Title Page
Abstract
Contents
Chapter 1. Overview 12
1.1. Imbalance of the immune system 13
1.1.1. Cancer 13
1.1.2. Autoimmune disease 14
1.2. Current status and limitations of immunotherapy 14
1.2.1. Immunotherapy for cancer 15
1.2.2. Immunotherapy for autoimmune disease 15
1.3. Nanomedicine for modulating immune cells 15
1.3.1. Nanomedicine for modulating T cells 16
1.3.2. Nanomedicine for modulating antigen-presenting cells 17
1.3.3. Advantage of nanomedicine for immune cell modulation 18
1.4. Scope of study 18
1.5. Reference 19
Chapter 2. Nanoparticle-mediated lipid metabolic reprogramming of T cells in tumor microenvironments for immunometabolic therapy 22
2.1. Introduction 23
2.2. Material and Method 25
2.2.1. Synthesis of amphiphilic poly (γ-glutamic acid) 25
2.2.2. Preparation of fenofibrate-loaded and anti-CD3 antibody-modified nanoparticles 26
2.2.3. Quantification of encapsulated fenofibrate 26
2.2.4. Characterization and stability studies 27
2.2.5. In vitro study of pH-dependent release 27
2.2.6. Animals 27
2.2.7. Evaluation of CD3 expression 27
2.2.8. In vitro study of nanoparticle uptake 28
2.2.9. In vitro cell viability study 28
2.2.10. In vitro study of PPARα, CD36 and fatty acid oxidation-associated gene expression 28
2.2.11. Assessment of mitochondrial activation 29
2.2.12. In vitro T cell proliferation test 30
2.2.13. In vitro measurement of lipid uptake by T cells 31
2.2.14. In vitro measurement of T cell secretion of fatty acid metabolites 31
2.2.15. In vitro assessment of the cancer cell-killing activity of nanoparticle-stimulated T cells 31
2.2.16. In vitro study of FoxP3 expression 32
2.2.17. In vivo T cell targeting of nanoparticles 33
2.2.18. In vivo PPARα expression measurement 33
2.2.19. In vivo lipid uptake and metabolite imaging in T cells 34
2.2.20. In vivo assessment of fatty acid oxidation-associated gene expression 34
2.2.21. In vivo antitumor efficacy 35
2.2.22. Detection of tumor-infiltrating lymphocytes and cytokines 35
2.2.23. In vivo toxicity study 35
2.2.24. Statistics 36
2.3. Results 36
2.3.1. Characterization of aCD3/F/ANs 36
2.3.2. Uptake of aCD3/F/ANs by T cells 37
2.3.3. Effect of aCD3/F/ANs on the expression of fatty acid metabolism-related genes and lipid uptake in T cells 38
2.3.4. T cell mitochondrial activation by aCD3/F/ANs 41
2.3.5. In vitro proliferation of metabolically reprogrammed T cells 42
2.3.6. In vitro cancer cell-killing activity of aCD3/F/AN-treated T cells 44
2.3.7. In vivo distribution of aCD3/F/ANs to T cells in tumor tissues 45
2.3.8. Reprogramming of in vivo fatty acid metabolism in aCD3/F/AN-treated T cells 48
2.3.9. In vivo antitumor efficacy of metabolically reprogrammed T cells 50
2.4. Discussion 53
2.5. Conclusion 56
2.6. Reference 57
Chapter 3. Lipid nanoparticle-mediated lymphatic delivery of immunostimulatory nucleic acids 62
3.1. Introduction 63
3.2. Material and Method 64
3.2.1. Preparation of PIC-Loaded M-NP 64
3.2.2. Characterization and Complexation Study of Nanoparticles 65
3.2.3. Animals 65
3.2.4. Isolation of Bone Marrow-Derived Dendritic Cells (BMDC) 65
3.2.5. In Vitro Cytotoxicity Assay 66
3.2.6. In Vitro Study of DC Targeting 66
3.2.7. In Vitro Study of DC Maturation 66
3.2.8. In Vivo Lymph Node Targeting of Nanoparticles 67
3.2.9. DC Maturation Study in Lymph Node 67
3.2.10. In Vivo Toxicity Study 67
3.2.11. Statistics 67
3.3. Results 68
3.3.1. Characterization of PIC/M-NP 68
3.3.2. Cytotoxicity of PIC/M-NP 68
3.3.3. Cellular Uptake of PIC/M-NP 69
3.3.4. In Vitro BMDC Maturation Effect of PIC/M-NP 70
3.3.5. In Vivo Lymph Node Targeting of PIC/M-NP 71
3.3.6. In Vivo DC Maturation Effect 72
3.4. Discussion 73
3.5. Conclusion 74
3.6. Reference 75
Chapter 4. Tannic acid-based nanomaterials for tolerogenic immunotherapy of rheumatoid arthritis 78
4.1. Introduction 79
4.2. Material and Method 81
4.2.1. Preparation of nanoparticles 81
4.2.2. Characterization of nanoparticles 82
4.2.3. Quantification of components in nanoparticles 83
4.2.4. Measurement of abatacept conjugated onto AbaCitDTN 84
4.2.5. Evaluation of radical-scavenging effect 84
4.2.6. In vitro study of nanoparticle uptake by DC 85
4.2.7. In vitro tannic acid core-mediated modulation of inflammatory signaling 85
4.2.8. In vitro study of tolerogenic DC induction 86
4.2.9. In vitro ROS-scavenging effect of nanoparticles on DC 87
4.2.10. In vitro blockade of the interaction between DC and T cells 87
4.2.11. Animals 88
4.2.12. In vivo biodistribution study 88
4.2.13. In vivo immune tolerance study 88
4.2.14. Suppression of CitP-specific splenocytes by AbaCitDTN 89
4.2.15. Detection of cytokine and chemokine levels in CIA mice 89
4.2.16. Analysis of antigen-specific immune responses 89
4.2.17. Analysis of immune cell profiles in CIA mice 90
4.2.18. Microcomputed tomography 91
4.2.19. Histology and immunohistochemical study 91
4.2.20. Defensive immune responses against hemagglutinin. 92
4.2.21. Statistical analysis 92
4.3. Results 92
4.3.1. Characterization of nanoparticles 92
4.3.2. Cellular uptake of nanoparticles and CD80/CD86-blocking study 98
4.3.3. In vitro tolerogenic DC induction and ROS-scavenging effect 100
4.3.4. Inhibition of co-stimulatory molecule interaction, T-cell expansion, and IL-2 secretion 103
4.3.5. In vitro suppression of CitP-specific splenocytes by AbaCitDTN 106
4.3.6. In vivo biodistribution of nanoparticles 107
4.3.7. Prophylactic efficacy of tolerogenic nanoparticles in CIA mice 109
4.3.8. Antigen-specific regulation in CIA mice 112
4.3.9. Immune cell profiles in CIA mice 114
4.3.10. Amelioration of joint destruction by AbaCitDTN 115
4.3.11. Maintenance of defensive immune responses after tolerogenic vaccination 118
4.4. Discussion 119
4.5. Conclusion 124
4.6. Reference 124
Chapter 5. Summary 129
요약 131
Chapter 4 8
Table 1. Primer sequences for quantitative real time-PCR 86
Chapter 1 9
Figure 1. The immunosuppressive factors that inhibit functions of immune cells in the TME are shown. 14
Figure 2. Strategy to enhance anticancer effect of TILs in the hypoglycemic TME by nanomedicine is illustrated. 16
Figure 3. Nanomedicine-mediated DC modulation for immunotherapy. A) Activation of immune response can be achieved by delivering immunogenic... 17
Chapter 2 9
Figure 1. Metabolic reprogramming of T cells by aCD3/F/ANs. A) Schematic illustration of the aCD3/F/AN preparation process. B)... 25
Figure 2. Characterization of nanoparticles. A) Schematic illustration of ANs, aCD3/ANs, F/ANs and aCD3/F/ANs. B) Morphology of aCD3/F/ANs,... 37
Figure 3. Uptake of nanoparticles by T cells. Mouse spleen-derived T cells were incubated with various fenofibrate-containing nanoparticle... 38
Figure 4. The cellular mechanism underlying aCD3/F/AN-induced enhancement of lipid metabolism is illustrated. The left half of the... 39
Figure 5. Fatty acid metabolism-associated gene expression and lipid uptake in T cells. A, C) The corresponding levels of PPARα protein in T... 40
Figure 6. Mitochondrial morphology, membrane potential, and fatty acid metabolism in T cells. T cells were treated with aCD3/F/ANs in low-glucose... 42
Figure 7. Enhanced T cell survival and proliferation induced by metabolic reprogramming. T cells were activated and treated with various nanoparticle... 43
Figure 8. In vitro anticancer activity of aCD3/F/AN-treated T cells. T cells were treated with various nanoparticle preparations and co-incubated with... 45
Figure 9. In vivo T cell-targeting ability of aCD3/F/ANs. T cell targeting ability was evaluated by intratumorally injecting mice (n=3) with various... 47
Figure 10. In vivo PPARα expression and lipid uptake by T cells in tumor tissues. A) B16F10 tumor-bearing mice were intratumorally injected with... 49
Figure 11. Antitumor effects of aCD3/F/ANs in vivo. A) B16F10 tumor-bearing mice were intratumorally injected with various nanoparticle... 51
Figure 12. In vivo cytokine production by T cells in tumor tissues. A) B16F10 tumor-bearing mice were injected with nanoparticle preparations on... 52
Chapter 3 10
Figure 1. Dendritic cell (DC)-mediated lymph node targeting by polyinosinic:polycytidylic acid (PIC)/mannose-modified cationic lipid... 64
Figure 2. Characterization of PIC/M-NP. A) Morphology of polyIC/M-NP 10 was observed by TEM. B) Mean particle sizes of nanoparticles in a... 68
Figure 3. PIC/M-NP cytotoxicity on bone marrow-derived dendritic cells (BMDC). BMDC were treated with nanoparticles in a naked form or PIC-... 69
Figure 4. Intracellular uptake of PIC/M-NP by BMDC. BMDC were treated with PIC/M-NP with mannose densities (0 to 10%) for 4 h. A) Intracellular... 70
Figure 5. In vitro activation of BMDC by PIC/M-NP. BMDC was treated with various PIC-loaded nanoparticles. After 48 h, the expression of CD86... 71
Figure 6. In vivo lymph node-targeting ability of PIC/M-NP. Mice were injected subcutaneously with Cy5-labeled nanoparticles. After 24 h,... 72
Figure 7. In vivo DC maturation in inguinal lymph nodes. Mice were injected subcutaneously with nanoparticles in a naked form or PIC-... 73
Chapter 4 10
Figure 1. Tolerogenic reprogramming of DC by AbaCitDTN. A) Synthesis procedure of AbaCitDTN. B) Proposed mechanism through which... 81
Figure 2. Characterization of TN. A) Procedure for synthesizing TN. B) FT-IR spectra of tannic acid, PVA, PEG, and TN, as analyzed by FT-IR spectrometry. 93
Figure 3. Radical scavenging activity of nanoparticles. A) Proposed radical scavenging mechanism of TN is illustrated. B) Superoxide anion scavenging... 94
Figure 4. Characterization of nanoparticles. A) Schematic illustration of various formulations. B) Morphology of AbaCitDTN, as visualized by TEM.... 96
Figure 5. Quantification of tannic acid and CitP in nanoparticles. A) Standard curve of tannic acid. B) Amount of tannic acid in nanoparticles, as... 97
Figure 6. DC targeting and endocytosis of AbaCitDTN via co-stimulatory molecules. A) Nanoparticles were labeled with Cy5 (red) and lysosomes... 99
Figure 7. Tolerogenic DC induction by nanoparticles. A) Representative molecular markers of immunogenic DC and tolerogenic DC are illustrated.... 101
Figure 8. Inhibition of inflammatory signaling pathway by nanoparticles through tannic acid core-mediated ROS scavenging. A) Schematic... 103
Figure 9. Co-stimulatory signal blockade by nanoparticles. A) Illustration of co-stimulatory signal blockade between DC and T cells. B) DC-specific... 105
Figure 10. Suppression of CitP-specific immune responses by nanoparticles at cellular level. A) The experimental schedule for the suppression of CitP-... 106
Figure 11. In vivo lymph node targeting ability of AbaCitDTN. A) DBA/1 mice were subcutaneously administered with Cy5-labeled nanoparticles at... 108
Figure 12. Prophylactic efficacy of nanoparticles in CIA mouse model. A) Experimental schedule for CIA mouse model establishment and... 109
Figure 13. Bone architecture and bone erosion observation. A) Experimental schedule for CIA mouse model establishment, nanovaccine... 111
Figure 14. Cytokine and chemokine evaluation in serum and synovial fluid. A) Experimental schedule for CIA mouse model establishment, nanovaccine... 112
Figure 15. Regulation of antigen-specific immune responses by the tolerogenic vaccine. A) Experimental schedule of CIA mouse model... 114
Figure 16. Immune cell profiling in lymphoid organs. A) Experimental schedule of CIA mouse model establishment, nanovaccine administration,... 115
Figure 17. Histological analysis of knee joints. At day 42 after the first administration of nanovaccines, mice were sacrificed and knee joints were... 117
Figure 18. Defensive immune responses against hemagglutinin. A) Experimental schedule for the administration of the nanovaccine and... 119