Development of the Scattering Chromatogram Analysis and Its Applications in Studies of the Endocytosis of Gold Nanoparticles

• Main Authors: Wang, Sheng Hann, 王盛翰
• Other Authors: Tseng, Fan Gang
• Format: Others
• Language: zh-TW
• Published: 2015
• Online Access: http://ndltd.ncl.edu.tw/handle/26439544723530430538

博士 === 國立清華大學 === 工程與系統科學系 === 104 === In this dissertation, we developed a chromatogram analysis based on the scattering color of gold nanoparticles (Au NPs), and applying it to the studies of intracellular interaction of Au NPs. In Chapter 1, we introduced the application, optical property of NPs, as well as consequence caused by their distribution and aggregation in biomedicine. In Chapter 2, we introduced the methods and instructions that we used in this thesis. In Chapter 3, we present a method to directly observe three-dimensional (3D) distribution of aggregated Au NPs in live cells by using chromatogram analysis, which based on the transformation of the color space from RGB to HSV (Hue, Saturation and Value). Owing to the localized surface plasmon resonance (LSPR) coupling effect of Au NPs and calibration of the known number of Au NPs by scattering and scanning electron microscopy (SEM) images, the Hue can distinguish Au NPs from scattering images of cellular organelles. The Value, the scattering intensity, can define the locations of Au NPs. The V/H ratio was applied to estimate the numbers of in aggregated Au NPs. This method is in good agreement with mass spectroscopic measurement in Au NPs number counting. Compared to conventional methods, this approach provides a simple observation of 3D distribution of Au NPs and their aggregation states in living cells. The chromatogram analysis was further applied to study the evolution of Au NP clustering in living cells in Chapter 4, which demonstrated its ability in the the 5 dimensional (location (x,y,z), time (t) and aggregation (n)) study of Au NPs. During endocytosis, Au NP clusters undergo fantastic color changes, from green to yellow-orange due to the plasmonic coupling effect. V/H ratio helps estimate the numbers of Au NPs in the clusters with time. The clusters were further categorized into four groups within the endocytosis. As the results, the late endosomes had increased number of large clusters with time, while clustered numbers in secondary and tertiary groups were first increased and then decreased that indicates the fusion and fission of the endocytic vesicles. Their time constants are calculated by an integrated rate equation, and show a positive correlation with the size of the Au NP cluster. The endocytic efficiency of Au NP is about 50% for normal cells, while 75% for cancer cells. Compared to normal cells, cancer cells show a larger number in uptake, while faster rate in removal. The propose method helps the kinetic study of endocytosed NPs in physiological conditions. We also discussed the dose-dependent distribution, aggregation and cytotoxicity of treated dose of Au NPs in Chapter 5. By using the scatter images and chromatic analysis, the dose-dependent cellular uptake, distribution and aggregation were revealed. With the 0.01 and 0.05 nM treatments, Au NPs were mostly endocytosed and clustered in cells. When the dose was increasing to 0.1, 0.2 nM, numbers of Au NPs were stuck on the membrane and formed two scattering color bands, yellow, larger aggregates in cells and green, individual on the membranes. As for the 0.5 nM, increased Au NPs were stuck on the membrane. Furthermore, owing the periodic lamellipodial contraction on the membrane, Au NPs were then transferred to and aggregated on the top of cells. Two yellow scatter color band were then shown. SEM images revealed the dose-dependently stuck density of Au NPS on the membrane. Dual-beam focused ion beam (DBFIB) and tilted SEM images were used to discuss the 2-D covering and 3-D stacking of the aggregated Au NPs on membrane and inside endocytic vesicles. Cytotoxicity test indicates the stuck Au NPs on the membrane would also efficiently impact the cell viability. In summary, we demonstrated the ability of the chromatogram analysis in the 5-D (location (x,y,z), time (t) and aggregation (n)) study of Au NPs. By using the chromatogram analysis, the optical system further realized the study in the long-term quantification and orientation of the intracellular progression of Au NPs. For the NP-based gene/drug delivery or contrast agents, this dissertation helps the understanding of the overall undergoing of the nano-carrier with cells and their following triggered cellular responses.