Detection of TiO₂ nanoparticles in cells by flow cytometry

Evaluation of the potential hazard of man‐made nanomaterials has been hampered by a limited ability to observe and measure nanoparticles in cells. In this study, different concentrations of TiO₂ nanoparticles were suspended in cell culture medium. The suspension was then sonicated and characterized by dynamic light scattering and microscopy. Cultured human‐derived retinal pigment epithelial cells (ARPE‐19) were incubated with TiO₂ nanoparticles at 0, 0.1, 0.3, 1, 3, 10, and 30 μg/ml for 24 hours. Cellular reactions to nanoparticles were evaluated using flow cytometry and dark field microscopy. A FACSCalibur™ flow cytometer was used to measure changes in light scatter after nanoparticle incubation. Both the side scatter and forward scatter changed substantially in response to the TiO₂. From 0.1 to 30 μg/ml TiO₂, the side scatter increased sequentially while the forward scatter decreased, presumably due to substantial light reflection by the TiO₂ particles. Based on the parameters of morphology and the calcein‐AM/propidium iodide viability assay, TiO₂ concentrations below 30 μg/ml TiO₂ caused minimal cytotoxicity. Microscopic analysis was done on the same cells using an E‐800 Nikon microscope containing a xenon light source and special dark field objectives. At the lowest concentrations of TiO₂ (0.1–0.3 μg/ml), the flow cytometer could detect as few as 5–10 nanoparticles per cell due to intense light scattering by TiO₂. Rings of concentrated nanoparticles were observed around the nuclei in the vicinity of the endoplasmic reticulum at higher concentrations. These data suggest that the uptake of nanoparticles within cells can be monitored with flow cytometry and confirmed by dark field microscopy. This approach may help fulfill a critical need for the scientific community to assess the relationship between nanoparticle dose and cellular toxicity Such experiments could potentially be performed more quickly and easily using the flow cytometer to measure both nanoparticle uptake and cellular health. Published 2010 Wiley‐Liss, Inc.