Effects of agglomeration and sedimentation of colloidal particles on endocytosis mechanism of silver nanoparticles

Recently, nanomaterials have attracted increasing interest and widely used in many applications, such as nanomedicine as well as consumer products. However, their accumulation in the environment raises concerns on the potential hazards of nanomaterials on biological and environmental system. Silver nanoparticles (Ag NPs) are widely used as antimicrobial agents, laundry detergents, wall paint and clothing such as underwear and socks. Notwithstanding the increasing use of Ag NPs in consumer products and potential risk of human exposure, there is no quantitative understanding on their cellular association mechanism and resultant toxicity. To better understand these phenomena, it is prerequisite to develop measurement method and perform quantitative analysis of intracellular Ag NPs. In part �� of this thesis, we propose a simple approach for quantitatively estimating the number of cellular silver (Ag) NPs in lung cancer cells (A549), based on their side scattering (SSC) intensities measured by flow cytometry (FCM). To develop a measurement method of cellular Ag NPs, we collected and normalized the SSC intensity signals of A549 cells exposed to various types of silver NPs (40���200 nm, 5 different core sizes with positive surface charge), then found their relationships with the number concentrations of cellular Ag NPs, which were also measured by the well-established, although labour-intensive and time-consuming, inductively coupled plasma mass spectrometry (ICPMS) technique as a cross-validation method. Various exposure conditions (e.g., upright and inverted configurations, media heights of 3, 6, 9 mm) were tested to confirm that the FCM���SSC intensities are highly correlated with the amount of cellular Ag NPs and their core sizes, rather than other biological variations in cellular size, shape, and subcellular organelles��� size/shape, which might be caused due to the differences in growth/exposure conditions. Moreover, it was also demonstrated that the number of Ag NPs associated with A549 cells can be estimated from their FCM���SSC intensities over a significantly wider linear range of ~80,000 Ag NPs/cell and ~23 nSSC, which is ~20 fold wider than that of previous studies on Au NPs associated with HeLa cells. In part��� of this thesis, The purpose of this study is to investigate the influence of nanoparticle sedimentation on their cellular association and related endocytosis mechanism, when A549 cells were exposed to Ag NPs. We have measured the dispersion stability, hydrodynamic sizes, and surface charges of Ag NPs using UV-Vis spectroscopy, dynamic light scattering (DLS) and zeta-potential. We also performed flow cytometry (FCM) and inductively coupled plasma mass spectrometry (ICPMS) measurements to quantify cellular Ag NPs under various exposure conditions. Inhibitor assays on endocytosis mechanisms were also conducted and confirmed with flow cytometry (FCM) and bio-transmission electron microscopy (bio-TEM), which provided us useful insight on the cellular uptake mechanisms of Ag NPs in A549 cells.Maste