Amanda M. Schrand

Unique cellular interaction of silver nanoparticles: size-dependent generation of reactive oxygen species.

The rapid advancement of nanotechnology has created a vast array of engineered nanomaterials (ENMs) which have unique physical (size, shape, crystallinity, surface charge) and chemical (surface coating, elemental composition and solubility) attributes. These physicochemical properties of ENMs can produce chemical conditions to induce a pro-oxidant environment in the cells, causing an imbalanced cellular energy system dependent on redox potential and thereby leading to adverse biological consequences, ranging from the initiation of inflammatory pathways through to cell death. The present study was designed to evaluate size-dependent cellular interactions of known biologically active silver nanoparticles (NPs, Ag-15 nm, Ag-30 nm, and Ag-55 nm). Alveolar macrophages provide the first defense and were studied for their potential role in initiating oxidative stress. Cell exposure produced morphologically abnormal sizes and adherence characteristics with significant NP uptake at high doses after 24 h. Toxicity evaluations using mitochondrial and cell membrane viability along with reactive oxygen species (ROS) were performed. After 24 h of exposure, viability metrics significantly decreased with increasing dose (10-75 microg/mL) of Ag-15 nm and Ag-30 nm NPs. A more than 10-fold increase of ROS levels in cells exposed to 50 microg/mL Ag-15 nm suggests that the cytotoxicity of Ag-15 nm is likely to be mediated through oxidative stress. In addition, activation of the release of traditional inflammatory mediators were examined by measuring levels of cytokines/chemokines, including tumor necrosis factor (TNF-alpha), macrophage inhibitory protein (MIP-2), and interleukin-6 (IL-6), released into the culture media. After 24 h of exposure to Ag-15 nm nanoparticles, a significant inflammatory response was observed by the release of TNF-alpha, MIP-2, and IL-1beta. However, there was no detectable level of IL-6 upon exposure to silver nanoparticles. In summary, a size-dependent toxicity was produced by silver nanoparticles, and one predominant mechanism of toxicity was found to be largely mediated through oxidative stress.

Metal-based nanoparticles and their toxicity assessment

Nanoparticles (NPs) can potentially cause adverse effects on organ, tissue, cellular, subcellular, and protein levels due to their unusual physicochemical properties (e.g., small size, high surface area to volume ratio, chemical composition, crystallinity, electronic properties, surface structure reactivity and functional groups, inorganic or organic coatings, solubility, shape, and aggregation behavior). Metal NPs, in particular, have received increasing interest due to their widespread medical, consumer, industrial, and military applications. However, as particle size decreases, some metal-based NPs are showing increased toxicity, even if the same material is relatively inert in its bulk form (e.g., Ag, Au, and Cu). NPs also interact with proteins and enzymes within mammalian cells and they can interfere with the antioxidant defense mechanism leading to reactive oxygen species generation, the initiation of an inflammatory response and perturbation and destruction of the mitochondria causing apoptosis or necrosis. As a result, there are many challenges to overcome before we can determine if the benefits outweigh the risks associated with NPs.

Silver Nanoparticle Induced Blood-Brain Barrier Inflammation and Increased Permeability in Primary Rat Brain Microvessel Endothelial Cells

The current report examines the interactions of silver nanoparticles (Ag-NPs) with the cerebral microvasculature to identify the involvement of proinflammatory mediators that can increase blood-brain barrier (BBB) permeability. Primary rat brain microvessel endothelial cells (rBMEC) were isolated from adult Sprague-Dawley rats for an in vitro BBB model. The Ag-NPs were characterized by transmission electron microscopy (TEM), dynamic light scattering, and laser Doppler velocimetry. The cellular accumulation, cytotoxicity (6.25-50 μg/cm(3)) and potential proinflammatory mediators (interleukin [IL]-1β, IL-2, tumor necrosis factor [TNF] α, and prostaglandin E(2) [PGE(2)]) of Ag-NPs (25, 40, or 80 nm) were determined spectrophotometrically, cell proliferation assay (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide) and ELISA. The results show Ag-NPs-induced cytotoxic responses at lower concentrations for 25 and 40 nm when compared with 80-nm Ag-NPs. The proinflammatory responses in this study demonstrate both Ag-NPs size and time-dependent profiles, with IL-1B preceding both TNF and PGE(2) for 25 nm. However, larger Ag-NPs (40 and 80 nm) induced significant TNF responses at 4 and 8 h, with no observable PGE(2) response. The increased fluorescein transport observed in this study clearly indicates size-dependent increases in BBB permeability correlated with the severity of immunotoxicity. Together, these data clearly demonstrate that larger Ag-NPs (80 nm) had significantly less effect on rBMEC, whereas the smaller particles induced significant effects on all the end points at lower concentrations and/or shorter times. Further, this study suggests that Ag-NPs may interact with the cerebral microvasculature producing a proinflammatory cascade, if left unchecked; these events may further induce brain inflammation and neurotoxicity.

Silver nanoparticles disrupt GDNF/Fyn kinase signaling in spermatogonial stem cells

Silver nanoparticles (Ag-NPs) are being utilized in an increasing number of fields and are components of antibacterial coatings, antistatic materials, superconductors, and biosensors. A number of reports have now described the toxic effects of silver nanoparticles on somatic cells; however, no study has examined their effects on the germ line at the molecular level. Spermatogenesis is a complex biological process that is particularly sensitive to environmental insults. Many chemicals, including ultrafine particles, have a negative effect on the germ line, either by directly affecting the germ cells or by indirectly acting on the somatic cells of the testis. In the present study, we have assessed the impact of different doses of Ag-NPs, as well as their size and biocompatible coating, on the proliferation of mouse spermatogonial stem cells (SSCs), which are at the origin of the germ line in the adult testis. At concentrations >OR= 10 microg/ml, Ag-NPs induced a significant decline in SSCs proliferation, which was also dependent on their size and coating. At the concentration of 10 microg/ml, reactive oxygen species production and/or apoptosis did not seem to play a major role; therefore, we explored other mechanisms to explain the decrease in cell proliferation. Because glial cell line-derived neurotrophic factor (GDNF) is vital for SSC self-renewal in vitro and in vivo, we evaluated the effects of Ag-NPs on GDNF-mediated signaling in these cells. Although the nanoparticles did not reduce GDNF binding or Ret receptor activity, our data revealed that already at a concentration of 10 microg/ml, silver nanoparticles specifically interact with Fyn kinase downstream of Ret and impair SSC proliferation in vitro. In addition, we demonstrated that the particle coating was degraded upon interaction with the intracellular microenvironment, reducing biocompatibility.

Assessment of Metal Nanoparticle Agglomeration, Uptake, and Interaction Using High-Illuminating System

In the present study, an ultrahigh-resolution system was applied as a simple and convenient technique to characterize the extent of metal nanoparticle agglomeration in solution and to visualize nanoparticle agglomeration, uptake, and surface interaction in three cell phenotypes under normal culture conditions. The experimental results demonstrated that silver (25, 80, 130 nm); aluminum (80 nm); and manganese (40 nm) particles and agglomerates were effectively internalized by rat liver cells (BRL 3A), rat alveolar macrophages (MACs), and rat neuroendocrine cells (PC-12). Individual and agglomerated nanoparticles were observed within the cells and agglomerates were observed on the cell surface membranes. The particles were initially dispersed in aqueous or physiological balanced salt solutions and agglomeration was observed using the Ultra Resolution Imaging (URI) system. Different methods, such as sonication and addition of surfactant (0.1% sodium dodecyl sulfate [SDS]) reduced agglomeration. Due to effects of SDS itself on cell viability, the surfactant could not be directly applied during cell exposure. Therefore, following addition of 0.1% SDS, the particles were washed twice with ultrapure water, which reduced agglomeration even further. Reducing the agglomeration of the nanoparticles is important for studying their uptake and in applications that benefit from individual nanoparticles such as diagnostics. In summary, this study demonstrates a simple technique to characterize the extent of nanoparticle agglomeration in solution and visualize nanoparticle (40 nm and larger) uptake and interaction with cells. Additionally, an example application of nanoparticle labeling onto the surface and neurite extensions of murine neuroblastoma cells (N2A) is presented as a potential imaging tool.

Preparation of cells for assessing ultrastructural localization of nanoparticles with transmission electron microscopy

We describe the use of transmission electron microscopy (TEM) for cellular ultrastructural examination of nanoparticle (NP)-exposed biomaterials. Preparation and imaging of electron-transparent thin cell sections with TEM provides excellent spatial resolution (∼1 nm), which is required to track these elusive materials. This protocol provides a step-by-step method for the mass-basis dosing of cultured cells with NPs, and the process of fixing, dehydrating, staining, resin embedding, ultramicrotome sectioning and subsequently visualizing NP uptake and translocation to specific intracellular locations with TEM. In order to avoid potential artifacts, some technical challenges are addressed. Based on our results, this procedure can be used to elucidate the intracellular fate of NPs, facilitating the development of biosensors and therapeutics, and provide a critical component for understanding NP toxicity. This protocol takes ∼1 week.

Interaction of silver nanoparticles with Tacaribe virus

BackgroundSilver nanoparticles possess many unique properties that make them attractive for use in biological applications. Recently they received attention when it was shown that 10 nm silver nanoparticles were bactericidal, which is promising in light of the growing number of antibiotic resistant bacteria. An area that has been largely unexplored is the interaction of nanomaterials with viruses and the possible use of silver nanoparticles as an antiviral agent.ResultsThis research focuses on evaluating the interaction of silver nanoparticles with a New World arenavirus, Tacaribe virus, to determine if they influence viral replication. Surprisingly exposing the virus to silver nanoparticles prior to infection actually facilitated virus uptake into the host cells, but the silver-treated virus had a significant reduction in viral RNA production and progeny virus release, which indicates that silver nanoparticles are capable of inhibiting arenavirus infection in vitro. The inhibition of viral replication must occur during early replication since although pre-infection treatment with silver nanoparticles is very effective, the post-infection addition of silver nanoparticles is only effective if administered within the first 2-4 hours of virus replication.ConclusionsSilver nanoparticles are capable of inhibiting a prototype arenavirus at non-toxic concentrations and effectively inhibit arenavirus replication when administered prior to viral infection or early after initial virus exposure. This suggests that the mode of action of viral neutralization by silver nanoparticles occurs during the early phases of viral replication.

Uptake of gold nanoparticles in murine macrophage cells without cytotoxicity or production of pro-inflammatory mediators

Abstract More information characterizing the biological responses to nanoparticles is needed to allow the U.S. Food and Drug Administration to evaluate the safety and effectiveness of products with nano-scale components. The potential cytotoxicity and inflammatory responses of Au NPs (60 nm, NIST standard reference materials) were investigated in murine macrophages. Cytotoxicity was evaluated by MTT and LDH assays. Cytokines (IL-6, TNF-α), nitric oxide, and ROS were assayed to assess inflammatory responses. Morphological appearance and localization of particles were examined by high resolution illumination microscopy, transmission electron microscopy (TEM), and scanning TEM coupled with EDX spectroscopy. Results showed no cytotoxicity and no elevated production of proinflammatory mediators; however, imaging analyses demonstrated cellular uptake of Au NPs and localization within intracellular vacuoles. These results suggest that 60 nm Au NPs, under the exposure conditions tested, are not cytotoxic, nor elicit pro-inflammatory responses. The localization of Au NPs in intracellular vacuoles suggests endosomal containment and an uptake mechanism involving endocytosis.

Effects of copper nanoparticles on rat cerebral microvessel endothelial cells

AIM The purpose of the current study was to determine whether copper nanoparticles (Cu-NPs) can induce the release of proinflammatory mediators that influence the restrictive characteristics of the blood-brain barrier. MATERIAL & METHODS Confluent rat brain microvessel endothelial cells (rBMECs) were treated with well-characterized Cu-NPs (40 or 60 nm). Cytotoxicity of the Cu-NPs was evaluated by cell proliferation assay (1.5-50 µg/ml). The extracellular concentrations of proinflammatory mediators (IL-1β, IL-2, TNF-α and prostaglandin E(2)) were evaluated by ELISA. RESULTS The exposure of Cu-NPs at low concentrations increases cellular proliferation of rBMECs, by contrast, high concentrations induce toxicity. Prostaglandin E(2) release was significantly increased (threefold; 8 h) for Cu-NPs (40 and 60 nm). The extracellular levels of both TNF-α and IL-1β were significantly elevated following exposure to Cu-NPs. The P-apparent ratio, as an indicator of increased permeability of rBMEC was approximately twofold for Cu-NPs (40 and 60 nm). CONCLUSION These data suggest that Cu-NPs can induce rBMEC, proliferation at low concentrations and/or induce blood-brain barrier toxicity and potential neurotoxicity at high concentrations.

Brain microvessel endothelial cells responses to gold nanoparticles: In vitro pro-inflammatory mediators and permeability

Abstract This report examined blood-brain barrier (BBB) related proinflammatory mediators and permeability changes in response to various sized gold nanoparticles (Au-NPs) (3, 5, 7, 10, 30 and 60 nm) in vitro using primary rat brain microvessel endothelial cells (rBMEC). The Au-NPs were characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS) and laser Doppler velocimetry (LDV). The accumulation of Au-NPs was determined spectrophotometrically. The rBMEC cytotoxicity of Au-NPs was evaluated by cell proliferation assay (XTT) (concentration range 0.24–15.63 μg/cm2, for 24 h). The time-dependent changes (0, 2, 4 and 8 h) of several proinflammatory mediators (IL-1β, IL-2, TNFα and PGE2) were evaluated by ELISA. The smaller Au-NPs (3–7 nm) showed higher rBMEC accumulation compared to larger Au-NPs (10–60 nm), while only moderate decreased cell viability was observed with small Au-NPs (3 nm) at high concentrations (≥ 7.8 μg/cm2). Even though slight changes in cell viability were observed with small Au-NPs, the basal levels of the various proinflammatory mediators remained unchanged with all treatments except LPS (positive control). rBMEC morphology appeared unaffected 24 h after exposure to Au-NPs with only mild changes in fluorescein permeability indicating BBB integrity was unaltered. Together, these data suggest the responses of the cerebral microvasculature to Au-NPs have a significant relationship with the Au-NPs unique size-dependent physiochemical properties.