Evaluation of the potential hazard of man-made nanomaterials has been hampered by a limited ability to observe and measure nanoparticles in cells. In this study, different concentrations of TiO 2 nanoparticles were suspended in cell culture medium. The suspension was then sonicated and characterized by dynamic light scattering and microscopy. Cultured human-derived retinal pigment epithelial cells (ARPE-19) were incubated with TiO 2 nanoparticles at 0, 0.1, 0.3, 1, 3, 10, and 30 lg/ml for 24 hours. Cellular reactions to nanoparticles were evaluated using flow cytometry and dark field microscopy. A FACSCalibur TM flow cytometer was used to measure changes in light scatter after nanoparticle incubation. Both the side scatter and forward scatter changed substantially in response to the TiO 2 . From 0.1 to 30 lg/ml TiO 2 , the side scatter increased sequentially while the forward scatter decreased, presumably due to substantial light reflection by the TiO 2 particles. Based on the parameters of morphology and the calcein-AM/propidium iodide viability assay, TiO 2 concentrations below 30 lg/ml TiO 2 caused minimal cytotoxicity. Microscopic analysis was done on the same cells using an E-800 Nikon microscope containing a xenon light source and special dark field objectives. At the lowest concentrations of TiO 2 (0.1-0.3 lg/ml), the flow cytometer could detect as few as 5-10 nanoparticles per cell due to intense light scattering by TiO 2 . Rings of concentrated nanoparticles were observed around the nuclei in the vicinity of the endoplasmic reticulum at higher concentrations. These data suggest that the uptake of nanoparticles within cells can be monitored with flow cytometry and confirmed by dark field microscopy. This approach may help fulfill a critical need for the scientific community to assess the relationship between nanoparticle dose and cellular toxicity Such experiments could potentially be performed more quickly and easily using the flow cytometer to measure both nanoparticle uptake and cellular health. Published
The cellular uptake of different sized silver nanoparticles (AgNP) (10, 50, and 75 nm) coated with polyvinylpyrrolidone (PVP) or citrate on a human derived retinal pigment epithelial cell line (ARPE-19) was detected by flow cytometry following 24-h incubation of the cells with AgNP. A dose dependent increase of side scatter and far red fluorescence was observed with both PVP and citrate-coated 50 nm or 75 nm silver particles. Using five different flow cytometers, a far red fluorescence signal in the 700-800 nm range increased as much as 100 times background as a ratio comparing the intensity measurements of treated sample and controls. The citrate-coated silver nanoparticles (AgNP) revealed slightly more side scatter and far red fluorescence than did the PVP coated silver nanoparticles. This increased far red fluorescence signal was observed with 50 and 75 nm particles, but not with 10 nm particles. Morphological evaluation by dark field microscopy showed silver particles (50 and 75 nm) clumped and concentrated around the nucleus. One possible hypothesis to explain the emission of far red fluorescence from cells incubated with silver nanoparticles is that the silver nanoparticles inside cells agglomerate into small nano clusters that form surface plasmon resonance which interacts with laser light to emit a strong far red fluorescence signal. The results demonstrate that two different parameters (side scatter and far red fluorescence) on standard flow cytometers can be used to detect and observe metallic nanoparticles inside cells. The strength of the far red fluorescence suggests that it may be particularly useful for applications that require high sensitivity.
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 G 1 phase and an increase in the G 2 /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.
23As a component of sunscreen formulations, TiO 2 engineered nanomaterials (ENM) are coated to 24 prevent reactive oxygen species from causing damage to skin. We investigated the stability of an 25 Al(OH) 3 coating by exposing 25 nm Al(OH) 3 ·TiO 2 ENM to simulated swimming pool water 26 (SPW) for 45 minutes, 1, 3, 10, or 14 days. Electron microscopy and spectroscopy indicated that 27 exposure to SPW caused a redistribution of the Al(OH) 3 coating allowing photocatalytic 28 formation of hydroxyl radicals. Aged ENM showed significantly greater phototoxicity under 29 UVA irradiation than un-aged ENM in a human-derived retinal pigment epithelium cell line 30 (ARPE-19). Photocatalytic activity and phototoxicity of aged Al(OH) 3 ·TiO 2 was significantly 31 less than that of the positive control-uncoated P25 TiO 2 . In summary, the aging of 32 Al(OH) 3 ·TiO 2 ENM in SPW redistributed the coating and reduced its protective properties, 33 thereby increasing reactivity and potential phototoxicity.34 35 those of toxic effects, including acute phototoxicity in aquatic species. 41 They concluded that the 403 probability distributions for TiO 2 environmental exposures and sensitive effects were relatively 404 close, with only about one order of magnitude separation. Assessments of potential 405 environmental risks of nanomaterials to date, have not considered the combination of 406 environmental transformations (demonstrated here), long-term bioaccumulation, and 407 phototoxicity from co-exposure to UV wavelengths (or visible wavelengths in the case of doped 408 TiO 2 varieties).409 410 Acknowledgment 411
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 in vivo 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.
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