Title Page
ABSTRACT
Contents
Chapter 1. Research overview 19
1.1. Cancer therapy 19
1.1.1. Utilization of sonodynamic therapy and photodynamic therapy as non-invassive approach for cancer treatment 19
1.1.2. Combined chemotherapy with sonodynamic therapy and photodynamic therapy for synergistic anticancer effects 20
1.2. Exosomes as biocompatible nanocarriers for the efficient delivery of sonosensitizer and photosensitizer 20
1.3. Engineering exosome for efficient cancer targeting 21
1.2.1. Loading of small molecule drugs 21
1.2.2. Surface modification of exosomes for enhanced cancer-targeting 22
1.2.3. Stimuli-responsive-exosome for efficient and selective cancer therapy 22
1.2.4. Brain tumor therapy using exosomes 23
1.3. The scope and organization of dissertation 23
1.3.1. The scope of dissertation 23
1.3.2. The organization of dissertation 24
Chapter 2. Safe and targeted sonodynamic cancer therapy using biocompatible exosome-based nanosonosensitizers 29
2.1. Introduction 29
2.2. Experimental Section 31
2.2.1. Materials 31
2.2.2. Isolation and characterization of exosomes 31
2.2.3. Western blot analysis 32
2.2.4. Preparation and characterization of ICG-loaded exosomes (ExoICG) and FA-loaded ExoICG (FA-ExoICG) 32
2.2.5. Assessment of colloidal stability of ExoICG and FA-ExoICG 33
2.2.6. Assessment of aqueous stability of ICG in ExoICG and FA-ExoICG 33
2.2.7. In vitro drug release analysis 34
2.2.8. Cell culture 34
2.2.9. Cellular uptake analysis of free ICG, ExoICG, and FA-ExoICG 34
2.2.10. ROS generation by free ICG, ExoICG and FA-ExoICG upon US treatment 35
2.2.11. In vitro sonotoxicity evaluation 35
2.2.12. Preparation of MCF-7 tumor-bearing mice for in vivo experiments 36
2.2.13. In vivo biodistribution study by in vivo imaging systems (IVIS) 36
2.2.14. In vivo antitumor efficacy of FA-ExoICG under US irradiation 37
2.2.15. Statistical analysis 37
2.3. Result 37
2.3.1. Design, preparation, and characterization of the ICG-loaded exosomes 37
2.3.2. High colloidal stability of ICG-loaded exosomes 40
2.3.3. Long-term aqueous stability of ICG by encapsulation into exosomes 40
2.3.4. US-triggered release of ICG by ICG-loaded exosomes 41
2.3.5. Cancer-targeted cellular uptake of FA-ExoICG 42
2.3.6. Enhanced sonodynamic effects by ICG-loaded exosomes 42
2.3.7. In vitro cancer-targeted sonodynamic therapy using FA-ExoICG 44
2.3.8. Effective tumor accumulation of FA-ExoICG 45
2.3.9. Efficient in vivo sonodynamic effects by FA-ExoICG 46
2.4. Discussion 47
2.5. Conclusion 48
Chapter 3. Engineered extracellular vesicle-based sonotheranostics for dual stimuli-sensitive drug release and photoacoustic imaging-guided chemo-sonodynamic cancer therapy 63
3.1. Introduction 63
3.2. Experimental Section 66
3.2.1. Materials 66
3.2.2. Preparation and characterization of EVs 66
3.2.3. Fabrication and characterization of engineered EVs 67
3.2.4. Colloidal stability assessment of SBC-EV(ICG/PTX) 67
3.2.5. Assessment of aqueous stability of ICG in ICG-loaded EVs 68
3.2.6. In vitro drug release profiles of SBC-EV(ICG/PTX) 68
3.2.7. Cellular internalization analysis of free ICG and ICG-loaded EVs 68
3.2.8. Live cell imaging using confocal laser scanning microscopy (CLSM) 69
3.2.9. ROS generation using ICG-loaded EVs upon US treatment 69
3.2.10. In vitro sonotoxicity evaluation 70
3.2.11. In vitro apoptosis analysis 70
3.2.12. MCF-7 tumor-bearing mouse models for in vivo studies 71
3.2.13. In vivo biodistribution study using in vivo imaging systems (IVIS) 71
3.2.14. In vivo PA imaging 71
3.2.15. In vivo antitumor efficacy of SBC-EV(ICG/PTX) with US treatment 72
3.2.16. Statistical analysis 73
3.3. Result 73
3.3.1. Design, preparation, and characterization of drug-loaded EVs 73
3.3.2. High colloidal stability and storage stability of engineered EVs 75
3.3.3. Enhanced aqueous stability of ICG via encapsulation into EVs 75
3.3.4. pH-responsiveness of SBC-loaded EVs 76
3.3.5. Dual pH- and US-responsive drug release by SBC-loaded EVs 76
3.3.6. Acid-triggered CO₂ generation by SBC-loaded EVs 77
3.3.7. Efficient cellular uptake of ICG-loaded EVs 77
3.3.8. Enhanced cytoplasmic release of ICG by SBC-loaded EVs 78
3.3.9. US-triggered intracellular ROS generation by ICG-loaded EVs 79
3.3.10. In vitro SDT efficacy of SBC-EV(ICG/PTX) 80
3.3.11. In vivo PA imaging using ICG-loaded EVs 81
3.3.12. Enhanced in vivo SDT by pH-sensitive SBC-EV(ICG/PTX) 81
3.4. Discussion 82
3.5. Conclusion 83
Chapter 4. Bioreducible exosomes encapsulating glycolysis inhibitors potentiate mitochondria-targeted sonodynamic cancer therapy via cancer-targeted drug release and cellular energy depletion 100
4.1. Introduction 100
4.2. Experimental Section 102
4.2.1. Materials 102
4.2.2. Synthesis of TPP-Ce6 102
4.2.3. Synthesis of diselenide-bearing lipids (DSe) 103
4.2.4. Preparation and characterizations of E(T-Ce6/FX11) and DSe-E(T-Ce6/FX11) 103
4.2.5. Western blot 104
4.2.6. Evaluation of colloidal stability and long-term aqueous stability of DSe-E(T- Ce6/FX11) 105
4.2.7. In vitro drug release studies 105
4.2.8. Cellular internalization analysis of T-Ce6 and T-Ce6-loaded exosomes 106
4.2.9. Mitochondrial uptake analysis of T-Ce6 and T-Ce6-loaded exosomes 106
4.2.10. JC-1 staining assay 107
4.2.11. Evaluation of in vitro ROS generation 107
4.2.12. In vitro quantification of lactate and ATP levels 108
4.2.13. In vitro extracellular acidification quantification 108
4.2.14. In vitro apoptosis analysis 109
4.2.15. In vitro cytotoxicity evaluation 109
4.2.16. Establishment of MCF-7 tumor-bearing mouse model for in vivo studies 110
4.2.17. In vivo biodistribution study using in vivo imaging systems (IVIS) 110
4.2.18. Evaluation of in vivo antitumor activity by various exosomes samples 110
4.2.19. In vivo quantification of lactate and ATP levels 110
4.2.20. In vivo ROS quantification 111
4.2.21. In vivo extracellular acidification quantification 111
4.3. Result 111
4.3.1. Design of bioreducible exosomes loaded with mitochondria-targeting sonosensitizers for safe and targeted cancer therapy 111
4.3.2. Fabrication and characterization of DSe and drug-loaded bioreducible EVs 112
4.3.3. Enhanced aqueous stability of T-Ce6 via encapsulation into exosomes 114
4.3.4. Redox-responsiveness of DSe-E(T-Ce6/FX11) 115
4.3.5. Dual GSH- and US-responsive drug release by DSe-E(T-Ce6/FX11) 115
4.3.6. Efficient cellular uptake and cytoplasmic drug release by bioreducible exosomes 117
4.3.7. Mitochondrial accumulation of T-Ce6 118
4.3.8. US-triggered intracellular ROS generation using T-Ce6-loaded exosomes 119
4.3.9. Efficient glycolysis inhibition by T-Ce6/FX11-loaded exosomes 120
4.3.10. Efficient anticancer activity by mitochondria-targeted SDT combined with energy-depleting therapy 121
4.3.11. Effective tumor accumulation of DSe-E(T-Ce6/FX11) 122
4.3.12. Efficient in vivo tumor inhibition by DSe-E(T-Ce6/FX11) 122
4.4. Discussion 123
4.5. Conclusion 125
Chapter 5. Brain endothelial cell-derived extracellular vesicles with a mitochondria-targeting photosensitizer effectively treat glioblastoma by hijacking the blood‒brain barrier 145
5.1. Introduction 145
5.2. Experimental Section 148
5.2.1. Preparation and characterization of bEVs and TPP-Ce6-loaded bEVs 148
5.2.2. In vitro transcytosis of TPP-Ce6-loaded EVs using a transwell BBB model 148
5.2.3. Mechanism of transferrin receptor-mediated transcytosis of bEV(TPP-Ce6) 150
5.2.4. In vitro phototoxicity evaluation 150
5.2.5. Preparation of orthotopic GBM-xenografted mice 150
5.2.6. In vivo brain tumor uptake study of bEV(TPP-Ce6) 151
5.2.7. In vivo biodistribution study and brain-tumor accumulation of bEV(TPP-Ce6) using IVIS 151
5.2.8. In vivo anti-brain tumor efficacy of bEV(TPP-Ce6) with laser irradiation 152
5.2.9. Statistical analysis 152
5.3. Result 152
5.3.1. Design of mitochondria-targeting photosensitizer-loaded bEVs for safe and efficient in vivo GBM therapy 152
5.3.2. Synthesis of TPP-Ce6 and preparation of TPP-Ce6-loaded bEVs 153
5.3.3. High colloidal stability of bEV(TPP-Ce6) under physiological conditions 154
5.3.4. Long-term aqueous stability of Ce6 by encapsulation into EVs 154
5.3.5. Light-triggered release of TPP-Ce6 from bEV(TPP-Ce6) 155
5.3.6. Efficient BBB transcytosis of TPP-Ce6-Loaded bEVs 155
5.3.7. Transferrin-mediated brain targeting of bEVs 156
5.3.8. Enhanced mitochondrial uptake of Ce6 by conjugation with TPP 157
5.3.9. Light-triggered intracellular ROS generation using TPP-Ce6-loaded bEVs 158
5.3.10. In vitro PDT using mitochondria-targeting TPP-Ce6-loaded bEVs 158
5.3.11. Effective brain tumor accumulation of bEV(TPP-Ce6) 160
5.3.12. Efficient in vivo PDT effects of bEV(TPP-Ce6) 160
5.4. Discussion 161
5.5. Conclusion 163
Summary and Future Outlook 183
References 186
Table 2.1. Size and zeta potentials of various exosomes 50
Table 3.1. Drug encapsulation efficiency of ICG in SBC-EV(ICG/PTX) with different DMSO composition ratios (v/v) in PBS for the preparation. 85
Table 3.2. Size and surface charges of various EV-derived samples. 85
Table 4.1. Loading efficiency of T-Ce6 and FX11 in E(T-Ce6/FX11) and DSe-E(T-Ce6/FX11) 127
Table 4.2. Loading capacity of T-Ce6, FX11, and Se in E(T-Ce6/FX11) and DSe-E(T-Ce6/FX11). 127
Table 4.3. Sizes and surface charges of blank exosomes, E(T-Ce6/FX11), and DSe-E(T-Ce6/FX11). 127
Table 5.1. Sizes and zeta potentials of various EV samples. 164
Figure 1.1. Mechanisim of SDT and PDT. Mechanism of (A) sonodynamic therapy (SDT) and (B) photodynamic therapy (PDT). 27
Figure 1.2. Preparation of exosome for drug carrier. Illustration for engineered method to prepare exosome for drug carrier. 28
Figure 2.1. Prepapration of FA-ExoICG for efficient SDT. Schematic illustration of the fabrication and sonodynamic therapeutic action of folic acid-conjugated, ICG-loaded exosomes... 51
Figure 2.2. Characterizations of FA-ExoICG. (A) Absorbance spectra of blank exosome, free ICG, FA, FA-Exosome, and FA-ExoICG solutions. (B) TEM images of blank exosome and FA-... 52
Figure 2.3. Stabilities of FA-ExoICG in different conditions. (A) Fluorescence intensity changes of free ICG, ExoICG, and FA-ExoICG after incubation in PBS at 4 °C for 10 days. (B) Size... 53
Figure 2.4. Drug release profiles of FA-ExoICG. (A) ICG release profiles from ExoICG and FA-ExoICG in the absence or presence of US irradiation (1 min) at different incubation times. (B)... 54
Figure 2.5. Cellular uptake analysis of FA-ExoICG. Relative cellular uptake of ICG in hDFB and MCF-7 cells after incubation with free ICG, ExoICG, and FA-ExoICG for 4 h. 55
Figure 2.6. ROS analysis of FA-ExoICG. (A) ROS generation in cell-free system of free ICG, ExoICG and FA-ExoICG. (B) Intracellular ROS levels of MCF-7 cells treated with free ICG,... 56
Figure 2.7. Cell viability analysis of MCF-7 cells by various treatments. (A) Cell viabilities of MCF-7 cells treated with free ICG, ExoICG, and FA-ExoICG before and after US irradiation (0.3... 57
Figure 2.8. Cell viability analysis of MCF-7 cells and hDFB cells. Cell viabilities of (A) MCF-7 and (B) hDFB cells treated with blank exosomes at different concentrations. 58
Figure 2.9. In vivo biodistribution of FA-ExoICG. In vivo real-time biodistribution images from MCF-7 tumor-bearing mouse at different times following i.v. injection of Free ICG, ExoICG, and... 59
Figure 2.10. Ex vivo imaging of each organ at 24 hours after administration of Free ICG, ExoICG, and FA-ExoICG. 60
Figure 2.11. Tumor growth inhibition effects of FA-ExoICG. (A) Normalized tumor growth ratio of MCF-7 tumor-bearing mice (n＝4) as a function of time. The mice were irradiated by US... 61
Figure 2.12. H&E staining of major organ slides of the mice at 14 days post-injection with various samples under US irradiation. Scale bars indicate 200 μm. 62
Figure 3.1. Prepapration of SBC-EV(ICG/PTX) for chemo-sonodynamic combination cancer therapy. Schematic illustration of the sonotheranostic action of dual pH/US-responsive EVs co-... 86
Figure 3.2. Characterization of SBC-EV(ICG/PTX). (A) Absorption spectra of blank EV, free ICG, free PTX, EV(ICG), EV(ICG/PTX), and SBC-EV(ICG/PTX) samples. (B) TEM images of... 87
Figure 3.3. Stabilities of SBC-EV(ICG/PTX) in different conditions. (A) Changes in fluorescence intensity of free ICG, EV(ICG), SBC-EV(ICG), and SBC-EV(ICG/PTX) after incubation in PBS... 88
Figure 3.4. pH sensitivity of SBC-EV(ICG/PTX). (A) Change in the size of SBC-EV(ICG/PTX) under physiological (i.e., pH 7.4) and acidic (i.e., pH 6.0) conditions. After 48 h of incubation in... 89
Figure 3.5. Drug release analysis of SBC-EV(ICG/PTX) in different conditions. (A) PTX and (B) ICG release profiles from SBC-EV(ICG/PTX) at different pH and US (1 min of irradiation) conditions. 90
Figure 3.6. Drug release analysis of EV(ICG/PTX) and SBC-EV(ICG/PTX). PTX release profiles of EV(ICG/PTX) and SBC-EV(ICG/PTX) at different pH conditions after 8 h of incubation. 91
Figure 3.7. Relative levels of CO₂ generation by blank EV and SBC-EV under different pH conditions, determined by an acid-base titration method. 92
Figure 3.8. Relative internalization of ICG by MCF-7 cells after 4 h of treatment with free ICG, EV(ICG), and SBC-EV(ICG). 93
Figure 3.9. Live confocal microscopic imaging of MCF-7 cells treated with RB-labeled EV(ICG) and RB-labeled SBC-EV(ICG). LysoTracker (green dots) was used to stain endo/lysosomal... 94
Figure 3.10. Relative colocalization ratios of RB with LysoTracker in MCF-7 cells after incubation with RB-labeled EV(ICG) and RB-labeled SBC-EV(ICG) for different incubation times. 95
Figure 3.11. Intracellular ROS levels in MCF-7 cells treated with free ICG and ICG-loaded EV samples before and after US treatment. 96
Figure 3.12. Viabilities of MCF-7 cells after treatment with various EV samples before and after US irradiation (0.3 W/cm² for 1 min). 97
Figure 3.13. In vivo whole-body PA images of MCF-7-bearing mice at different time intervals after tail vein injection of EV(ICG/PTX) and free ICG (n＝3). (A) PA MAP and depth-resolved... 98
Figure 3.14. Tumor growth inhibition effect of SBC-EV(ICG/PTX). (A) Normailzed tumor growth ratio of MCF-7 tumor-bearing mice (n＝4) for 14 days after administration of various... 99
Figure 4.1. Preparation of DSe-E(T-Ce6/FX11) for cancer-targeted drug release and cellular energy depletion. Schematic illustration for the preparation of mitochondria-targeting T-Ce6- and... 128
Figure 4.2. Characterization of DSe-E(T-Ce6/FX11). (A) Absorption spectra of blank exosomes, FX11, T-Ce6, E(T-Ce6/FX11), and DSe-E(T-Ce6/FX11). (B) TEM images of blank exosomes... 129
Figure 4.3. EDS mapping of DSe-E(T-Ce6/FX11). Scale bars: 50 nm. 130
Figure 4.4. Stability of DSe-E(T-Ce6/FX11) in different conditions. (A) Fluorescence stability of T-Ce6, E(T-Ce6/FX11), and DSe-E(T-Ce6/FX11) in normal lighting conditions. (B) Changes... 131
Figure 4.5. Redox-responsiveness of DSe-E(T-Ce6/FX11). (A) Change in the size of DSe-E(T-Ce6/FX11) under physiological conditions (PBS, pH 7.4) and reductive conditions (PBS + 10... 132
Figure 4.6. Drug release analysis. (A) Cumulative release of FX11 from DSe-E(FX11) incubated with 10 mM GSH in PBS at various time points. The cumulative release profiles of FX11 were... 133
Figure 4.7. Relative internalization of T-Ce6 and DSe-E(T-Ce6/FX11) by MCF-7 cells. 134
Figure 4.8. Confocal micrographs of MCF-7 cells after treatment with RB-labeled E(T-Ce6/FX11) and RB-labeled DSe-E(T-Ce6/FX11). LysoTracker (green dots) and DAPI (blue dots) were... 135
Figure 4.9. Mander's overlap coefficients representing the colocalization ratios of RB signals with LysoTracker or T-Ce6, which were based on confocal micrographs (Figure 4.8). 136
Figure 4.10. Mitochondrial uptake analysis by confocal. (A) Confocal micrographs of MCF-7 cells after incubation with Ce6, T-Ce6, E(T-Ce6/FX11), and DSe-E(T-Ce6/FX11). MitoTracker... 137
Figure 4.11. Mitochondrial damage analysis. (A) Fluorescence images of JC-1 stained MCF-7 cells after exposure to various samples and US. Scale bars: 150 µm. (B) Relative ratios of red to... 138
Figure 4.12. US-triggered ROS production in MCF-7 cells after exposure to various samples. The cells were irradiated with 1 MHz US for 2 min (0.5 W/cm²). 139
Figure 4.13. Lactate and ATP quantification. Changes in the levels of (A) lactate and (B) ATP in MCF-7 cells after various treatments under US irradiation (1 MHz, 0.5 W/cm², 2 min). 140
Figure 4.14. Viabilities of MCF-7 cells after treatment with various samples before and after 2 min of 1 MHz US irradiation (0.5 W/cm²). 141
Figure 4.15. In vivo biodistribution images from tumor-bearing mice after i.v. administration of T-Ce6, E(T-Ce6/FX11), and DSe-E(T-Ce6/FX11). 142
Figure 4.16. Tumor growth inhibition effects by DSe-E(T-Ce6/FX11). (A) Relative tumor growth ratio of sample-injected MCF-7 tumor-xenograft mice (n＝4) for 14 days. 3 min of 1 MHz US... 143
Figure 4.17. In vivo lactate and ATP level analysis. Changes in the levels of (A) lactate and (B) ATP in the tumor of mice on day 14 after exposure to various samples. 144
Figure 5.1. Preparation of bEV(TPP-Ce6) for efficient glioblastoma treatment. Schematic illustration of the mitochondria-targeted PDT using bEV(TPP-Ce6) that crosses the BBB. After... 165
Figure 5.2. Characterization of bEV(TPP-Ce6). (A) Absorbance spectra of blank bEVs, Ce6, 2-hydroxyethyl TPP, TPP-Ce6, and bEV(TPP-Ce6) solutions. (B) TEM images of blank bEV and... 166
Figure 5.3. Stability of bEV(TPP-Ce6) in different conditions. (A) Size changes of bEV(TPP-Ce6) incubated in PBS-containing 10% FBS for 5 days, determined by NTA. (B) Changes in... 167
Figure 5.4. TPP-Ce6 release profiles from bEV(TPP-Ce6) before and after light irradiation (660 nm, 1 min) at different incubation times. 168
Figure 5.5. In vitro BBB model. (A) Schematic diagram of in vitro BBB model using bEnd.3 cells. (B) Apparent permeability coefficient (Papp) of TPP-Ce6 and bEV(TPP-Ce6) measured... 169
Figure 5.6. Relative Ce6 uptake of bEV(TPP-Ce6) by U87MG cells after crossing BBB layers. 170
Figure 5.7. Relative cellular uptake of bEV(TPP-Ce6) in U87MG cells following pre-treatment with Tf at different concentrations. 171
Figure 5.8. Relative mitochondrial uptake of Ce6, TPP-Ce6, and bEV(TPP-Ce6) in U87MG cells. 172
Figure 5.9. Mitochondrial accumulation analysis by confocal micrographs. Confocal micrographs displaying intracellular localization of Ce6 and TPP-Ce6 in U87MG cells. U87MG... 173
Figure 5.10. Relative ROS level in cell-free system treated by Ce6, TPP-Ce6 and bEV(TPP-Ce6) 174
Figure 5.11. Relative intracellular ROS levels in U87MG cells treated with Ce6, TPP-Ce6, and bEV(TPP-Ce6) before and after light irradiation (660 nm, 1 min). 175
Figure 5.12. Mitochondrial damge analysis. Fluorescence images JC-1 stained U87MG cells after various treatments: 1) control, 2) Ce6+L, 3) TPP-Ce6+L, and 4) bEV(TPP-Ce6)+L. The red JC-... 176
Figure 5.13. Viabilities of U87MG cells treated with Ce6, TPP-Ce6, and bEV(TPP-Ce6) before and after light irradiation (660 nm, 1 min). 177
Figure 5.14. Viabilities of U87MG cells treated with various concentrations of bEV. 178
Figure 5.15. In vivo biodistribution. In vivo real-time fluorescence imaging of orthotopic GBM-xenografted mouse model after i.v. administration of free Ce6, free TPP-Ce6, and bEV(TPP-Ce6).... 179
Figure 5.16. Ex vivo imaging of tumor-contained brain and major organs at 24 h post-injection of free Ce6, free TPP-Ce6, and bEV(TPP-Ce6). 180
Figure 5.17. Tumor growth inhibition effects by bEV(TPP-Ce6). (A) Time-lapse bioluminescence intensity (BLI) in the brain tumors of orthotopic GBM-xenografted mice after... 181
Figure 5.18. H&E stained brain sections of mice on day 9 after i.v. injection of PBS, Ce6, TPP-Ce6, and bEV(TPP-Ce6) following light irradiation. Tumor region of interest in the brain section... 182