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

Use of novel inhalation kinetic studies to refine physiologically-based pharmacokinetic models for ethanol in non-pregnant and pregnant rats

<div><p></p><p>Ethanol (EtOH) exposure induces a variety of concentration-dependent neurological and developmental effects in the rat. Physiologically-based pharmacokinetic (PBPK) models have been used to predict the inhalation exposure concentrations necessary to produce blood EtOH concentrations (BEC) in the range associated with these effects. Previous laboratory reports often lacked sufficient detail to adequately simulate reported exposure scenarios associated with BECs in this range, or lacked data on the time-course of EtOH in target tissues (e.g. brain, liver, eye, fetus). To address these data gaps, inhalation studies were performed at 5000, 10 000, and 21 000 ppm (6 h/d) in non-pregnant female Long-Evans (LE) rats and at 21 000 ppm (6.33 h/d) for 12 d of gestation in pregnant LE rats to evaluate our previously published PBPK models at toxicologically-relevant blood and tissue concentrations. Additionally, nose-only and whole-body plethysmography studies were conducted to refine model descriptions of respiration and uptake within the respiratory tract. The resulting time-course and plethysmography data from these <i>in vivo</i> studies were compared to simulations from our previously published models, after which the models were recalibrated to improve descriptions of tissue dosimetry by accounting for dose-dependencies in pharmacokinetic behavior. Simulations using the recalibrated models reproduced these data from non-pregnant, pregnant, and fetal rats to within a factor of 2 or better across datasets, resulting in a suite of model structures suitable for simulation of a broad range of EtOH exposure scenarios.</p></div