Title Page
Abstract
Contents
1. Introduction 14
2. Literature Survey 18
2.1. Hydroxyethyl cellulose for gene/drug delivery systems 18
2.2. Cationic polymer for gene delivery 20
2.2.1. Viral vector and non-viral vector 20
2.2.2. Necrosis process by cationic polymer 20
2.2.3. Research for overcoming the cytotoxicity of PEI 21
2.3. Bcl-2 siRNA for tumor therapy 23
2.3.1. RNA interference with siRNA for tumor therapy 23
2.3.2. Role of Bcl-2 protein in tumor 23
2.3.3. Synergistic antitumor effect by co-delivery of antitumor drugs and Bcl-2 siRNA 24
3. Materials and Methods 26
3.1. Materials 26
3.2. Methods 28
3.2.1. Synthesis of HECP2k (Polyethylenimine 2000Da-conjugated Hydroxyethyl cellulose) 28
3.2.2. Characterization of HECP2k 29
3.2.3. Characterization of HECP2k/pDNA polyplex 31
3.2.4. In vitro assay of HECP2k and HECP2k/pDNA polyplex 32
3.2.5. Antitumor effect of HECP2k@Dox/siRNA 37
4. Results and Discussion 40
4.1. Synthesis and Characterization of HECP2k 40
4.1.1. Synthesis of HECP2k 40
4.1.2. Structural analysis with ¹H NMR and primary amine quantification 40
4.1.3. Molecular weight measurements with GPC 45
4.1.4. Structural analysis with ¹³C NMR and FT-IR 47
4.2. Characterization of HECP2k/pDNA polyplex 50
4.2.1. pDNA condensing ability with agarose gel electrophoresis 50
4.2.2. Average size and zeta-potential measurements 50
4.3. In vitro assay of HECP2k and HECP2k/pDNA polyplex 54
4.3.1. Cell metabolic activity with MTT assay 54
4.3.2. Gene transfection efficiency with luciferase transgene expression assay 56
4.3.3. Cellular uptake measurements with Flow cytometry 59
4.3.4. Intracellular trafficking visualization with CLSM 59
4.3.5. Serum stability test 66
4.4. Antitumor effect of HECP2k@Dox/siRNA 68
4.4.1. Drug loading contents and drug loading efficiency measurements 68
4.4.2. Average size and zeta-potential measurements with DLS 68
4.4.3. Antitumor effect analysis with MTT assay 72
4.4.4. Apoptosis induction test with Annexin V staining 78
5. Conclusion 80
References 82
Abstract in Korean 89
Table 1. Molar amounts of primary amine per unit mass of polymers. 44
Table 2. Molecular weight of OxHEC, PEI2k, HECP2k 1X, HECP2k 5X, HECP2k 10X by GPC (PEG standard). 46
Table 3. Drug loading content and drug loading efficiency of HECP2k@Dox. 70
Table 4. Z-average size and zeta-potential values of HECP2k/siRNA polyplexes and HECP2k@Dox/siRNA nanocomplexes. 71
Table 5. Chart of Dox and Bcl-2 siRNA concentration for each condition. 75
Figure 1. Schematic illustration showing formation and antitumor effect of the nanocomplex. 17
Figure 2. Schematic diagram showing a apoptosis induction of Bcl-2 protein. 25
Figure 3. Synthesis scheme of HECP2k. 42
Figure 4. ¹H NMR spectra of (A) HEC, (B) PEI2k, (C) HECP2k 1X, (D) HECP2k 5X, (E) HECP2k 10X. 43
Figure 5. Calibration curve for primary amine quantification (PEI2k standard). 44
Figure 6. GPC chromatograms of OxHEC, PEI2k, HECP2k 1X, HECP2k 5X, HECP2k 10X. 46
Figure 7. ¹³C NMR spectra of HECP2k 10X. 48
Figure 8. FT-IR spectra of HEC, OxHEC, PEI2k, HECP2k 10X. 49
Figure 9. Agarose gel electrophoresis of (A) HECP2k 1X, (B) HECP2k 5X, (C) HECP2k 10X. 52
Figure 10. (A) Z-average size and (B) zeta-potential values of HECP2k/pDNA polyplexes. 53
Figure 11. MTT assay results in (A) HeLa and (B) HepG2 cells. 55
Figure 12. Luciferase assay results of PEI25k/pDNA and HECP2k/pDNA in HeLa cells at the various weight ratios of polymer/pDNA=10, 20, 30 : 1 w/w. 57
Figure 13. Luciferase assay results of PEI25k/pDNA and HECP2k/pDNA in HeLa cells at the various weight ratios of polymer/pDNA=30, 50, 70 : 1 w/w.... 58
Figure 14. Flow cytometry results of PEI25k/pDNA and HECP2k/pDNA in HeLa cells at various weight ratios of polymer/pDNA= 30, 50, 70 : 1 w/w.... 61
Figure 15. CLSM images of PEI25k/pDNA (A, C, E) and HECP2k/pDNA polyplexes (B, D, F, G) in HeLa cells. pDNA was labeled by YOYO-1 (green).... 65
Figure 16. Serum stability test results of PEI25k/pDNA and HECP2k/pDNA polyplexes by luciferase assay. 67
Figure 17. Calibration curve for Dox loading content and Dox loading efficiency measurements. 70
Figure 18. MTT assay results showing antitumor effects of free dox, free siRNA, PEI25k/siRNA, HECP2k@Dox, HECP2k/siRNA,... 74
Figure 19. Relative cell viability for free Dox, HECP2k/siRNA, HECP2k@Dox/siRNA nanocomplexes at a high concentration of Dox and... 75
Figure 20. Dose-effect curves of Dox and siRNA in (A) HeLa and (B) HepG2 cells. Median effect plots for Dox in (C) HeLa and (D) HepG2 cells. Median... 76
Figure 21. Combination index (CI) based on the median effect plots for (A) HeLa and (B) HepG2 cells. 77
Figure 22. Flow cytometry results by annexin V staining of (A) untreated cell and (B) HECP2k@Dox/siRNA. (FL1: Alexa® Fluor 488, FL2: PI)[이미지참조] 79