Citation:

Goldstein-Plesser, A., J. Nyffeler, C. Willis, J. Harrill, AND W. Boyes. Assessing the effects of silver nanoparticles on ARPE-19 cells via high content imaging-based phenotypic profiling. SETAC, Sacramento, California, November 04 - 08, 2018.

Impact/Purpose:

The purpose was to evaluate high content imaging as a method to study the potential effects of engineered nanomaterials

Description:

Efficient methods are needed to evaluate the human and environmental health effects of silver nanoparticles (AgNPs). A high content imaging-based phenotypic profiling approach was used to determine the effects of 12 types (4 sizes x 3 coatings) 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 an ionization control. The in vitro sedimentation, diffusion, and dosimetry (ISDD) 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 ISDD model predicted that the fraction of AgNP deposited on cells increased with particle size and differed based on coating (branched polyethyleneimine (BPEI) > citrate > Polyvinylpyrrolidone (PVP)). Phenotypic profiling showed that all AgNP types affected the morphology of each organelle in a concentration-dependent manner, with many effects observed below the threshold for cytotoxicity. The pattern of changes in cell morphology differed across capping agents and silver nitrate. For example, the effect of citrate on mitochondrial texture was opposite that of other AgNP coatings. 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. In summary, high content imaging assessed the cellular toxicity of AgNPs, and identified signature morphology patterns that differentiated between three common AgNP coatings. Further, this screening method may inform subsequent assay selection by highlighting the intracellular regions effected by AgNPs of interest

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