Citation:

Goldstein-Plesser, A., J. Nyffeler, C. Willis, J. Harrill, AND W. Boyes. Assessing the effects of Silver Nanoparticles on ARPE-19 Cells via Through High Content imaging-based phenotypic profiling. Society of Toxicology, Baltimore, MD, March 10 - 14, 2019.

Impact/Purpose:

This product illustrates that there are numerous effects of silver nanoparticles on cells at dose levels below those causing cytotoxicity, that the effects differ by particle coating, that the effects of silver particles are different from silver ions, and that high content screening may be a valuable tool for evaluating potential toxic effects of engineered nanomaterials

Description:

Evaluating the Toxicity of Silver Nanoparticles in ARPE-19 Cells Through High Content Morphometric Analysis A. Goldstein-Plesser, J. Nyffeler, C. Willis, J. Harrill, W. Boyes Efficient methods are needed to evaluate the human and environmental health effects of silver nanoparticle (AgNP) exposure. A high content imaging-based phenotypic profiling approach was used to determine the effects of 12 types (40,60, 80, and 100nm coated with Citrate, Polyvinylpyrrolidone (PVP), and branched polyethyleneimine (BPEI)) of AgNPs on organelle morphology in human retinal pigmented epithelial (ARPE-19) cells. AgNPs (0.1-30 µg/ml) were applied to the cells seeded in a 384-well format, with silver nitrate acting as silver ionization control. The Distorted Grid (DG) model was used to estimate the cellular delivered dose. After 24 hrs of treatment, cells were live-labeled with MitoTracker (mitochondria), fixed, permeabilized and labeled with Hoechst-33342 (nuclei), SYTO14 (nucleoli) and fluorescent conjugates of concanavalin A (ER), phalloidin (actin cytoskeleton), and wheat germ agglutinin (Golgi/plasma membrane). A multiplexed cell viability and apoptosis assay was run in parallel. Cells were imaged using an Opera Phenix High Content Screening System and profiled using Harmony High Content Analysis software. Approximately 1700 morphological features were measured per cell and summarized to the well level for analysis. The DG model predicted that the fraction of AgNP deposited on cells increased with particle size and differed based on coating (BPEI > citrate > PVP). Phenotypic profiling showed that all AgNP types affected the morphology of each organelle in a concentration-dependent manner, with over 600 outcomes below the benchmark dose for cytotoxicity. The pattern of changes in cell morphology differed across coating agents and silver nitrate. Citrate coated AgNPs showed the most pronounced effects below the cytotoxic threshold. 60 nm PVP had fewer effects on cell morphology than other coatings, yet showed apoptosis at an estimated delivered dose as low as 1.45 µg/ml. This screening method may inform subsequent assay selection by highlighting the intracellular regions effected by AgNPs of interest. This abstract does not necessarily reflect US EPA policy.

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