Intracellular accumulation and dissolution of silver nanoparticles in L-929 fibroblast cells using live cell time-lapse microscopy

Abstract

Cytotoxicity assessments of nanomaterials, such as silver nanoparticles, are challenging due to interferences with test reagents and indicators as well uncertainties in dosing as a result of the complex nature of nanoparticle intracellular accumulation. Furthermore, current theories suggest that silver nanoparticle cytotoxicity is a result of silver nanoparticle dissolution and subsequent ion release. This study introduces a novel technique, nanoparticle associated cytotoxicity microscopy analysis (NACMA), which combines fluorescence microscopy detection using ethidium homodimer-1, a cell permeability marker that binds to DNA after a cell membrane is compromised (a classical dead-cell indicator dye), with live cell time-lapse microscopy and image analysis to simultaneously investigate silver nanoparticle accumulation and cytotoxicity in L-929 fibroblast cells. Results of this method are consistent with traditional methods of assessing cytotoxicity and nanoparticle accumulation. Studies conducted on 10, 50, 100 and 200 nm silver nanoparticles reveal size dependent cytotoxicity with particularly high cytotoxicity from 10 nm particles. In addition, NACMA results, when combined with transmission electron microscopy imaging, reveal direct evidence of intracellular silver ion dissolution and possible nanoparticle reformation within cells for all silver nanoparticle sizes.

• Cellular uptake
• confocal microscopy
• ICP-MS
• image analysis
• nanosilver
• transmission electron microscopy
• silver ions
• silver nanoparticles

Acknowledgements

We acknowledge the US FDA White Oak Nanotechnology Core Facilities for use of TEM and Dr. Katherine Tyner for use of her confocal microscope and live cell incubation chamber.

Declaration of interest

The findings and conclusions in this paper have not been formally disseminated by the US Food and Drug Administration and should not be construed to represent any agency determination or policy. The mention of commercial products, their sources or their use in connection with material reported herein is not to be construed as either an actual or implied endorsement of such products by US Department of Health and Human Services.

This work was supported by U.S. Food and Drug Administration nanotechnology research funds. We acknowledge the Research Fellowship Program support administered by the Oak Ridge Associated University through a contract with the U.S. Food and Drug Administration.

Supplementary material available online

Notes

¹ICP-MS cannot distinguish between Ag ions and AgNPs. In ICP-MS, all silver is eventually ionized by a plasma torch and thus all silver is eventually reduced to its ionic form. There are methods capable of differentiating and quantifying Ag ions and AgNPs, e.g. single particle ICP-MS; however, these techniques were not utilized for this study.