Detection of large extracellular silver nanoparticle rings observed during mitosis using darkfield microscopy.

During studies on the absorption and interactions between silver nanoparticles and mammalian cells grown in vitro it was observed that large extracellular rings of silver nanoparticles were deposited on the microscope slide, many located near post-mitotic cells. Silver nanoparticles (AgNP, 80nm), coated with citrate, were incubated at concentrations of 0.3 to 30 μg/ml with a human-derived culture of retinal pigment epithelial cells (ARPE-19) and observed using darkfield and fluorescent microscopy, 24 h after treatment. Approximately cell-sized extracellular rings of deposited AgNP were observed on the slides among a field of dispersed individual AgNP. The mean diameter of 45 nanoparticles circles was 62.5 +/-12 microns. Ring structures were frequently observed near what appeared to be post-mitotic daughter cells, giving rise to the possibility that cell membrane fragments were deposited on the slide during mitosis, and those fragments selectively attracted and retained silver nanoparticles from suspension in the cell culture medium. These circular structures were observable for the following technical reasons: 1) darkfield microscope could observe single nanoparticles below 100 nm in size, 2) a large concentration (108 and 109) of nanoparticles was used in these experiments 3) negatively charged nanoparticles were attracted to adhesion membrane proteins remaining on the slide from mitosis. The observation of silver nanoparticles attracted to apparent remnants of cellular mitosis could be a useful tool for the study of normal and abnormal mitosis

Biophysical comparison of four silver nanoparticles coatings using microscopy, hyperspectral imaging and flow cytometry.

This study compared the relative cellular uptake of 80 nm silver nanoparticles (AgNP) with four different coatings including: branched polyethyleneimine (bPEI), citrate (CIT), polyvinylpyrrolidone (PVP), and polyethylene glycol (PEG). A gold nanoparticle PVP was also compared to the silver nanoparticles. Biophysical parameters of cellular uptake and effects included flow cytometry side scatter (SSC) intensity, nuclear light scatter, cell cycle distributions, surface plasmonic resonance (SPR), fluorescence microscopy of mitochondrial gross structure, and darkfield hyperspectral imaging. The AgNP-bPEI were positively charged and entered cells at a higher rate than the negatively or neutrally charged particles. The AgNP-bPEI were toxic to the cells at lower doses than the other coatings which resulted in mitochondria being transformed from a normal string-like appearance to small round beaded structures. Hyperspectral imaging showed that AgNP-bPEI and AgNP-CIT agglomerated in the cells and on the slides, which was evident by longer spectral wavelengths of scattered light compared to AgNP-PEG and AgNP-PVP particles. In unfixed cells, AgNP-CIT and AgNP-bPEI had higher SPR than either AgNP-PEG or AgNP-PVP particles, presumably due to greater intracellular agglomeration. After 24 hr. incubation with AgNP-bPEI, there was a dose-dependent decrease in the G1 phase and an increase in the G2/M and S phases of the cell cycle suggestive of cell cycle inhibition. The nuclei of all the AgNP treated cells showed a dose-dependent increase in nanoparticles following non-ionic detergent treatment in which the nuclei retained extra-nuclear AgNP, suggesting that nanoparticles were attached to the nuclei or cytoplasm and not removed by detergent lysis. In summary, positively charged AgNP-bPEI increased particle cellular uptake. Particles agglomerated in the peri-nuclear region, increased mitochondrial toxicity, disturbed the cell cycle, and caused abnormal adherence of extranuclear material to the nucleus after detergent lysis of cells. These results illustrate the importance of nanoparticle surface coatings and charge in determining potentially toxic cellular interactions

<i>In vitro</i> screening of metal oxide nanoparticles for effects on neural function using cortical networks on microelectrode arrays

<p>Nanoparticles (NPs) may translocate to the brain following inhalation or oral exposures, yet higher throughput methods to screen NPs for potential neurotoxicity are lacking. The present study examined effects of 5 CeO₂ (5– 1288 nm), and 4 TiO₂ (6–142 nm) NPs and microparticles (MP) on network function in primary cultures of rat cortex on 12 well microelectrode array (MEA) plates. Particles were without cytotoxicity at concentrations ≤50 µg/ml. After recording 1 h of baseline activity prior to particle (3–50 µg/ml) exposure, changes in the total number of spikes (TS) and # of active electrodes (#AEs) were assessed 1, 24, and 48 h later. Following the 48 h recording, the response to a challenge with the GABA_A antagonist bicuculline (BIC; 25 µM) was assessed. In all, particles effects were subtle, but 69 nm CeO₂ and 25 nm TiO₂ NPs caused concentration-related decreases in TS following 1 h exposure. At 48 h, 5 and 69 nm CeO₂ and 25 and 31 nm TiO₂ decreased #AE, while the two MPs increased #AEs. Following BIC, only 31 nm TiO₂ produced concentration-related decreases in #AEs, while 1288 nm CeO₂ caused concentration-related increases in both TS and #AE. The results indicate that some metal oxide particles cause subtle concentration-related changes in spontaneous and/or GABA_A receptor-mediated neuronal activity <i>in vitro</i> at times when cytotoxicity is absent, and that MEAs can be used to screen and prioritize nanoparticles for neurotoxicity hazard.</p