Title Page
ABSTRACT
Contents
Chapter 1. Introduction 13
1.1. Research background 13
1.2. Recent researches 13
1.3. Research purpose 15
Chapter 2. Efficient combination chemo/sonodynamic therapy using T-Ce6 and PL loaded tween80 nanoparticles 17
2.1. Materials 17
2.2. Method 17
2.2.1. Preparation and characterization of T80(T-Ce6) and T80(T-Ce6/PL) 17
2.2.2. Assessment of the stability of T80(T-Ce6/PL) 18
2.2.3. Drug release profile of T-Ce6 or PL from T80(T-Ce6/PL) 18
2.2.4. Cell culture 18
2.2.5. Cellular uptake evaluation of Ce6, T-Ce6 and T80(T-Ce6/PL) 19
2.2.6. Mitochondrial uptake evaluation of Ce6, T-Ce6, and T80(T-Ce6/PL) 19
2.2.7. US-triggered intracellular ROS generation by T80(T-Ce6/PL) 20
2.2.8. In vitro cytotoxicity evaluation 20
2.2.9. Statistical analysis 20
2.3. Results and Discussion 21
2.3.1. Characterization of T80(T-Ce6/PL) NPs 21
2.3.2. High colloidal stability of T80(T-Ce6/PL) under physiological conditions 24
2.3.3. In vitro drug release profiles of T-Ce6 or PL from T80(T-Ce6/PL) 26
2.3.4. Enhanced cellular uptake of T-Ce6 by T80-based NPs 28
2.3.5. Enhancement of mitochondrial uptake of T-Ce6 30
2.3.6. ROS generation of T80(T-Ce6/PL) under US irradiation 33
2.3.7. Cytotoxicity analysis 35
2.3.8. Challenges and solutions 40
Chapter 3. Enhanced sonodynamic therapy using T-Ce6 loaded MnO₂ nanoparticles 41
3.1. Materials 41
3.2. Method 41
3.2.1. Synthesis of hollow MnO₂ nanoparticles 41
3.2.2. Synthesis of MnO₂(T-Ce6) NPs 42
3.2.3. Cell culture 42
3.2.4. Quantification of intracellular O₂ generation 42
3.2.5. Colocalization of MnO₂(T-Ce6) in the mitochondria 43
3.2.6. Quantification of intracellular ROS 43
3.2.7. In vitro cytotoxicity evaluation 43
3.2.8. Statistical analysis 44
3.3. Result and discussion 45
3.3.1. Characteristic of MnO₂(T-Ce6) NPs 45
3.2.2. Quantification of intracellular O₂ generation 47
3.2.3. Efficient colocalization of MnO₂(T-Ce6) in the mitochondria 49
3.2.4. Quantification of intracellular ROS 51
3.2.5. In vitro cytotoxicity evaluation 53
3.2.6. Weaknesses and solutions 55
Chapter 4. Conclusion 56
References 57
국문 초록 64
Table. 2.1. Size and zeta potential of various T80-based NPs 23
Fig. 1.1. Graphical abstract depicting sonodynamic therapy via mitochondria-uptake using T80(T-Ce6/PL) and MnO₂(T-Ce6) NPs. 16
Fig. 2.1. (A) TEM image of T80(T-Ce6) NPs and (B) T80(T-Ce6/PL) NPs. Scale bars indicate 100 nm. (C) UV-Vis spectra of T-Ce6, PL, T80(T-Ce6), and T80(T-Ce6/PL) NPs. 22
Fig. 2.2. Size changes of T80(T-Ce6/PL) NPs were observed after incubation in 10% FBS-containing PBS for various periods of time. 25
Fig. 2.3. (A) T-Ce6 and (B) PL released from T80(T-Ce6/PL) in PBS (pH 7.4) at 37℃ before and after US irradiation (1 MHz, 0.3 W/㎠, 2 min). 27
Fig. 2.4. (A) Relative cellular uptake levels of Ce6, T-Ce6 and T80(T-Ce6/PL) in hDFB and (B) MCF-7 cells. 29
Fig. 2.5. (A) Relative mitochondrial uptake levels of Ce6, T-Ce6 and T80(T-Ce6/PL) in hDFB and (B) MCF-7 cells. 31
Fig. 2.6. (A) Confocal micrographs displaying the intracellular localization of Ce6, T-Ce6, and T80(T-Ce6/PL) in MCF-7 cells. Scale bars indicate 25 ㎛. (B) Mander's overlap coefficients... 32
Fig. 2.7. Relative intracellular ROS level in (A) hDFB and (B) MCF-7 cells treated with various samples before and after US irradiation (1 MHz, 0.3 W/㎠, 2 min). 34
Fig. 2.8. MCF-7 cells viability treated with empty T80 at various concentrations. 37
Fig. 2.9. (A) MCF-7 cells viability treated with Ce6 and (B) T-Ce6 at various concentrations. 38
Fig. 2.10. (A) hDFB and (B) MCF-7 cells viability treated with blank T80, PL, T-Ce6, T80(T-Ce6) (5 μM of T-Ce6) and T80(T-Ce6/PL) (5μM of T-Ce6 and 10 μM of PL) before and after US... 39
Fig. 3.1. (A) Step of synthesis of hollow-MnO₂ (B) TEM image of hollow MnO₂ and MnO₂(T-Ce6). The scale bar is 100 nm. (C) UV-Vis spectra of MnO₂, T-Ce6 and MnO₂(T-Ce6) 46
Fig. 3.2. Quantification of intracellular O₂ generation by T-Ce6, MnO₂ and MnO₂(T-Ce6). 48
Fig. 3.3. (A) Confocal micrographs displaying the intracellular localization of Ce6, T-Ce6, and MnO₂(T-Ce6) in MCF-7 cells. Scale bars indicate 25 ㎛. (B) Mander's overlap coefficients... 50
Fig. 3.4. Quantification of intracellular ROS levels in MCF-7 cells incubated with various samples under normoxic and hypoxic conditions. The cells were irradiated with US for 2 min (1... 52
Fig. 3.5. (A) Viability of MCF-7 cells after various treatments under hypoxic conditions. (B) Viability of MCF-7 cells after various treatments under normoxic conditions. 54