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.

Determination of Silver Nanoparticle Dose in vitro.

An important issue for interpreting in vitro nanomaterial testing is quantifying the dose delivered to target cells. Considerations include the concentration added to the culture, the proportion of the applied dose that interacts with the target cells, and the amount that is eventually absorbed by the target cells. Rapid and efficient techniques are needed to determine delivered doses. Previously, we demonstrated that TiO2 and silver nanoparticles (AgNP) were absorbed by cells in a dose dependent manner between 1 µg/ml and 30 µg/ml and were detected in cells by light scatter using a flow cytometer. Here, we compare four potential indices of the dose of AgNP to cells, including: inductively coupled plasma - mass spectrometry (ICP-MS); flow cytometry side scatter (SSC); and amount of silver deposited to the cell layer as estimated with both an integrated Volumetric Centrifugation Method - In Vitro Sedimentation, Diffusion and Dosimetry Model (VCM-ISDD) and a Distorted Grid (DG) model. A retinal pigment epithelial cell line was exposed to 20 nm or 75 nm citrate-coated AgNP for 24 hr. The relationships between particle sizes and internalized doses varied according to the dose metric. Twenty-four hours after exposure, the cell layer contained a greater mass of silver when treated with 75 nm AgNP than with 20 nm AgNP. When the dose was expressed as the number of particles or as the total surface area of absorbed particles, however, the reverse was true; the dose to the cells was higher after exposure to 20 than 75 nm AgNP. Flow cytometry SSC increased with dose for both sizes of AgNP, and was correlated with Ag in cells measured by ICP-MS. The rate of SSC increase was greater for 75 than for 20 nm AgNP, suggesting it could be used as an indicator of cellular dose after accounting for particle size and composition. Silver was detected by ICP-MS in re-suspended supernates of the isolated cell layer suggested that not all the silver deposited to the cell layer was absorbed by the cells. Both the VCM-ISDD and DG models estimated the proportion of Ag deposited to the cellular layer, which in both cases was greater than the amount of silver in the cells measured by ICP-MS. Modeled deposition more closely compared to the total Ag deposition by ICP-MS, i.e. mass of silver in the cells plus the resuspended, unabsorbed Ag from the cell layer. ICP-MS indicated the mass of silver in cells from AgNP treatment, but not whether the Ag was in the form of particles or dissolved ions. Deposition models predicted the amount of AgNP deposited to the cell layer, but not cellular uptake. Flow cytometry SSC was correlated to cellular uptake of particle-form AgNP and could be calibrated against ICP-MS to indicate mass of cellular uptake. Therefore, a combination of approaches may be required to accurately understand cellular dosimetry of in vitro nanotoxicology experiments. In summary, cellular dosimetry is an important consideration for nanotoxicology experiments, and not necessarily related to the applied dose.

In vitro screening of silver nanoparticles and ionic silver using neural networks yields differential effects on spontaneous activity and pharmacological responses.

Silver nanoparticles (AgNPs) are used in a wide range of consumer and medical products because of their antimicrobial and antifungal properties, and can translocate to the brain following exposure. Therefore, to screen AgNPs for potential impacts on human health, it is essential to examine neural function. The present study examined AgNPs (3 citrate coated, 3 PVP coated; 10-75nm) and AgNO3 effects on spontaneous and pharmacologically-induced neural network function in rat primary cortical cells on multi-well microelectrode array (mwMEA) plates. Baseline activity (1h) was recorded prior to exposure to non-cytotoxic concentrations of AgNPs and AgNO3 (0.08-0.63 and 0.08-1.7µg/ml, respectively). Changes in number of total extracellularly-recorded action potential spikes (total spikes; TS) and active electrodes (AE), relative to controls, were assessed 1, 24, and 48h after exposure to AgNP suspensions or AgNO3. After the 48h recording, the response to a pharmacological challenge with the GABAA antagonist, bicuculline (BIC), was assessed. Only two particles altered neural network function. Citrate coated 10nm AgNP caused concentration-related increases in AEs at 24h. After BIC treatment, PVP coated 75nm AgNP caused concentration-dependent increases in AE. AgNO3 effects differed from AgNPs, causing a concentration-related decrease in AEs at 24 and 48h, and a concentration-related decrease in TS following BIC challenge. Importantly, the direction of AgNO3 effects on neural activity was opposite those of 10nm Ag citrate at concentrations up to 0.63µg/ml, and different from 75nm Ag PVP, indicating ionic silver does not mediate these effects. These results demonstrate that non-cytotoxic concentrations of 10nm citrate- and 75nm PVP-coated Ag NPs alter neural network function in vitro, and should be considered for additional neurotoxicity hazard characterization.

Neurophysiological assessment of auditory, peripheral nerve, somatosensory, and visual system function after developmental exposure to gasoline, E15, and E85 vapors.

The use of gasolines blended with a range of ethanol concentrations may result in inhalation of vapors containing a variable combination of ethanol with other volatile gasoline constituents. The possibility of exposure and potential interactions between vapor constituents suggests the need to evaluate the possible risks of this complex mixture. Previously we evaluated the effects of developmental exposure to ethanol vapors on neurophysiological measures of sensory function as a component of a larger project evaluating developmental ethanol toxicity. Here we report an evaluation using the same battery of sensory function testing in offspring of pregnant dams exposed during gestation to condensed vapors of gasoline (E0), gasoline blended with 15% ethanol (E15) or gasoline blended with 85% ethanol (E85). Pregnant Long-Evans rats were exposed to target concentrations 0, 3000, 6000, or 9000 ppm total hydrocarbon vapors for 6.5h/day over GD9 - GD20. Sensory evaluations of male offspring began as adults. The electrophysiological testing battery included tests of: peripheral nerve (compound action potentials, nerve conduction velocity [NCV]), somatosensory (cortical and cerebellar evoked potentials), auditory (brainstem auditory evoked responses), and visual functions. Visual function assessment included pattern elicited visual evoked potentials (VEP), VEP contrast sensitivity, dark-adapted (scotopic) electroretinograms (ERGs), light-adapted (photopic) ERGs, and green flicker ERGs. The results included sporadic statistically significant effects, but the observations were not consistently concentration-related and appeared to be statistical Type 1 errors related to multiple dependent measures evaluated. The exposure concentrations were much higher than can be reasonably expected from typical exposures to the general population during refueling or other common exposure situations. Overall the results indicate that gestational exposure of male rats to ethanol/gasoline vapor combinations did not cause detectable changes in peripheral nerve, somatosensory, auditory, or visual function when the offspring were assessed as adults.

In vitro screening of metal oxide nanoparticles for effects on neural function using cortical networks on microelectrode arrays.

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.

Characterization, detection, and counting of metal nanoparticles using flow cytometry.

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 µm 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 µm reference bead. To insure that the concentrations were in the proper counting range, the nanoparticle samples were mixed with a known concentration of 1 µm counting beads. Sequential dilutions of metal nanoparticles in a 1 µm 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 × 10(6) beads/mL) of 1 µm polystyrene reference beads.