You are here:
Detection of Ti02 nanoparticles using flow cytometry and optimized darkfield microscopy
Citation:
ZUCKER, R. M., E. J. MASSARO, W. K. BOYES, L. DEGN, AND K. SANDERS. Detection of Ti02 nanoparticles using flow cytometry and optimized darkfield microscopy. Presented at XXV Congress of the Internatonial Society for Advancement of Cytometry, Seattle, WA, May 08 - 12, 2010.
Impact/Purpose:
Flow cyometry of nanoparticles showing dose response --parameter is light scatter.
Description:
Evaluation of the potential hazard of man made nanomaterials has been hampered by a limited ability to observe and measure nanoparticles in cells. In the present study, TiOz nanoparticles (rutile, 30-40 nm) were suspended in DMEM/F12 with 10% fetal bovine serum, sonicated, and characterized by dynamic light scattering and microscopy. Nano-TiOz was added to a humanderived retinal pigment epithelium cell line (ARPE-19) for 24 hrs at concentrations of 0.1, 0.3, 1, 3, 10, and 30 ug/ml. The cells were then evaluated using a flow cytometer and an E-800 Nikon microscope containing a xenon light source with optimized dark field objectives. A FACSCalibur flow cytometer was used to measure light scatter in cells exposed to TiOz. Presumably due to high light reflection by the TiOz, the side scatter increased by 10 folds and forward scatter decreased by 5 folds in a dose-dependent manner between 0.1 and 30 ug/ml. Using cell morphology and a ca1cein-AM/PI viability assay, minimal toxicity was observed below 30 ug/ml TiOz. Microscopic observation of the same population of cells analyzed by flow cytometry indicated that approximately 5 single nanoparticles or aggregates per cell were detected at 0.1 ug/ml and about 10 single particles or aggregates per cell at 0.3 ug/ml. At concentrations above 1 ug/ml, individual particles in cells could not be counted due to agglomeration. Cells grown on glass chamber slides were used for microscopic analysis of nanoparticles. Specific organelle dyes and an Invitrogen transfection kit were used to stain the endoplasmic reticulum(ER), Golgi apparatus, nucleus, mitochondria, and lysosomes. Observations using fluoresence and dark field microscopy suggest that nanoparticles enter cells by endocytosis and accumulate in clumps and single particles in the ER surrounding the nucleus and in the Golgi. These nanoparticles remain in a configuration that apparently cause minimal damage to cell viability over the course of 24 hours. At higher concentrations (l0-30 ug/ml), particles occupied the entire cytoplasm and inhibit cell division and block the cell cycle in S phase. These data suggest that the uptake of nanoparticles into cells can be monitored using both flow cytometry and microscopy. This enables an efficient measurement of the intracellular dose of nanoparticles during in vitro experiments. The capability to determine cellular dose by a combination of flow cytometry and microscopy will enhance the critical ability to link tissue dose to outcomes in evaluations of potential nanomaterial toxicity. This abstract does not reflect EPA policy.