奈米金粒子散射光色譜分析法之發展及其在研究奈米顆粒胞吞作用之應用

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

王盛翰(Wang, Sheng Hann)

指導教授 ： 曾繁根 ； 魏培坤

國立清華大學/原子科學院/工程與系統科學系/博士(2015年)

https://doi.org/10.6843/NTHU.2015.00515

摘要

本研究主要為發展散射光色譜法及其應用在觀測奈米金粒子與活體細胞之交互作用。奈米粒子近年來在生物醫學上的應用與發展受到國際間的相當矚目。因此，於本文之第一章，我們簡單介紹了奈米粒子之光學特性及現階段在生物醫學上之應用，並整理了現今已被美國食品藥品監督管理局(FDA)核准上市或正在進行安全評估研究之奈米粒子之相關生醫產品。此外，我們簡單介紹了奈米粒子的聚集和分佈對於在生物細胞系統上所造成的影響及其重要性。在第二章中，我們介紹了於此研究中所需用到之儀器、藥品及研究方法。
於第三章中，我們研究並發展可直接觀測奈米金粒子在活體細胞中的三維分佈及其聚集程度之觀測分析法–散射光色譜分析法。藉由搭配暗場光學切片顯微及散射光色譜分析，我們可以同時定位且定量分析在活體細胞內之奈米金粒子(群)。此方法主要乃基於散射光彩色影像在RGB及HSV色彩空間上的轉換。搭配奈米金粒子獨特的局域表面電漿共振及耦合效應，彩度(H, Hue)可用來分辨奈米金粒子(群)與細胞體，亮度(V, Value)則可用來定位奈米金粒子(群)。再者，藉由已校正的已知的奈米金粒子聚集數目及其對應的V/H 值，我們便可快速估算出在活體細胞內奈米金粒子(群)的位置及聚集數量。且此方法所估算之奈米金粒子總數量與利用感應耦合電漿質譜儀所得之值相符合。相較於其他分析儀器，散射光色譜分析提供了奈米金粒子於活體細胞上之定位及定量研究上另一個更為簡易的方法。
在第四章裡，我們利用了散色光色譜分析法來探討奈米金粒子在細胞胞吞作用中的動態發展與分佈。由於胞吞作用的影響，奈米金粒子會在細胞內經歷一連續的聚集與分離，並因此使得其散射光顏色有著明顯之變化。藉由散射光色譜分析，我們可得知在每個時期下，奈米金粒子的聚集程度。基於聚集程度的不同，我們簡單將其分為奈米金粒子於胞吞做用中的四個階段–細胞膜上貼附、胞吞小泡、初級內體以及次級內體。結果發現，貼附在細胞膜上的數量會隨著時間而下降，在胞吞小泡及初級內體裡則是會先短暫的增加後再隨即下降，而在次級內體內的數量則是會隨著時間增加，所有的數量在一段時間後皆會達到穩定值。藉由自然指數函數的擬合，便可得知奈米金粒子在各個胞吞階段的半衰期且其值與奈米金粒子之聚集程度有正相關。再者，奈米金粒子在細胞所能攝取的總數量及移除的速率與細胞的表面積相關。但相較於正常細胞(50%)，癌化細胞還是有著較高的胞吞效率(75%)。同時，我們也驗證了色譜分析法在研究奈米金粒子動態分布和聚集的可行性。
在第五章裡，我們探討了隨著施加的劑量的不同，奈米金粒子在細胞內所造成的分佈，聚集程度以及細胞毒性的差異。藉由動態散射觀測及散色光色譜分析，我們發現當施加低濃度(0.01及0.05 nM)的奈米金粒子時，絕大部分的奈米金粒子會為細胞所胞吞。而當施加濃度開始增加時(0.1及0.2 nM)，部分奈米金粒子會開始堵塞在細胞膜上。且因為片狀偽足的收縮作用，金粒子會被運送至細胞頂部，並在細胞的暗場影像上產生兩區色帶：位於細胞內之金黃色及附蓋於細胞上之綠色。當濃度增加至0.5 nM時，堵塞在膜上的奈米金粒子會隨著增加，且開始聚集並造成散射光顏色之改變，並使得在膜上的綠色散射光也變成了與細胞內之散射光一樣的金黃色。藉由掃描式電子顯微鏡(SEM)，我們可發現即便在低濃度下，奈米金粒子也並無法完全為細胞所胞吞。而其堆積在細胞頂部的奈米金粒子的數量與密度則是與所施加之濃度成正相關。藉由雙聚焦離子束(DBFIB)的細胞切片觀測，可發現不同於在細胞膜上的二維空間的群聚，奈米金粒子在細胞內為一三維空間的堆疊。最後，細胞毒性測試發現除了在細胞內的奈米金粒子外，隨著聚集在細胞膜上的奈米金粒子增加，所造成細胞毒性也會同時增加。
總結，在此論文中，我們使用所發展的散射光色譜分析法，實現了利用光學影像系統達到長時間觀測且定量及定位的活體細胞與奈米粒子交互作用目的。對以奈米粒子為基礎的藥物或基因傳遞治療或是生醫影像觀測，此研究可望為其提供另一分析研究平台，以利研發或改進其效率。

關鍵字

奈米金粒子 ； 表面電漿共振 ； 散射影像 ； 色譜分析 ； 胞吞作用 ； 細胞毒性

並列摘要

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.

並列關鍵字

Gold nanoparticles ； Local surface plasmon resonance ； Scattering images ； Chromatogram analysis ； Endocytosis ； Cytotoxicity

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