Title Page
Contents
ABSTRACT 11
Ⅰ. Introduction 13
Ⅱ. Isolation and application of various nature-derived exosomes (EXOs) 15
2.1. Materials and methods 15
2.1.1. Materials 15
2.1.2. Isolation and characterization of exosomes (EXOs) from cells 15
2.1.3. Isolation of bovine milk-derived EXOs (mEXOs) 17
2.1.4. Characterization of mEXOs 18
2.1.5. Isolation of ginseng-derived EXOs (gEXOs) 18
2.1.6. Characterization of gEXOs 19
2.2. Results and discussions 20
2.2.1. Characterization of cell-derived EXOs 20
2.2.2. Characterization of mEXOs 24
2.2.3. Characterization of gEXOs 27
2.3. Conclusion 30
Ⅲ. Engineering EXOs for targeted drug delivery 31
3.1. Materials and methods 31
3.1.1. Materials 31
3.1.2. Formulation and characterization of drug-loaded EXOs 31
3.1.3. Anticancer effect of DOX/EXOs 33
3.1.4. Preparation and characterization of 3D spheroid 33
3.1.5. Formulation and characterization of PEGylated EXOs (PEG-EXOs) 34
3.1.6. Spheroid penetration assay 35
3.1.7. Preparation of nonporous PLGA MS 36
3.1.8. Surface modification of PLGA MS with OVA and EXO 36
3.1.9. Intracellular uptake of EXO-Dopa MS 37
3.2. Results and discussions 38
3.2.1. Formulation and characterization of drug-loaded EXOs 39
3.2.2. Characterization of 3D spheroid 44
3.2.3. Spheroid penetration assay 47
3.2.4. Preparation of MS with different levels of surface roughness 51
3.2.5. Characterization of EXO-Dopa MS 53
3.2.6. Intracellular uptake of EXO-Dopa MS 56
3.3. Conclusion 60
Ⅳ. Formulation of lipid nanoparticles (LNPs) for gene therapy 61
4.1. Materials and methods 61
4.1.1. Materials 61
4.1.2. Preparation of LNPs 61
4.1.3. Characterization of LNPs 62
4.2. Results and discussion 64
4.2.1. Preparation of LNPs 64
4.2.2. Characterization of LNPs 66
4.3. Conclusion 70
Ⅴ. Conclusion 71
References 73
Abstract (in Korean) 81
〈Table 2-1〉 Characteristics of various nature-derived exosomes (EXOs) 23
〈Table 2-2〉 Flowchart of isolation procedure of mEXOs and sEXOs. 25
〈Table 2-3〉 Flowchart of isolation procedure of ginseng-derived EXOs (gEXOs) 28
〈Figure 2-1〉 Characterization of cell-derived EXOs prepared by ultracentrifugation (UC) method. (a) Hydrodynamic size by DLS analysis and... 21
〈Figure 2-2〉 Characterization of cell-derived EXOs prepared by UC combined with ExoQuick (UC+EQ) method. (a) Hydrodynamic size and (b)... 22
〈Figure 2-3〉 Characterization of mEXOs by UC and UC+EDTA. (a) The amount of exosomal protein per mL of milk measured by BCA assay. (b) Hydrodynamic size of... 26
〈Figure 2-4〉 Characterization of gEXOs. (a) The amount of exosomal protein per g of ginseng and (b) the total amount of protein in individual... 29
〈Figure 3-1〉 Loading capacity of GW4869-incorporated EXOs (GW4869/EXOs) prepared by sonication. As control, simple mix of drug... 41
〈Figure 3-2〉 Characterization of SR717-incorporated EXOs (SR717/EXOs). (a) The amount of SR717 in supernatant after incubation... 42
〈Figure 3-3〉 Characterization of DOX-incorporated EXOs (DOX/EXOs). (a) Loading capacity of DOX in EXOs prepared by incubation. As control,... 43
〈Figure 3-4〉 Clustering behavior of cells according to the types of cancer cells and existence of fibroblasts. Cancer monoculture and... 45
〈Figure 3-5〉 Morphology of spheroids observed by (a) inverted fluorescence microscopy (upper: bright field, bottom: green fluorescence)... 46
〈Figure 3-6〉 Characterization of PEGylated EXOs (PEG-EXOs). (a) Hydrodynamic size of particles. (b) The yield of incorporated DSPE-PEG-... 48
〈Figure 3-7〉 Confocal images for (a) DiD and (b) green fluorescent protein (GFP) in the A549-GFP/fibroblast coculture spheroids after... 49
〈Figure 3-8〉 Fluorescence intensity of each z-stack slice in confocal images of spheroids (n=4) for (a) DiD and (b) GFP, respectively. (c)... 50
〈Figure 3-9〉 Schematic illustration for the preparation of dopamine-coated PLGA microspheres (Dopa MS) and exosome-coated Dopa MS... 52
〈Figure 3-10〉 Characterization of EXO-Dopa MSs. (A) Scanning electron microscopy (SEM) images of MS before surface modification. Scale bar=... 55
〈Figure 3-11〉 (a) Fluorescence microscopy images and (b) quantitative analysis of coumarin-labeled EXO-Dopa MS (EXO/MS weight ratios=0, 5,... 58
〈Figure 3-12〉 Fluorescence intensity and images of coumarin-labeled MS according to the existence of EXOs internalized by DC2.4 cells. 59
〈Figure 4-1〉 Schematic illustration for the preparation of endosome escaper-incorporated lipid nanoparticles (LNPs) delivering... 65
〈Figure 4-2〉 Hydrodynamic size of endosome escaper-incorporated LNPs to determine the ratio of incorporating EE into LNPs. 67
〈Figure 4-3〉 Characterization of LNPs, pHis/LNPs, EE#1/LNPs, and EE#2/LNPs with Gb3 synthase siRNA. (a) Hydrodynamic size by DLS... 68
〈Figure 4-4〉 Quantification of encapsulated (a) siRNA and (b) endosome escaper in LNPs. 69