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.

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.