Citation:

Zucker, R., W. Boyes, L. Degn, AND J. Ortenzio. Hyperspectral Imaging, Flow cytometry and Microscopic Morphology of Silver Nanoparticle within Cells. CYTO 2017, Boston, MA, June 10 - 14, 2017.

Impact/Purpose:

The ability to detect and track silver nanoparticles (AgNP) that enter cells is important to understand the potential biological and toxicological actions of AgNP.

Description:

The ability to detect and track silver nanoparticles (AgNP) that enter cells is important to understand the potential biological and toxicological actions of AgNP. The uptake and fate in cells of four different types of AgNP was studied in a retinal pigment epithelial cell line (ARPE-19) using flow cytometry, hyperspectral imaging and microscopic morphology. The four types of 80 nm AgNP were capped and stabilized with the following: PVP, Citrate, PEG and bPEI (branched polyethyleneimine). Surface charge of the branched was positive while the 3 other surfaces were negative. At concentrations between 0.1 µg/ml and 3 µg/ml, the AgNP with positively charged coating were taken into cells more that the negatively charged materials tested. Cellular organelles including: Golgi, nuclei, endoplasmic reticulum, endosomes, lysosomes and actin were stained using BacMam 2.0 (Molecular Probes) organelle transfection products and fluorescent dye stains. A Nikon upright microscope, was optimized for acquiring darkfield and fluorescence images, was used to acquire 3 fluorescence images and one darkfield image on a sample. The results showed that AgNP incorporated into cells, migrated quickly to the endoplasmic reticulum and became encapsulated in lysosomes. A PARISS hyperspectral imager showed increases in the spectral wavelength profiles of nanoparticle clusters after they entered the cells and migrated to the ER and lysosomes. Longer wavelengths of scattered light suggested that the NP agglomerated into larger structures. A number of other observations were made from these experiments: 1) AgNP enter cells in a dose dependent manner which can be quantified by FCM side scatter and observed microscopically. 2) Clumping of AgNP in the ER can be measured by the predominance of longer wavelength NP light scatter. AgNP that is located closer to the nucleus scatter longer wavelengths. Hyperspectral imaging showed the clumping status of the particles within the cell. 3) The surface of the AgNP influences the uptake and toxicity. Positively charged particles enter the cell more than negatively charged particles. Mitochondria morphology are effected by bEPI AgNP at low concentrations and show selective mitochondrial toxicity. 4) Lysosomes sequester AgNP from the cellular cytoplasm apparently generating cellular protection. 5) AgNP (>3ug/ml) used for toxicological endpoints observed using darkfield microscopy are too concentrated. During cellular division, a circular pattern outlined with nanoparticles was demonstrated. It was hypothesized that cytoplasmic membrane adhesion proteins selectively bound free AgNP to form this circular pattern. This abstract does not represent USEPA policy.

Record Details: