Detection of TiO₂ nanoparticles in cells by flow cytometry

Abstract

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 TiO 2 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 TiO 2 nanoparticles at 0, 0.1, 0.3, 1, 3, 10, and 30 lg/ml for 24 hours. Cellular reactions to nanoparticles were evaluated using flow cytometry and dark field microscopy. A FACSCalibur TM 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 TiO 2 . From 0.1 to 30 lg/ml TiO 2 , the side scatter increased sequentially while the forward scatter decreased, presumably due to substantial light reflection by the TiO 2 particles. Based on the parameters of morphology and the calcein-AM/propidium iodide viability assay, TiO 2 concentrations below 30 lg/ml TiO 2 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 TiO 2 (0.1-0.3 lg/ml), the flow cytometer could detect as few as 5-10 nanoparticles per cell due to intense light scattering by TiO 2 . 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

Quantifying Nanoparticle Cellular Uptake: Which Method is Best?

“…These techniques require a fluorescence signal from the NPs which can derive from an intrinsic property of NPs exhibiting the capacity to emit the light, for examplesemiconductor nanocrystals such as quantum dots [15] and titanium dioxide NPs [16] or by the use of fluorescence tags [17]. These types of NPs can be detected and quantified by fluorescence spectroscopy, flow cytometry, or fluorescence imaging techniques.…”

“…Forward scattered light is useful to determine the volume of the cell, thus the reference volume is defined in this specific technique. It is a fast method to obtain the relative amount of cells loaded and/or associated with fluorescently labeled NPs (or possessing fluorescent properties) [16,17]. In addition, the cells have to be labeled in order to define the reference volume.…”

High throughput toxicity screening and intracellular detection of nanomaterials

“…It was demonstrated that TiO 2 78, 79, 80 and Ag81 NMs induce an increase of SS signal in cell populations after exposure to both NMs. SS indicates the granularity of the cells and is strongly linked to their content of NMs.…”

“…SS indicates the granularity of the cells and is strongly linked to their content of NMs. As few as 5–10 NMs per cell can be detected 79. Recently, SS signal analysis was successfully applied for detecting carboxylated nanodiamonds and tungsten carbide‐cobalt (WC‐Co) NMs in cells, results being validated by Raman and confocal microscopy associated with 3D reconstruction 82, 83.…”

An Image Cytometric MTT Assay as an Alternative Assessment Method of Nanoparticle Cytotoxicity

“…15 Compared to the conventional cell based assays measuring cellular responses collectively, the cytometric approach measuring individual cellular information seems to have huge potential as an alternative assay protocols that can circumvent or overcome various limitations of nanotoxicity assessments. For instance, recent flow cytometry studies by Zucker et al 16 have provided useful insight into the detection of cellular NP associations and resultant toxicities, while Suzuki et al 17 have proposed scattering-based flow cytometry as a simple and easy method to evaluate cellular association of NPs that can be used as a cost-effective technique for initial screening of the nanotoxicity. Thus, we believe that the image cytometry based MTT cell viability assay protocols proposed in this study also provided a simple, reliable, and unbiased method to evaluate nanotoxicity of TiO 2 NPs and can be further modified and expanded for the cytotoxicity assessment of various manufactured nanomaterials.…”