Cancer Cell Uptake Behavior of Au Nanoring and Its Localized Surface Plasmon Resonance Induced Cell Inactivation Efficiency

在本論文中，我們將侷域表面電漿子共振波長為1058奈米的金奈米環分成生物鍵結上針對SAS口腔癌細胞膜上受體的抗體和未鍵結兩組，加到培養皿中與此癌細胞培養，隨後以波長為1065奈米的雷射激發金奈米環的侷域表面電漿子共振，利用其產生的光熱效應使癌細胞死亡。透過不同金奈米環加入癌細胞的培養時間，我們可得到不同細胞攝取金奈米環的數量，進而得到不同滅活細胞所需最小的臨界雷射強度。對於每個培養時間，我們試著利用感應耦合電漿質譜分析儀去測量吸附在細胞膜上和被內化入細胞的金奈米環的數量和攝取的百分比，為達到此目的，我們收集以生理食鹽水沖洗細胞的殘餘液得到懸浮的金含量，以碘化鉀和碘的混合液將吸附在細胞膜表面的奈米環蝕刻，以生理食鹽水沖洗收集得到吸附在細胞膜的金含量，並以細胞溶液來得到內吞的金含量，透過掃描式電子顯微鏡估量出奈米環之大小，進而分析出此三部分的金奈米環數量。我們以圈圖的方式估算出在不同雷射強度照射下，在沖洗前，沖洗後，以及金蝕刻後的臨界雷射強度。對於其上有抗體的金奈米環，癌細胞攝取的速度較快，且隨著時間的增加，細胞內吞的金奈米環的量越多。但觀察吸附在細胞膜上的金奈米環，發現加入金奈米環後十二小時達到峰值。對於其上沒有抗體鍵結的金奈米環，細胞攝取的速度較慢，在同樣時間下攝取金奈米環的量明顯較少，且吸附在細胞膜上的奈米環的數量在二十小時後才達到峰值。以吸附和內吞的金奈米環和臨界雷射強度交叉比對後，我們發現吸附的金奈米環對於細胞滅活的效果較好。當細胞吸附最多金奈米環時會得到最小臨界雷射強度。Au nanorings (NRIs), which have the LSP resonance wavelength around 1058 nm, either with linked antibody (NRI-AB) or without antibody (NRI-control), are applied to SAS oral cancer cell for cell inactivation through the localized surface plasmon (LSP)-induced photothermal effect when it is illuminated by a laser of 1065 nm in wavelength. Various incubation times after the application of Au NRIs to cell are considered for observing the variations of cell uptake of Au NRI and hence the threshold laser intensity for cancer cell inactivation. In each case of incubation time, the SAS cell is washed for evaluating the Au NRI number per cell adsorbed and internalized by the cells. Also, the Au NRIs adsorbed (remaining on cell membrane) are etching with KI/I2 to evaluate the Au NRI number per cell internalized by the cells. The threshold laser intensities before washout, after washout, and after KI/I2 etching are calibrated from the circular area sizes of inactivated cells around the illuminated laser spot center under the illuminations of various laser power levels. The adsorbed and internalized Au NRIs per cell are obtained from inductively coupled plasma mass spectrometry measurements of the flushed solution in the KI/I2 etching process and the remaining cell solution, respectively. By using Au NRIs with antibody, it is found that the internalized Au NRI number per cell increases with incubation time. However, the adsorbed Au NRI number per cell reaches a maximum at 12 hrs in incubation time. The cell uptake behaviors of Au NRIs without antibody are similar to those with antibody. Nevertheless, the NRI numbers per cell are significantly smaller. Meanwhile, the incubation time for the maximum adsorbed NRI number per cell is delayed to 20 hrs. By comparing the variation of threshold laser intensity for cell inactivation, it is found that the adsorbed NRIs can cause more effective cancer cell inactivation, when compared with the internalized NRIs. The minimum threshold laser intensities after cell solution washout in either case with or without antibody are observed before KI/I2 etching under the aforementioned incubation time conditions of maximum adsorbed NRI number per cell.誌謝 II 中文摘要 III Abstract V Content VII Chapter 1 Introduction 1 1.1 Localized Surface Plasmon 1 1.2 Review of Gold Nanoparticle 3 1.2.1 Gold Nanosphere 3 1.2.2 Gold Nanorod 5 1.3 Characteristics of Gold Nanoring 8 1.4 Process of Cell Uptake of Au NPs 12 1.5 Photothermal Therapy 15 1.6 Research Motivations 17 Chapter 2 Materials and Methods 29 2.1 Fabrication of Bio-conjugated Au Nanorings 29 Chapter 3 Cell Inactivation by Localized Surface Plasmoninduced Photothermal Effect 44 3.1 Experimental Procedures 44 3.2 Experimental Results 48 3.3 Discussions 54 References 6