臺灣博碩士論文加值系統

結直腸癌（CRC）發生率在世界癌症排名中位居第三，而且近期不斷增加。Irinotecan是治療結腸直腸癌的主要化療藥物之一，另外，加強癌細胞對化學治療藥物的敏感性能夠增加結腸直腸癌治療的效果並且降低化學治療藥物的副作用，所以在本研究中，使用固體脂質奈米粒子(SLNs)包覆功能性的微小RNA (microRNA)，希望能進一步增加Irinotecan的細胞毒性。然而，抗癌藥物在體內非選擇性分布常會損害正常細胞，因此利用微脂體遞送化療藥物能夠透過被動或主動標的將藥物確實地運送至腫瘤部位。於是我們建構了一個多功能微脂體載體運送Irinotecan，此載體同時具有pH敏感的特性，也在粒子表面修飾上具有各式功能的胜肽，以增加藥物積聚在腫瘤的效果，另一方面，將microRNA有效的包覆在SLN當中，與包覆irinotecan的微脂體合併給藥。胜肽修飾的微脂體和帶正電的固體脂質納米粒子皆為球形顆粒且粒子分布均勻，我們發現胜肽修飾的微脂體比沒有修飾的微脂體細胞攝取量更多，因此將修飾的微脂體包覆irinotecan給予細胞之後，能夠有效的降低結腸直腸癌細胞的存活率。當合併給予microRNA更能夠增加irinotecan處理的結腸直腸癌細胞之毒性。其抑制細胞生長的效果可能來自於irinotecan本身抑制細胞生長的能力以及功能性的microRNA兩者組合後的效應，我們推測誘導細胞凋亡是此合併給藥最主要造成細胞毒性的原因。此外，miR-b能夠調節EMT途徑並且阻止癌細胞遷移。總而言之，microRNA和irinotecan包覆雙功能脂質體的合併治療可以提供作為抑制結腸癌細胞生長的新方法。

The incidence of colorectal cancer (CRC) is the third in cancers around the world and keeps increasing recently. Irinotecan is one of the main chemotherapeutic agents for CRC. Sensitizing cancer cells to chemotherapeutic drugs enhanced the treatment toward colorectal cancer and decreased side effects along with chemotherapeutic drugs. Using microRNA entrapped in solid lipid nanoparticles (SLNs) would further increase the cytotoxicity of irinotecan. However, non-selective anticancer drugs usually damage normal cells. Thus, liposomes are able to deliver drugs to the tumor sites by passive or active targeting. In the present study, we constructed irinotecan-loaded multi-functional liposomes that respond to the extracellular low pH. miRNA entrapped in SLNs were combined with the liposomes to enhance the cytotoxic effects. The peptide-modified liposomes and positively charged solid lipid nanoparticles (SLNs) displayed sphere particles and narrow size distribution. Greater cellular uptake was found in peptide-conjugated liposomes than that of liposomes without modification. The cell viability of colorectal cancer cells treated with these liposomes was reduced. Delivery of microRNA by SLNs would be able to further increase the cytotoxicity of colorectal cancer cells treated with irinotecan peptide-modified liposomes. Cell growth inhibition attributes to the combined effect of irinotecan and miR-b. We found that apoptosis was accounted for the cytotoxic effect of the combined therapy. Furthermore, miRNA additionally modulated the EMT pathway and downregulated the cancer cell migration. Collectively, the combination therapy of microRNA and irinotecan-loaded dual-functional liposomes may provide a novel approach to inhibit colon cancer cell growth.

Contents
中文摘要 i
Abstract ii
Contents iii
Figure contents viii
Table contents ix
Chapter 1 Introduction 1
1. Colorectal cancer 1
2. Irinotecan 1
2.1. Irinotecan and its induction of apoptosis 1
2.2. Irinotecan and multidrug resistance 2
3. MicroRNA (miRNA or miR) 2
4. miR-b 3
4.1. miR-b regulates epithelial-mesenchymal transition (EMT) pathway 3
4.2. miR-b increases sensitivity of cancer cells to chemotherapeutic drug 3
4.3. miR-b correlates with WNT/β-catenin signaling pathway 4
5. Delivery of genes and chemical drugs 4
6. Solid lipid nanoparticle (SLN) 5
7. Liposomes (Lip) 5
7.1. The EPR effect and passive tumor targeting 6
7.2. PEGylation 7
7.3. Onyvide® (irinotecan liposome injection) 7
7.4. Active targeting 8
7.5. Cell penetrating peptides (CPPs) 8
7.6. RF peptides (RF) 9
7.7. K peptides 10
7.8. H peptides 10
8. Stimulus-responsive carriers 11
8.1. pH-sensitive drug release 11
Chapter 3 Experimental design 14
Chapter 4 Materials 15
1. Preparation of SLNs 15
2. Preparation of liposomes 15
3. microRNA 16
4. Drug 16
5. Peptides 16
6. HPLC-UV 16
7. Encapsulation efficiency 16
8. Transmission electron microscopy (TEM) 17
9. In vitro release 17
10. Cell culture 17
11. Cellular uptake and pathway 17
12. Intracellular accumulation 18
13. Transfection efficiency 18
14. Hemolysis test 18
15. Cytotoxicity 19
16. Apoptosis analysis 19
17. Western blot 19
18. Caspase activity assay 19
19. Realtime PCR 19
20. Migration assay 20
21. Primary antibody 20
22. Secondary antibody 21
23. Instruments 21
Chapter 5 Methods 22
1. Synthesis of DSPE-PEG-peptide 22
2. Synthesis of DSPE-imine-omPEG 22
3. Preparation of solid lipid nanoparticles (SLN) 22
4. Preparation and characterization of liposomes 23
5. Stability of liposome 24
6. pH-triggered release (In vitro release study). 24
7. Encapsulation efficiency (EE %) 24
8. pH-induced size and zeta potential change 25
9. Cell lines 25
10. Transfection efficiency 25
11. Cellular uptake 25
12. Identification of cellular uptake pathways 26
13. Intracellular localization in different pH condition 26
14. Hemolysis assay 26
15. Cell viability assays: SRB assay 27
16. Cell apoptosis 27
17. Detection of cell cycle by propidium iodide (PI) staining 27
18. Caspase activity assay 28
19. Real-time PCR quantification of mRNA 28
20. Migration assay 28
21. Western blot assay 29
22. Statistical analysis 29
Chapter 6 Results 30
1. Cytotoxicity of peptide-modified liposomes in CT26 cells. 30
2. Synthesis of DSPE-PEG-peptide and DSPE-omPEG 30
3. Characterization of solid lipid nanoparticles (SLNs) 31
4. Characterization of liposomes (Lip) 31
5. The stability of liposomes 32
6. In Vitro release of irinotecan pH-responsive liposomes 32
7. Characterization of pH-responsive liposomes (omLip-RFKH) 33
8. Cellular uptake of pH-responsive peptide-modified liposomes 33
9. Intracellular distribution of pH-responsive liposomes 34
10. Cellular uptake of peptide-modified liposomes 34
11. Cellular uptake mechanism of RF, K and H-conjugated liposomes 35
12. Transfection efficiency of miRNA (miR-b-FAM) 36
13. Hemolysis assay 37
14. Cytotoxic effect of blank formulations 37
15. Cytotoxic effect of different formulations 37
16. Apoptosis analysis 38
17. Cell cycle analysis 39
18. mRNA expression levels of apoptosis 39
19. Protein expression levels of apoptosis 40
20. Activity assay of apoptosis-related proteins 40
21. Protein expression levels of Wnt/β-catenin pathway 41
22. Protein expression levels of MDR pathway 41
23. Migration assay and the protein expression levels of EMT pathway 42
Chapter 7 Discussion 44
1. Conjugation of PEGylated lipids and peptides 44
2. Characterization of solid lipid nanoparticles and liposomes 44
3. pH-responsive liposomes as tumor site selective delivery systems 45
4. Endosomal escape of SLNs and omLip-RFKH 46
5. Cellular uptake mechanism of Lip-RFKH 47
6. SLNs as transfection reagents 48
7. Toxicological study 49
8. Irinotecan as an apoptosis inducer 50
9. miR-b as a migration inhibitor 51
10. miR-b as a chemotherapeutic sensitizer 52
Chapter 8 Conclusions 54
Chapter 9 References 55
Chapter 10 Figures 60

Figure contents
Figure 1. Cytotoxicity of peptide-modified liposomes on CT26 cells. 60
Figure 2. Characterization of peptide-modified PEG-lipids and pH-sensitive lipid. 62
Figure 3. Characterization of solid lipid nanoparticles (SLNs) and peptide-modified liposomes loaded with irinotecan. 64
Figure 4. Characterization of peptide-modified liposomes loaded with irinotecan. 65
Figure 5. Characterization of pH-sensitive liposomes. 68
Figure 6. Cellular uptake of DiI labeled peptide-modified liposomes 69
Figure 7. Transfection efficiency of miR-b-FAM in HCT116 cells. 70
Figure 8. The toxicity of liposomal formulation 71
Figure 9. Cytotoxic effects of peptide-modified liposomes. 73
Figure 10. Apoptosis analysis of the combination of miR-b and irinotecan-loaded peptide-modified liposomes in HCT116 cells. 74
Figure 11. Effects of the combination of miR-b and irinotecan-loaded liposomes on mRNA and protein expression levels of apoptosis pathway. 75
Figure 12. Effects of the combination of miR-b and irinotecan-loaded peptide-modified liposomes on protein expression levels of Wnt/β-catenin and MDR pathway. 76
Figure 13. Effects of the combination of miR-b and irinotecan-loaded peptide-modified liposomes on the migration width and the relative protein expression levels. 77

Table contents
Table 1. Characterization of solid lipid nanoparticles (SLNs) and peptide-modified liposomes loaded with irinotecan. 63
Table 2. Characterization of pH-responsive peptide-modified liposomes under pH 6.5. 66

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