, 109894-109914, 2017). FIG. 2B illustrates the total protein contents of RBC-Exo and RBC-EM, which are prepared from the same number of red blood cells. As illustrated in FIG. 2B, RBC-EM prepared from the same number of red blood cells showed a significantly higher protein concentration (about 65 times) than that of RBC-Exo (P<0.001).

(33) From the result, it can be seen that RBC-EM can be mass-produced more efficiently than naturally made RBC-Exo.

Example 2. Labeling of Exosome Mimetics Derived From Red Blood Cells (RBC-EM) With .SUP.99m.Tc and Labeling Purity

(34) 2-1. Labeling Exosome Mimetics Derived From Red Blood Cells (RBC-EM) With .sup.99mTc

(35) Since only .sup.99mTc reduced to a low oxidation state is firmly bound to hemoglobin, particularly, the beta-chain of hemoglobin, RBC-EM was incubated along with tin (II) chloride in order to reduce .sup.99mTc. The RBC-EM obtained in Example 1-1 and the same amount of 0.01% tin (II) chloride (Sigma, USA) were incubated in a shaker at 37° C. for 5 min, and the exosome mimetics derived from red blood cells were labeled with a radioactive material (.sup.99mTc) by adding technetium-99m (.sup.99mTc) to RBC-EM (RBC-EM (100 μg): .sup.99mTc (111 MBq)) and incubating the resulting mixture in a shaker at 37° C. for 20 min, and the labeled exosome mimetics derived from red blood cells were named as .sup.99mTc-RBC-EM. When a radiochemical purity was less than 95%, an ultracentrifugation was performed at 4° C. and 100,000 g for 1 hour in order to purify .sup.99mTc-RBC-EM.

(36) FIG. 3 is a schematic view schematically illustrating a process of labeling RBC-EM with .sup.99mTc.

(37) 2-2. .sup.99mTc Labeling Purity

(38) The labeling purity of RBC-EM with .sup.99mTc was measured using instant thin-layer chromatography (TLC) using a 0.9% NaCl solution as each column eluent, and the radioactivity of the column was counted using a radio-TLC imaging scanner (AR-2000, Bioscan, Poway, Calif., United States). The stability was determined by a rate of change in radiochemical purity of .sup.99mTc-RBC-EM according to the passage of time.

(39) FIG. 4 illustrates the radiochemical purity of .sup.99mTc-RBC-EM over time. As illustrated in FIG. 4, the radiochemical purity immediately after purification by ultracentrifugation (0 hour) was 100%. The proportion of RBC-EM labeled with .sup.99mTc was 99.57% (3 hours), 97.07% (6 hours), 96.34% (12 hours), and 94.98% (24 hours). The radiochemical purity was analyzed by employing free .sup.99mTc as a control.

(40) Through the aforementioned result, it can be seen that exosome mimetics derived from red blood cells (.sup.99mTc-RBC-EM) labeled with .sup.99mTc as a radioactive material were stable.

(41) 2-3. Measurement of Serum Stability

(42) .sup.99mTc-RBC-EM was cultured in a PBS solution including 20% FBS in a CO.sub.2 incubator at 37° C. The stability of .sup.99mTc-RBC-EM was measured in 0, 1, 3, and 24 hours after the culture using a radio-TLC imaging scanner (AR-2000, Bioscan, Poway, Calif., United States).

(43) FIG. 5 illustrates the stability of .sup.99mTc-RBC-EM over time. As illustrated in FIG. 5, it can be seen that .sup.99mTc-RBC-EM is stable up to 24 hours.

Example 3. Analysis of Characteristics of RBC-EM and .SUP.99m.Tc-RBC-EM

(44) 3-1. Nanoparticle Tracking Analysis (NTA)

(45) Particle sizes of RBC-Exo and RBC-EM prepared by the method in Example 1-1 and .sup.99mTc-RBC-EM prepared by the method in Example 2-1 were analyzed using Nano Sight LM 10 (Malver) according to the provided protocol. Each sample was diluted 1000 times with Milli-Q water and infused into a chamber using a sterilized syringe, and measurement was performed in the same manner as the previously publicly known method (Gangadaran et al., J. Control. Release 264, 112-126, 2017).

(46) 3-2. Field Emission Transmission Electron Microscopy (FE-TEM) and Scanning Transmission Electron Microscopy (STEM)

(47) Pellets of RBC-Exo, RBC-EM, and .sup.99mTc-RBC-EM samples were resuspended in 100 μl of 2% paraformaldehyde. 5 μl of each sample was attached (drop) to a formvar/carbon-coated EM grid (Electron Microscopy Sciences, USA), and dried in an open space for 20 min. For washing, 50 μl of PBS was added to a parafilm sheet, and the grid was floated upside down on the PBS balls using sterilized forceps. The grid was transferred to 50 μl of 1% glutaraldehyde, incubated for 5 min and washed with distilled water for 2 min. RBC-EM and .sup.99mTc-RBC-EM on the grid were stained with 10 μl of 2% uranyl acetate, and then the grid was again washed 7 times with PBS. Thereafter, the grid was completely dried. All the processes were performed at room temperature.

(48) In order to observe the sizes of RBC-Exo, RBC-EM, and .sup.99mTc-RBC-EM and perform an element analysis, the sample was observed in a Titan G2 ChemiSTEM (FEI Company) with a Cs Probe (FEI company, Netherlands). In order to measure the sizes of RBC-Exo, RBC-EM, and .sup.99mTc-RBC-EM, a HT 7700 transmission electron microscope (Hitachi, Tokyo, Japan) was used.

(49) 3-3. Characteristics of RBC-EM and .sup.99mTc-RBC-EM

(50) The size distribution of RBC-Exo, RBC-EM, and .sup.99mTc-RBC-EM was measured by the method in Example 3-1. FIG. 6A illustrates the size distribution of RBC-Exo, RBC-EM, and .sup.99mTc-RBC-EM, and FIG. 6B illustrates the average sizes of RBC-Exo, RBC-EM, and .sup.99mTc-RBC-EM. As illustrated in FIG. 6, as a result of NTA analysis, the size of RBC-Exo was within a range of 30 nm to 450 nm, and the average size thereof was 209.1±19.8 nm. Further, the unlabeled RBC-EM had an average diameter of 201.3±16 nm, and .sup.99mTc-RBC-EM labeled with .sup.99mTc had an average diameter of 195.0±11.1 nm. The sizes of RBC-Exo, RBC-EM, and .sup.99mTc-RBC-EM did not show a significant difference (all the P values were 0.05 or more).

(51) From this, it can be seen that the size of the exosome mimetics derived from red blood cells is similar to that of red blood exosomes, and the change in size was not generated by a .sup.99mTc radioactive label.

(52) In order to confirm whether the radioactive label changed the shape and morphology of RBC-EM and analyze whether .sup.99mTc was present therein or in the membrane, FE-TEM was performed by the method in Example 3-2. FIG. 7 illustrates an image in which RBC-EM and .sup.99mTc-RBC-EM are observed by field emission transmission electron microscopy (FE-TEM). As illustrated in FIG. 7, it was confirmed that there was no morphological change caused by the .sup.99mTc label because both the unlabeled RBC-EM and .sup.99mTc-RBC-EM maintained a similar circular shape.

(53) The aforementioned result shows that the radioactive label of .sup.99mTc does not change the size and shape of RBC-EM.

(54) FIG. 8 illustrates an image in which RBC-EM and .sup.99mTc-RBC-EM were observed by high-angle annular dark field scanning transmission electron microscopy (HAADF-STEM). Table 1 shows the results of quantifying the amounts of iron and technetium present in the cytosol of RBC-EM and .sup.99mTc-RBC-EM using the STEM image of FIG. 8. As in the results of Table 1 and FIG. 8, it can be seen that .sup.99mTc is successfully included in the inside (cytosol) of RBC-EM because .sup.99mTc is significantly abundantly present in the cytosol of .sup.99mTc-RBC-EM.

(55) TABLE-US-00001 TABLE 1 RBC-EM .sup.99mTc-RBC-EM Weight Error in Weight Error in (wt). % wt. % (wt). % wt. % Iron 90.17 21.94 40.80 18.89 Technetium 9.92 9.53 59.20 34.59

(56) 4. In Vivo Distribution of .sup.99mTc-RBC-EM

(57) 4-1. In Vivo Gamma Camera Imaging

(58) In vivo gamma camera imaging was performed on 5-week-old male C57BL/6 mice (Hamamatsu, Shizuoka). The gamma camera images were captured and taken for 10 min using a 2 mm pinhole collimator (Infinia II, GE Healthcare, Milwaukee, Wis., USA). A 200 μl volume of 37 MBq .sup.99mTC-RBC-EM was infused into the tail vein of mice. As a control, the same amount of free .sup.99mTc was infused. During the imaging, mice were continuously anesthetized using 2.5% isoflurane (Merial, Lyon, France). The gamma camera images were obtained by capturing the gamma camera images 1 hour and 3 hours after infusing .sup.99mTc-RBC-EM and free .sup.99mTc.

(59) The region of interests (ROIs) in mice into which free .sup.99mTc was infused were the thyroid gland and the stomach, and the region of interests (ROIs) in mice into which .sup.99mTc-RBC-EM was infused were the thyroid gland and the liver/spleen. The right thigh part was used as a control of ROI. The average ROI count per pixel was calculated by dividing the same mice by the control ROI.

(60) After .sup.99mTc and .sup.99mTc-RBC-EM were injected into the vein of mice, the in vivo gamma camera images were obtained. FIG. 9A illustrates an in vivo gamma camera image of free .sup.99mTc and .sup.99mTc-RBC-EM, and FIG. 9B illustrates quantitative values of the measured radiation. As illustrated in FIG. 9, the in vivo gamma camera images show that .sup.99mTc-RBC-EM may be clearly detected from living mice. The image obtained 1 hour after injecting free .sup.99mTc shows a strong uptake of free .sup.99mTc in the thyroid gland, the stomach, and the bladder (left upper panel of FIG. 9A). Simultaneously, the uptake of .sup.99mTc-RBC-EM was obtained in the liver, the spleen, and the bladder 1 hour after injecting .sup.99mTc-RBC-EM (right upper panel of FIG. 9A). Unlike free .sup.99mTc, the absorption of .sup.99mTc-RBC-EM in the thyroid gland or stomach of mice was not observed. Further, the gamma camera image 3 hours after the infusion showed a pattern similar to the image captured 1 hour after the infusion (right lower and left lower panels of FIG. 9A). However, the absorption of free .sup.99mTc in the thyroid gland and the stomach was significantly (P<0.001) increased 3 hours after the infusion compared to that 1 hour after the infusion (left graph of FIG. 9B), whereas the uptake of .sup.99mTc-RBC-EM in the thyroid gland, the liver, and the spleen 3 hours after the infusion was not significantly increased (P>0.05) compared to that 1 hour after the infusion (right graph of FIG. 9B).

(61) Free .sup.99mTc is easily taken up by the thyroid gland and the stomach due to chemical characteristics via different mechanisms. However, for .sup.99mTc-RBC-EM, since .sup.99mTc is bound to hemoglobin (Hb) present in the cytosol of RBC-EM, radiation may be detected by .sup.99mTc, but .sup.99mTc-RBC-EM behaves in vivo as characteristics of RBC-EM. Accordingly, RBC-EM is taken up by the reticuloendothelial system such as the liver or the spleen. As illustrated in FIG. 9B, since the uptake of .sup.99mTc-RBC-EM in the thyroid gland was not increased even 3 hours after the infusion likewise 1 hour after the infusion, it means that free .sup.99mTc freed from .sup.99mTc-RBC-EM is scarcely present up to 3 hours, and through this, it can be seen that the bond of .sup.99mTc and RBC-EM is stable.

(62) 4-2. Biodistribution of .sup.99mTc-RBC-EM

(63) .sup.99mTC-RBC-EM or free .sup.99mTC was infused into mice via intravenous injection. One hour later, blood samples were collected, and the mice were sacrificed. The uptake value was measured using a gamma counter in the organ such as the lungs, the heart, the liver, the stomach, the spleen, the intestines, the kidneys, the muscle, and the thyroid gland. The resulting values were expressed as a percentage of the amount injected per gram of tissue (% ID/g).

(64) FIG. 10 illustrates results of measuring the biodistribution of .sup.99mTc and .sup.99mTc-RBC-EM after free .sup.99mTc and .sup.99mTc-RBC-EM are administered to mice via intravenous injection. In FIG. 10, the upper graph illustrates the radiation dose measured from different organs, specifically illustrating the radiation dose measured in the lower left panel and right panel. As illustrated in FIG. 10, the absorption of .sup.99mTc-RBC-EM in the thyroid gland was low similarly (P<0.01) compared to that of free .sup.99mTc. A similar uptake tendency was also observed from the stomach (P<0.05). The absorption of .sup.99mTc-RBC-EM in the liver (P<0.001), the spleen (P<0.01), and the kidneys (P<0.05) was 20 to 40 times higher than the uptake of free .sup.99mTc in these organs. Further, as illustrated in FIG. 10, the absorption of .sup.99mTc-RBC-EM in the brain (P<0.05), the blood (P<0.05), the lungs (P<0.05), the muscle (P<0.05), and the bones (P<0.05) is also higher than that of free .sup.99mTc. These results show that exosome mimetics derived from red blood cells labeled with a radioactive material (.sup.99mTc-RBC-EM) may be visualized in living animals.

(65) 4-3. Fluorescent Imaging of RBC-EM DID In Vivo and Ex Vivo

(66) After RBC-EM and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate (DiD; Thermo Fisher Scientific) were cultured together at room temperature for 20 min, the cultured product was washed with PBS, and RBC-EM.sup.DiD was separated by performing a two-step Opti-Prep density gradient ultracentrifugation in the same manner as in Example 1-1. C57BL/6 mice were anesthetized with isoflurane, and RBC-EM.sup.DiD or PBS was infused into the tail vein via intravenous injection. 1 minute and 1 hour after the infusion, fluorescent imaging was performed using an in vivo imaging system (IVIS Lumina III instrument, PerkinElmer) [wavelengths: Excitation—644 nm and emission—665 nm; imaging parameters: binning—4, smoothing—3×3, Field of subject(stage—D): 12.5 cm, height of subject image: 1.5 cm]. Ex vivo fluorescent imaging was performed by collecting the liver and spleen after the imaging, and a quantitative analysis was performed using IVIS software (Living Image Software, PerkinElmer).

(67) In order to examine the biodistribution of RBC-EM, RBC-EM was labeled with DiD by the aforementioned method, and then administered intravenously to the mice. FIG. 11A and FIG. 11B illustrate the results in which fluorescent imaging is performed on RBC-EM.sup.DiD. Specifically, FIG. 11A illustrates a fluorescent image of RBC-EM.sup.DiD in vivo. FIG. 11B illustrates a fluorescent image of RBC-EM.sup.DiD in the liver and spleen collected from mice and a result of quantifying the same. As illustrated in the drawing, immediately after the administration (0 hour), a strong fluorescent signal was shown in the liver, and 1 hour after the administration, stronger fluorescent signals were shown in the regions of the liver and spleen. In contrast, as illustrated in FIG. 11A, the control (administration of PBS) mice did not show any signal. As illustrated in FIG. 11B, it can be seen that RBC-EM.sup.DiD is significantly distributed in the liver and spleen through in vivo and ex vivo fluorescent imaging.

(68) 4-4. Immunofluorescence Assay

(69) In order to analyze the location of RBC-EM in more detail, the liver tissue was cryo-sectioned and subjected to immunofluorescence (IF) assay. The liver sections of mice into which RBC-EM.sup.DiD or PBS was infused were stained with anti-rabbit CD68 (Abcam), and then stained with goat anti-rabbit FITC (Abcam). The liver tissues were mounted using a VECTASHIELD mounting medium (Vector Laboratories, Burlingame, Calif., United States). The immunofluorescence (IF)-stained sections were observed under a confocal microscope (LSM 5 Exciter, Zeiss, Oberkochen, Germany). The observer counted 6 fields. The number of CD68 positive (CD68+; green) cells were counted from RBC-EM.sup.DiD or PBS-infused mouse liver sections. And then, DiD positive was counted from CD68+ or CD68− cells.

(70) FIG. 12A and FIG. 12B illustrate the results of performing an immunofluorescence assay on RBC-EM.sup.DiD in the section of a liver tissue. Specifically, FIG. 12A illustrates an immunofluorescence assay image on RBC-EM.sup.DiD in the section of a liver tissue, and FIG. 12B illustrates immunofluorescence results for CD68+ and DiD with numerical values. As illustrated in the drawing, it was confirmed that there was no significant (P=0.1856) change in CD68 positive cells (Kupffer cells, liver resident macrophages) in the liver section. Further, as a result of immunofluorescence assay, it can be seen that a considerable number of RBC-EM.sup.DiD coexist with Kupffer cells. As illustrated in the drawing, about 75% of RBC-Em.sup.DiD were restricted to Kupffer cells. CD68+ cells of the control mice did not exhibit any DiD signal.

(71) By the aforementioned result, it can be seen that RBC-EM is mainly distributed in the liver and is endogenous to Kupffer cells (macrophages).

Example 5. Preparation of Exosome Mimetics Derived From Red Blood Cells Loaded With Drug

(72) Retroorbital blood samples were collected from Sprague Dawley rats (150 g; 6 weeks old; Hana Corp., Korea). Serum were separated by performing centrifugation at 4° C. and 200 g for 15 min to 20 min, and a mixture (PBS:ACD=9:1 volume ratio) of PBS and acid citrate dextrose (ACD) as an anticoagulant was mixed with blood at a ratio of 4:1.

(73) The mixture was extruded through a 1-μm pore size polycarbonate membrane filter (Nuclepore, Whatman, Inc., Clifton, N.J., USA) using a mini-extruder (Vanti Polar Lipids, Birmingham, Ala., USA). The extruded sample was diluted by adding 20×PBS to the sample, and centrifuged at 4,000 g for 10 min in order to remove RBC, larger vesicles, and debris. The centrifuged sample was filtered by a 0.22 μm syringe filter, and ultra-centrifuged at 4° C. and 100,000 g for 1 hour. After the ultracentrifugation, an obtained pellet was subjected to two-step density gradient ultracentrifugation at 4° C. using iodixanol (OptiPrep™ Density Gradient Medium, Sigma-Aldrich, USA). The exosome mimetics derived from red blood cells (RBC-EM) were obtained at the intersection point of a 60% iodixanol layer and a 20% iodixanol layer, and proteins of RBC-EM were quantified by a BCA analysis method.

(74) A drug at a concentration of 10 to 20 uM/ml was mixed with RBC-EM, and the resulting mixture was incubated at room temperature for 2 hours. As the drug, dexamethasone and curcumin were used. Thereafter, pellets obtained by performing ultracentrifugation at 100,000 g for 1 hour were washed with PBS, and a two-step density gradient ultracentrifugation was performed at 4° C. using iodixanol. A loading efficiency of the drug was calculated using UV spectrometry. The loading efficiency of the drug was about 2%.

(75) FIG. 13 illustrates a result in which RBC-EM is successfully loaded with dexamethasone or curcumin. As illustrated in FIG. 13, it can be observed by the unaided eye that as the concentration of drug added is increased, the number of RBC-EMs loaded with the drug is increased. Based on the result, it can be seen that exosome mimetics derived from red blood cells may be successfully loaded with a drug.

Example 6. Cell Labeling Using .SUP.99m.Tc-RBC-EM

(76) 6-1. Labeling of Tumor Cells With .sup.99mTc Using .sup.99mTc-RBC-EM

(77) A human atypical thyroid carcinoma 8505C cell line (Deutsche Sammlung von Mikroorganismen and Zellkulturen (DSMZ)) was cultured under a humidified atmosphere of 5% CO.sub.2 at 37° C. in Dulbecco's Modified Eagle Medium (DMEM, Invitrogen, Grand Island, N.Y., USA) supplemented with 10% fetal bovine serum (FBS), 10 U/ml penicillin, and 10 μg/ml streptomycin (Invitrogen). The 8505C cells were cultured together with .sup.99mTc-RBC-EM with various doses (0, 1, 2, and 4 μCi). 24 hours later, free .sup.99mTc-RBC-EM, which had not been absorbed in cells, were washed using PBS. Thereafter, the 8505C cells were cultured in a 20% FBS solution in a 37° C. CO.sub.2 incubator for 1, 6, 12, and 18 hours. The uptake values were measured by a gamma-counter (Cobra II, 1 Hewlett Packard, USA) at all the time points, and the amount of radiation measured was shown as a count per minute (cpm) unit. In order to compare the aforementioned results with the results of .sup.99mTc-RBC-EM (4 μCi), it was measured whether tumor cells were labeled with .sup.99mTc-RBC-EM using free .sup.99mTc (4 μCi).

(78) FIG. 14A illustrates amounts of radiation of 8505C cells according to the culture time and the dose of .sup.99mTc-RBC-EM. As the result illustrated in FIG. 14A, the amounts of radiation were gradually increased according to the dose of .sup.99mTc-RBC-EM and the time. Free .sup.99mTc absorbed in cells was analyzed as a control. FIG. 14B illustrates amounts of .sup.99mTc-RBC-EM and free .sup.99mTc with the same dose (4 μCi) absorbed in cells. As illustrated in FIG. 14B, cells rarely absorbed free .sup.99mTc, and as the time is increased, the absorption of free .sup.99mTc was not increased.

(79) This result shows that .sup.99mTc-RBC-EM has an ability to deliver .sup.99mTc to cells, and thus cells may be labeled with a radioactive material using .sup.99mTc-RBC-EM.

(80) 6-2. Labeling of White Blood Cells (WBCs) With .sup.99mTc Using .sup.99mTc-RBC-EM

(81) White blood cells (WBCs) were separated from a buffy coat of a blood sample collected in the same manner as in Example 1-1. The separated sample was centrifuged at 250 g for 10 min. Thereafter, in order to remove the contamination of RBCs, a cell pellet was re-suspended in 3 ml of a lysis buffer (0.83% (w/v) NH.sub.4Cl, 10 mM HEPES-NaOH, pH 7.0), and incubated at 37° C. for 7 min. Thereafter, the WBCs were obtained by performing a centrifugation at 250 g at room temperature for 10 min, and used in a subsequent experiment.

(82) In order to label WBCs with .sup.99mTc using .sup.99mTc-RBC-EM, the separated WBCs were cultured along with .sup.99mTc-RBC-EM in a 37° C. and CO.sub.2 incubator for 6 hours. In consideration of the short half-life of WBC, WBCs were labeled with .sup.99mTc by the same method for a culture time of 6 hours.

(83) After the culture, free .sup.99mTc-RBC-EM was removed by performing a centrifugation at 250 g for 10 min, and white blood cells labeled with a radioactive material were constructed by dissolving the collected pellet in PBS, and were named as .sup.99mTc-WBC. .sup.99mTc-WBC in which white blood cells were labeled with .sup.99mTc using .sup.99mTc-RBC-EM was additionally used in an in vivo acute inflammation tracking experiment.

Example 7. Tracking of Tumor Using Acute Inflammation Animal Model

(84) 7-1. Establishment and Confirmation of Acute Inflammation Mouse Model

(85) 6-Week-old female BALB/c nude mice were purchased from Hamamatsu (Shizuoka, Japan). In order to establish an acute inflammation mouse model, 100 μl of 1% carrageenan was injected subcutaneously into the left foot of the BALB/c nude mouse (n=15). 6 hours after the injection, characteristics of acute inflammation in which the left foot was swollen and turned red were observed.

(86) In order to confirm the establishment of acute inflammation, the .sup.18F-FDG PET/CT imaging was performed. During the injection and imaging, .sup.18F-FDG 11.1 MBq (300 μCi) was injected intravenously into mice under general anesthesia with 1% to 2% isoflurane in 100% O.sub.2, and then .sup.18F-FDG PET/CT was performed. The images were reconstructed with a 2-dimensional ordered-subsets expectation maximization algorithm (OSEM). No correction for attenuation or scattering was performed.

(87) FIG. 15 illustrates a .sup.18F-FDG PET/CT image of acute inflammatory mice. As illustrated in FIG. 15, it was confirmed that acute inflammation had been established in mice by confirming that the glucose metabolism on the left foot was strongly increased and the glucose metabolism was increased in the lymph node of the groin part.

(88) 7-2. In Vivo Gamma Camera Imaging

(89) 200 μl of 3.7 MBq .sup.99mTc-WBC was infused into the tail vein of the acute inflammation mouse model constructed in Example 7-1. Gamma camera images were obtained using a 2 mm pinhole collimator (Infinia II, GE Healthcare, Milwaukee, Wis., USA) for 10 min. During the imaging, mice were continuously anesthetized using 2.5% isoflurane. Gamma camera images were obtained by taking pictures 5 min, 3 hours, 6 hours, 12 hours, and 24 hours after administration of .sup.99mTc-WBC.

(90) 7-3. Analysis of Biodistribution of .sup.99mTc-WBC in Acute Inflammation Mouse Model

(91) Mice were sacrificed immediately after the gamma camera imaging of Example 7-2. The uptake amounts of .sup.99mTc-WBC in the organ such as the brain, thyroid gland, lungs, heart, liver, spleen, stomach, intestines, bilateral kidneys, bones, muscle, left foot, and right foot were measured by a gamma counter (Cobra II, 1 Hewlett Packard, USA). The resulting values were expressed as a percentage of the amount injected per gram of tissue (% ID/g).

(92) 7-4. Tracking of Inflammation Using .sup.99mTc-WBC

(93) FIGS. 16A to 16D illustrate that in an acute inflammation mouse model, red blood cells labeled with .sup.99mTc may be effectively used to track inflammation. Specifically, FIG. 16A illustrates a gamma camera image in an acute inflammation mouse model into which .sup.99mTc-WBC is infused via the tail vein. FIG. 16B illustrates amounts of radiation measured from the left and right feet of the acute inflammation mouse model to which .sup.99mTc-WBC is injected. FIG. 16C illustrates the biodistribution of .sup.99mTc-WBC. FIG. 16D illustrates the amounts of .sup.99mTc-WBC uptaken by the left and right legs.

(94) As illustrated in FIG. 16A, the image obtained within 5 min after injection of .sup.99mTc-WBC shows that .sup.99mTc-WBC is strongly taken up in the liver, spleen, and bladder. As in the result illustrated in FIG. 16B, .sup.99mTc-WBC was significantly accumulated in the left leg 3 hours after the injection, and the cpm values were statistically higher in the left foot than those in the right foot. It could be seen that cpm 24 hours after injection was also higher in the left foot than that in the right foot. As illustrated in FIG. 16C, in an ex vivo study, the activities of the liver and the kidneys were significantly higher in the right foot than those in the left foot, the activities of the brain, heart, intestines, muscle, and thyroid gland were significantly lower in the right foot than those in the left foot, but the activities of the liver and kidneys were rapidly decreased 3 hours after the injection. As illustrated in FIG. 16D, the uptake amount of .sup.99mTc-WBC in the left foot was higher than that in the right foot at all the time points. 24 hours after the injection, the amount of radiation in the left foot was highest of those of the other organs, and was about 20 times higher than that in the right foot.

(95) The result as described above shows that WBCs may be successfully labeled with .sup.99mTc using RBC-EM, and the position of inflammation may be successfully visualized in an animal with an acute inflammation using WBCs labeled with .sup.99mTc.

Example 8. Analysis of Position of Exosomes Derived From Red Blood Cells in Rheumatoid Arthritis Animal Model

(96) 8-1. Construction of Exosome Mimetics Derived From Red Blood Cells Labeled With Radioactive Material (.sup.99mTc) and Fluorescent Material (DiD)

(97) After the RBC-EM obtained in Example 1-1 and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate (DiD; Thermo Fisher Scientific) were cultured together at room temperature for 20 min, the cultured product was washed with PBS, and DiD-RBC-EM was separated by performing a two-step Opti-Prep density gradient ultracentrifugation in the same manner as in Example 1-1.

(98) Since only .sup.99mTc reduced to a low oxidation state is firmly bound to hemoglobin, particularly, the beta-chain of hemoglobin, DiD-RBC-EM was incubated along with tin (II) chloride in order to reduce .sup.99mTc. Exosome mimetics derived from red blood cells loaded with the radioactive material (.sup.99mTc) and the fluorescent material (DiD) were constructed by incubating DiD-RBC-EM and the same amount of 0.01% tin (II) chloride (Sigma, USA) in a shaker at 37° C. for 5 min, adding technetium-99m (.sup.99mTc) to DiD-RBC-EM (DiD-RBC-EM (100 μg):.sup.99mTc (111 MBq)) and incubating the resulting mixture in a shaker at 37° C. for 20 min, and the exosome mimetics were named as .sup.99mTc-DiD-RBC-EM. When a radioactive purity was less than 95%, an ultracentrifugation was performed at 4° C. and 100,000 g for 1 hour in order to purify .sup.99m Tc-DiD-RBC-EM.

(99) 8-2. Construction of Arthritis Animal Model

(100) A murine collagen-induced arthritis model was performed as previously reported (Journal of Controlled Release 252 (2017): 62-72; PloS one 12.4 (2017)). Bovine type-II collagen (CII; Chondrex, Redmond, Wash., USA) was dissolved at a concentration of 2 mg/ml in 10 mM acetic acid. For primary inoculation (immunization), CII (100 μg) emulsified with the same amount of a complete Freund's adjuvant was injected intradermally into the tail base of 6- to 8-week-old DBA/1J male mice (Japan SLC, Inc., Hamamatsu, Japan). An additional inoculation (booster injection) was performed on day 21 after the primary inoculation of CII (100 μs) in the complete Freund's adjuvant. Clinical characteristics of arthritis of mice were observed daily from day 22. Two independent observers observed clinical characteristics of arthritis three times per week from day 22 after the primary inoculation. The clinical severity of arthritis was recorded according to a grade from 0 to 4 as previously reported (Arthritis & Rheumatism 65.7 (2013)). When arthritis occurred, mice were randomly selected and used in a subsequent experiment.

(101) 8-3. Gamma Camera Imaging and Fluorescent Imaging

(102) The gamma camera images were captured and taken for 10 min using a 2 mm pinhole collimator (Infinia II, GE Healthcare, Milwaukee, Wis., USA). 200 μl of ˜MBq .sup.99mTc-DiD-RBC-EM was infused into the tail vein of mice. As a control, the same amount of free .sup.99mTc was infused. During the imaging, mice were continuously anesthetized using 2.5% isoflurane. The gamma camera images were taken 1, 3, 12, or 24 hours after .sup.99mTc-DiD-RBC-EM and free 99mTc were infused.

(103) C57BL/6 mice were anesthetized with isoflurane, and .sup.99mTc-DiD-RBC-EM or PBS was infused into the tail vein via intravenous injection. 1, 3, 12, or 24 hours after the infusion, fluorescent imaging was performed using an in vivo imaging system (IVIS Lumina III instrument, PerkinElmer) [wavelengths: Excitation—644 nm and emission—665 nm; imaging parameters: binning—4, smoothing—3×3, Field of subject (stage—D): 12.5 cm, height of subject image: 1.5 cm].

(104) 8-4. Analysis of Position of .sup.99mTc-DiD-RBC-EM

(105) FIGS. 17A and 17B illustrate the results of performing gamma camera imaging after .sup.99mTc-DiD-RBC-EM is infused into the articular cavity of an animal model with rheumatoid arthritis. Specifically, FIG. 17A illustrates a gamma camera image of mice with rheumatoid arthritis, and FIG. 17B quantitatively illustrates amounts of measured radiation. The left graph of FIG. 17B illustrates absolute values of amounts of radiation measured (background corrected counts) in a portion injected into the arthritic lesion, and the right graph illustrates the amounts of radiation (retention rate) remaining in the injected lesion after radiation decay correction in the same data. That is, FIG. 17 illustrates how much radiation remains in the injected lesion when the radiation dose on hour 1 is seen as 100% (corresponding to 1.0 in the graph).

(106) As illustrated in FIGS. 17A and 17B, it could be confirmed that .sup.99mTc-DiD-RBC-EM remained in the articular cavity without significantly moving to other organs up to 24 hours after the infusion.

(107) As a result of performing fluorescent imaging after .sup.99mTc-DiD-RBC-EM is infused into the articular cavity of the animal model with rheumatoid arthritis, FIG. 18A illustrates a fluorescent image over time, and FIG. 18B quantitatively illustrates amounts of fluorescent material measured. The No. 2 mouse died on day 2, but it can be seen that .sup.99mTc-DiD-RBC-EM remains in the articular cavity up to day 15 in the IVIS images of the No. 1 mouse. It is determined as autofluorescence that fluorescence is observed in addition to the articular cavity on 12th hour, day 3, day 6, day 8, day 10, and day 15 in the IVIS images of the No. 1 mouse. As illustrated in FIG. 18, .sup.99mTc-DiD-RBC-EM are remaining in the articular cavity without significantly moving to other organs in the same manner as in the gamma camera imaging result.

Example 9. Analysis of Position of Exosomes Derived From Red Blood Cells in Tumor Animal Model

(108) 9-1. Construction of Tumor Animal Model

(109) Nude mice transplanted subcutaneously with Ca162/effluc (5×10.sup.6 cells) in the lower right region were grown for 6 weeks. After the mice were anesthetized with 2.5% isofluorane and 100 μl of D-luciferin (3 mg/mouse; Caliper) was injected intraperitoneally for bioluminescence images, a bioluminescence imaging (hereinafter, referred to as BLI) was performed using the IVIS Lumina III imaging system. Subsequently, BLI was observed up to 6 weeks, and the mice were used in an additional experiment.

(110) 9-2. Construction of Exosome Mimetics Derived From Red Blood Cells Labeled With Fluorescent Material (Porphyrin)

(111) Red blood cells were obtained by the method described in Example 1-1, diluted by adding PBS to red blood cells (RBC:PBS=1:9 by volume ratio), and porphyrin was added thereto so as to have a concentration of 0.1%. The mixture was extruded once to four times through a 1-μm pore size polycarbonate membrane filter (Nuclepore, Whatman, Inc., Clifton, N.J., USA) using a mini-extruder (Vanti Polar Lipids, Birmingham, Ala., USA).

(112) The labeled exosome mimetics derived from red blood cells labeled with porphyrin were obtained at the intersection point of a 60% iodixanol layer and a 20% iodixanol layer, and were immediately used without an additional treatment. The obtained exosome mimetics derived from red blood cells labeled with porphyrin were named as porphyrin-RBC-exosome mimetics (porphyrin-RBC-EM).

(113) 9-3. Fluorescent Imaging

(114) C57BL/6 mice were anesthetized with isofluorane, and porphyrin-RBC-EM or free porphyrin was infused into the tumor of the tumor animal model and into the hypodermis on the tendon side. 1 to 288 hours after the infusion, fluorescent imaging was performed using an in vivo imaging system (IVIS Lumina III instrument, PerkinElmer) [wavelengths: Excitation—644 nm and emission—665 nm; imaging parameters: binning—4, smoothing—3×3, Field of subject (stage—D): 12.5 cm, height of subject image: 1.5 cm]. Ex vivo fluorescent imaging was performed by collecting the subcutaneous tissue, liver, spleen, heart, lungs, kidneys, and tumors after the imaging, and a quantitative analysis was performed using IVIS software (Living Image Software, PerkinElmer).

(115) 9-4. Position of Porphyrin-RBC-EM

(116) RBC-EM was loaded with porphyrin, and then injected into the hypodermis and tumors. FIG. 19 illustrates a fluorescent imaging over time when RBC-EM-porphyrin is infused into the tumor. The HRS described in FIG. 19 means time. FIG. 20 illustrates amounts of porphyrin measured subcutaneously and in the tumor when RBC-EM-porphyrin is infused subcutaneously and into the tumor. As illustrated in FIGS. 19 and 20, it can be confirmed in an in vivo IVIS imaging that RBC-EM-porphyrin remains in the tumor up to day 12.

(117) On 12 days, fluorescent imaging was performed by sacrificing mice and collecting subcutaneous tissues, liver, spleen, heart, lungs, kidneys, and tumor tissues. FIG. 21 illustrates an ex-vivo fluorescent image of various organs and the amount of porphyrin quantitatively measured. As illustrated in FIG. 21, it can be seen that RBC-EM-porphyrin remains in the tumor without significantly moving to other organs in the same manner as in the results because the amount of porphyrin in the hypodermis and tumor is remarkably high.

(118) Statistical Analysis

(119) The experimental result values are shown as mean±standard deviation (SD). The statistical significance is determined (student t-test) by GraphPad Prism5 software version 7.04 (GraphPad Software, Inc., La Jolla, Calif., USA). P values less than 0.05 were considered statistically significant.