There is a need to accurately detect, characterize, and quantify nanoparticles in suspensions. This study helps to understand the complex interactions between similar types of nanoparticles. Before initiating a study of metal nanoparticles, five submicron PS beads with sizes between 200 nm and 1 mm were used to derive a reference scale that was useful in evaluating the flow cytometer for functionality, sensitivity, resolution, and reproducibility. Side scatter intensity (SSC) from metal nanoparticles was obtained simultaneously from 405 nm and 488 nm lasers. The 405 nm laser generally yielded histogram distributions with smaller CVs, less side scatter intensity, better separation indices between beads and decreased scatter differences between different sized particles compared with the 488 nm laser. Submicron particles must be diluted to 10 6 and 10 7 particles/mL before flow cytometer analysis to avoid coincidence counting artifacts. When particles were too concentrated the following occurred: swarm, electronic overload, coincidence counting, activation of doublet discrimination and rejection circuitry, increase of mean SSC histogram distributions, alterations of SSC and pulse width histogram shape, decrease and fluctuations in counting rate and decrease or elimination of particulate water noise and 1 mm reference bead. To insure that the concentrations were in the proper counting range, the nanoparticle samples were mixed with a known concentration of 1mm counting beads. Sequential dilutions of metal nanoparticles in a 1 mm counting bead suspension helped determine the diluted concentration needed for flow cytometer analysis. It was found that the original concentrated nanoparticle samples had to be diluted, between 1:10,000 and 1:100,000, before characterization by flow cytometry. The concentration of silver or gold nanoparticles in the undiluted sample were determined by comparing them with a known concentration (1.9 3 10 6 beads/mL) of 1 mm polystyrene reference beads. Published 2015 Wiley Periodicals Inc., on behalf of ISAC
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
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 GABAA 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 GABAA receptor-mediated neuronal activity in vitro at times when cytotoxicity is absent, and that MEAs can be used to screen and prioritize nanoparticles for neurotoxicity hazard.
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