Title Page
Abstract
Contents
Chapter 1. Introduction 12
1.1. Cancer immunotherapy 12
1.2. Extracellular vesicles 15
1.3. Immune cell-derived extracellular vesicles 17
1.4. Surface engineering of extracellular vesicles 18
Chapter 2. Material and Methods 23
2.1. Cell culture 23
2.2. Macrophage polarization and EV isolation from cell culture media 23
2.3. Transient transfection for preparation of lentivirus 24
2.4. Preparation of M1EV-IL2 constructed through endogenous modification 24
2.5. Preparation of DBCO-IL-2 for click chemistry reaction 25
2.6. Metabolic glycoengineering of macrophage to obtain M1EV-IL2 constructed through click chemistry reaction 25
2.7. Insertion of DSPE-PEG-N₃ into M1EV for the construction of M1EV-N₃ before the click chemistry reaction 25
2.8. Characterization of M1EV and M1EV-IL2 26
2.9. Reprogramming function of M1EV and M1EV-IL2 26
2.10. Activation function on CD4+ T cells induced by M1EV and M1EV-IL2 27
2.11. Proliferation and activation functions on CD8+ T cells induced by M1EV and M1EV-IL2 27
2.12. RNA isolation, cDNA synthesis and reverse transcription polymerase chain reaction (RT-PCR) analysis 27
2.13. Statistical analysis 28
Chapter 3. Results and Discussion 31
3.1. Genetically modification through lentiviral transduction to obtain M1-type macrophage-derived extracellular vesicles with surface-displayed IL-2 31
3.1.1. Polarization and infection by lentiviruses of M1-type macrophage 31
3.1.2. Isolation of M1EV-IL2 constructed through genetic modification 34
3.1.3. Characterization of M1EV-IL2 constructed through genetic modification 35
3.2. Directly modification through chemical engineering to obtain M1-type macrophage-derived extracellular vesicles with surface displayed IL-2 38
3.2.1. Metabolic glycoengineering of macrophages through the treatment of Ac4ManNAz and polarization into M1-type macrophages 38
3.2.2. Characterization of M1EV-IL2 constructed through metabolic glycoengineering and click chemistry reaction 40
3.3. Directly modifying agents through lipid insertion into EV membrane to obtain M1-type macrophage-derived extracellular vesicles with surface-displayed IL-2 43
3.4. Reprogramming function of M1-type macrophage-derived extracellular vesicles with surface-displayed IL-2 44
3.5. CD4+ T cell activation function of M1-type macrophage-derived extracellular vesicles with surface displayed IL-2 48
3.6. CD8+ T cells proliferation and activation functions of M1-type macrophage-derived extracellular vesicles with surface displayed IL-2 48
Chapter 4. Conclusion 53
References 55
국문초록 64
Table 2.1. Primer sequence for qRT-PCR analysis 29
Figure 1. Schematic of composition of immune cells in tumor microenvironment [4]. M2-type tumor-associated macrophages (TAMs) are abundant and pivotal in the tumor microenvironment (TME), whereas T cells and M1-type macrophages are depleted. The non-immunogenic... 14
Figure 2. Schematic of extracellular vesicles as key player of intercellular communication [25]. Extracellular vesicles (EVs) contain a diverse range of bioactive molecules, such as lipids,... 16
Figure 3. Various strategies for engineering EVs to enhance therapeutic effects [55]. The current emphasis in preclinical research largely centers on augmenting their application through various engineering approaches. These approaches encompass genetic engineering, membrane... 21
Figure 4. Schematic of constructing M1EV-IL2 and evaluating the functions of macrophage reprogramming and T cells (CD4+ and CD8+) activation. Multi-functional EVs were constructed by surface-modifying IL-2, which has T cells activation function, on M1EV, which has a... 22
Figure 5. The process of lentiviral transduction and the selection of infected monocytes. (A) Lentiviruses, generated by transfecting HEK293 cells with a lentiviral transfer plasmid containing... 32
Figure 6. Polarization and IL-2 mRNA expression of infected M1-type macrophages. THP-1 cells, selected with puromycin, underwent differentiation into macrophages following PMA treatment.... 33
Figure 7. Isolation of EVs from normal and infected M1-type macrophages. (A) Illustration of M1EV derived from infected M1-type macrophages, denoted as M1EV-IL2, produced through endogenous modification. EVs were isolated from (B) normal M1-type macrophages... 36
Figure 8. Characterization of M1EV-IL2 construction through genetic modification. For size information analysis, NTA was employed to measure (A) size distribution, (B) concentration, and... 37
Figure 9. Metabolic glycoengineering through the treatment of Ac4ManNAz on macrophages and polarization into M1-type macrophages. (A) Illustration of M0 macrophage treated with Ac4ManNAz. (B) Schematic of EDC/NHS reaction to construct DBCO-IL-2. (C) The expression... 39
Figure 10. Isolation of EVs derived from metabolic engineered M1-type macrophage after click chemistry reaction. (A) Schematic of M1EV-IL2 derived from M1-type macrophages labeled with an azide group. (B) M1EV and M1EV-IL2 were concentrated and purified through... 41
Figure 11. Characterization of M1EV-IL2 constructed through metabolic glycoengineering and click chemistry reaction. For size information analysis, NTA was employed to measure (A) size... 42
Figure 12. Lipid insertion into EV membrane to obtain M1EV-IL2. (A) Schematic of the insertion of DSPE-PEG-N₃ into M1EV. The results of NTA analysis of M1EV and M1EV-IL2 are the results of (A, C) size distribution, (B, D) concentration. 45
Figure 13. Characterization of M1EV-IL2 constructed through lipid insertion. For size information analysis, NTA was employed to measure (A) mean size for both M1EV and M1EV-... 46
Figure 14. Reprogramming function of M1EV and M1EV-IL2. (A) Schematic of the reprogramming of M2-type macrophage into M1-type macrophage. RT-PCR was performed for analysis to confirm the relative expression of (B, C) M1 marker and (D)M2 marker. Polarization into... 47
Figure 15. CD4+ T cells activation function of M1EV-IL2. (A) Schematic of CD4+ T cell activation by M1EV-IL2. (B) RT-PCR was performed for analysis to confirm the relative... 50
Figure 16. CD8+ T cells proliferation function of M1EV-IL2. (A) Schematic of CD8+ T cell activation. (B) WST-1 assay was performed for analysis to confirm the proliferation function by... 51
Figure 17. CD8+ T cells activation function of M1EV-IL2. (A) RT-PCR was performed for analysis to confirm the relative expression of activation markers. (B)The quantity of the T cell... 52