Influence of Submicro-Grooved Surface and Zwitterionic Molecule-Modified PEI on Gene Transfection

基因治療是利用分子生物學方法將DNA或RNA導入細胞內，其在癌症、免疫疾病、組織工程以及基因缺陷的治療上被廣泛的應用與討論。在過去的研究中，細胞的型態對基因轉殖的影響是比較少被探討的。在本實驗中，我們利用不同次微米的聚苯乙烯(PS)表面觀察小鼠成肌細胞C2C12的細胞形態，期望透過地形的改變來影響細胞基因轉殖的結果。我們發現，延展形態的細胞會造成基因轉殖效率降低，顯示細胞核形態會影響非病毒載體的基因轉殖。另外，我們利用兩性離子分子將作為基因轉殖載體的聚乙烯亞胺做修飾，希望降低其細胞毒性，並能提高基因轉殖的效率。 首先，將小鼠成肌細胞C2C12培養在PS表面上，使用聚乙烯亞胺與DNA的複合物來做基因轉殖，隔天將細胞裂解並測量螢光蛋白質的螢光強度來決定基因轉殖的效率。結果顯示生長在有溝脊地形的細胞其基因轉殖效率會小於平坦表面的基因轉殖效率，且隨著溝脊的加深與加寬而更加明顯。接著，我們將會探討在不同的次微米地形上，細胞核的型態對非病毒基因轉殖的影響，結果顯示，在有溝脊的表面上細胞核會有變形的現象，且溝脊較寬的表面細胞核的形變量也較大。我們同時也發現細胞骨架的排列度會是影響基因轉殖的重要因素。 另外，利用兩性離子分子CBAAester修飾的聚乙烯亞胺，其細胞毒性有所下降，也提高基因轉殖的效率。在不同的接枝率中，又以6%接枝率的基因轉殖效果最好。Gene therapy is the use of molecular biology to treat disease by delivering therapeutic DNA into cells, which is well application and discussion on cancer, immune disease, tissue engineering and gene defects. Previous research has less discussion on cell morphology and gene transfection. In this work, we used different sub-micro polystyrene (PS) surface to observe the morphology of mouse myoblast cell C2C12, expecting to affect gene transfection efficiency by varying topography. We found that the transfection efficiency to elongated cells on the grooved surface was lower compared to the efficiency of the cells on the flat surface. The results indicate that nuclei morphology might affect the gene transfection by non-viral cationic polymeric vehicles. Then, we used zwitterionic molecule to modify Polyethylenimine (PEI) which was used as a carrier for gene transfection, hoping to decrease cell toxicity and increase gene transfection efficiency. First, C2C12 cells were seeded on grooved polystyrene. PEI and DNA complexes for were applied on gene transfection. Lysing cells and quantified the fluorescence intensity of fluorescent Proteins to define gene transfection efficiency. The results indicated that the gene transfection efficiency of the cells on the grooved surface is less than which on the flat surface, and the trend became more obviously as ridge and width increased. Then, we will discuss the influence of different submicron surface topography on nucleus morphology and non-viral gene transfection. The results showed that nucleus deformed on the grooved surfaces, and the deformation became larger on the grooved surfaces. We also suggested that the arrangement of cytoskeleton was the important factor of gene transfection. In addition, the zwitterionic molecule CBAAester was used to modify PEI, the cell viability was decrease and the gene transfection efficiency was enhanced. The grafting ratio of 6% has better gene transfection efficiency than other grafting ratios.致謝 i 摘要 iii Abstract iv Content vi List of Tables viii List of Figures ix Chapter 1 Introduction 1 1.1 Gene transfection 1 1.1.1 Gene transfer and delivery strategies 1 1.1.2 PEI and the application on gene delivery 5 1.2 Endosomal escape pathways and proton sponge effect for delivery 7 1.3 The Properties and delivery applications of zwitterion 12 1.4 The effect of topography on cell morphology and gene delivery 14 1.5 Motive and Aims 20 1.6 Research framework 21 Chapter 2 Materials and Methods 23 2.1 Chemicals 23 2.1.1 Sub-micron surface fabrication 23 2.1.2 Cell culture 23 2.1.3 Gene transfection 24 2.1.4 Cell staining 25 2.1.5 Cell viability 26 2.1.6 Cell number determination 26 2.1.7 Synthesis of zwitterionic molecule-modified PEI 26 2.2 Experimental instrument and materials 27 2.2.1 Experimental instrument 27 2.2.2 Experimental materials 28 2.3 Solution formula 29 2.4 Methods 31 2.4.1 The fabrication of PDMS molds 31 2.4.2 The fabrication of PS substrates and plasma pretreatment 32 2.4.3 Water contact angle measurement 35 2.4.4 Cell culture 35 2.4.5 The observation of cell morphology 36 2.4.6 F-actin staining and vimentin staining 36 2.4.7 The analysis of nuclear circularity 37 2.4.8 The analysis of F-actin arrangement and vimentin density 37 2.4.9 The quantification of cell number 38 2.4.10 Gene transfection on pattern surface 39 2.4.11 Determination of DNA uptake 39 2.4.12 Synthesis of CBAAester and zwitterionic molecule-modified PEI 41 2.4.13 Measurement of size and zeta potential 43 2.4.14 Determination of cytotoxicity 43 2.4.15 Gene transfection of PEI-CBAAester 43 2.4.16 Statistic analysis 44 Chapter 3 Influence of Surface Topography on Gene Transfection 45 3.1 Surface characterization 45 3.2 Cellular morphology 46 3.3 Nuclear circularity 47 3.4 F-actin filament alignment 49 3.5 Vimentin observation 49 3.6 Gene transfection efficiency and cell proliferation 50 3.7 DNA uptake 51 3.8 Discussion 52 Chapter 4 Influence of Zwitterionic Modified PEI on Gene Transfection 77 4.1 NMR characterization 77 4.2 Measurement of size and zeta potential 78 4.3 Cytotoxicity of PEI-CBAAester 79 4.4 Gene transfection efficiency 79 4.5 Discussion 80 Chapter 5 Conclusion and Future work 91 Reference 93 Supporting Information 10