Extinction-to-Absorption Ratio for Sensitive Determination of the Size and Dielectric Function of Gold Nanoparticles.

Gold nanoparticles (AuNPs) have become an essential tool for a variety of fields across the biological, physical, and chemical sciences. The characterization of AuNPs by UV-vis spectroscopy is simple and commonly used but remains prone to error because of size and shape polydispersity and uncertainties in the dielectric function. We here propose and demonstrate a method to significantly improve this routine characterization technique by measuring not only the extinction but also the absorption spectrum. Specifically, we show that by considering the ratio of the extinction to absorption spectra, denoted η, we are able to determine the volume of AuNPs with a significant increase in accuracy compared to the UV-vis extinction method. We also prove an important property of η: it is independent of particle shape within the quasi-static/dipolar approximation, typically for particle sizes up to 100 nm. This shape independence results in very strong constraints for the theoretical predictions to agree with the experiments. We show that the spectral shape of η can therefore be used to discriminate between different proposed data sets for the dielectric function of gold, a long-standing challenge in plasmonics research.

Bioactivity of nanosilver in Caenorhabditis elegans: Effects of size, coat, and shape.

The in vivo toxicity to eukaryotes of nanosilver (AgNP) spheres and plates in two sizes each was assessed using the simple model organism Caenorhabditis elegans. For each shape, smaller AgNP size correlated with higher toxicity, as indicated by reduced larval growth. Smaller size also correlated with significant increases in silver uptake for silver nanospheres. Citrate coated silver spheres of 20 nm diameter induced an innate immune response that increased or held steady over 24 h, while regulation of genes involved in metal metabolism peaked at 4 h and subsequently decreased. For AgNP spheres, coating altered bioactivity, with a toxicity ranking of polyethylene glycol (PEG) > polyvinylpyrrolidone (PVP) ≅ branched polyethyleneimine (BPEI) > citrate, but silver uptake ranking of PEG > PVP > citrate > BPEI. Our findings in C. elegans correlate well with findings in rodents for AgNP size vs. uptake and toxicity, as well as for induction of immune effectors, while using methods that are faster and far less expensive, supporting the use of C. elegans as an alternative model for early toxicity screening.

Protein binding modulates the cellular uptake of silver nanoparticles into human cells: implications for in vitro to in vivo extrapolations?

Nanoparticles (NP) absorbed in the body will come in contact with blood proteins and form NP/protein complexes termed protein coronas, which may modulate NP cellular uptake. This study quantitated human epidermal keratinocyte (HEK) uptake of silver (Ag) NP complexed to different human serum proteins. Prior to HEK dosing, AgNP (20nm and 110nm citrate BioPure™; 40nm and 120nm silica-coated) were preincubated for 2h at 37°C without (control) or with physiological levels of albumin (44mg/ml), IgG (14.5mg/ml) or transferrin (3mg/ml) to form protein-complexed NP. HEK were exposed to the protein incubated AgNP for 3h, rinsed and incubated for 24h, rinsed in buffer and lysed. Ag was assayed by inductively-coupled plasma optical emission spectrometry. Uptake of Ag in HEK was <4.1% of applied dose with proteins suppressing citrate, but not silica coated Ag uptake. IgG exposure dramatically reduced 110nm citrate AgNP uptake. In contrast, greatest uptake of 20nm silica AgNP was seen with IgG, while 110nm silica AgNP showed minimal protein effects. Electron microscopy confirmed cellular uptake of all NP but showed differences in the appearance and agglomeration state of the NP within HEK vacuoles. This work suggests that NP association with different serum proteins, purportedly forming different protein coronas, significantly modulates Ag uptake into HEK compared to native NP uptake, suggesting caution in extrapolating in vitro uptake data to predict behavior in vivo where the nature of the protein corona may determine patterns of cellular uptake, and thus biodistribution, biological activity and toxicity.

In vitro biodistribution of silver nanoparticles in isolated perfused porcine skin flaps.

Nanomaterials increasingly are playing a role in society for uses ranging from biomedicine to microelectronics; however, pharmacokinetic studies, which will be necessary for human health risk assessments, are limited. Currently the most widely used nanoparticle in consumer products is silver (Ag). The objective of the present study was to quantify the local biodistribution of two types of Ag nanoparticles, Ag-citrate and Ag-silica, in the isolated perfused porcine skin flap (IPPSF). IPPSFs were perfused for 4 h with 0.84 µg ml(-1) Ag-citrate or 0.48 µg ml(-1) Ag-silica followed by a 4-h perfusion with media only during a washout phase. Arterial and venous concentrations of Ag were measured in the media by inductively coupled plasma optical emission spectrometry (ICP-OES). Venous concentrations of Ag for both types of nanoparticles were best fit with a two compartment model. The normalized volumes of distribution estimated from the noncompartmental analysis of the venous concentrations indicated distribution of Ag greater than the vascular space; however, because total Ag was measured, the extravascular distribution could be attributed to diffusion of Ag ions. The estimated clearance for both types of Ag nanoparticles was 1 ml min(-1) , which was equal to the flap perfusion rate, indicating no detectable elimination of Ag from the system. Four hours after infusion of the Ag nanoparticles, the recovery of Ag in the venous effluent was 90 ± 5.0% and 87 ± 22% of the infused Ag for Ag-citrate and Ag-silica, respectively.

Silver nanoparticles do not influence stem cell differentiation but cause minimal toxicity.

AIMS: To evaluate the toxicity and cellular uptake of both undifferentiated and differentiated human adipose-derived stem cells (hASCs) exposed to silver nanoparticles (Ag-NPs), and to assess their effect on hASC differentiation. MATERIALS & METHODS: hASC were exposed to 10- or 20-nm Ag-NPs at concentrations of 0.1, 1.0, 10.0, 50.0 and 100.0 µg/ml either before or after differentiation down the adipogenic or osteogenic pathways. RESULTS: Exposure of hASC to either 10- or 20-nm Ag-NPs resulted in no significant cytotoxicity to hASC, and minimal dose-dependent toxicity to adipogenic and osteogenic cells at 10 µg/ml. Each of the hASC, adipogenic and osteogenic cells showed cellular uptake of both 10- and 20-nm Ag-NPs, without causing significant ultrastructural alterations. Exposure to 10- or 20-nm Ag-NPs did not influence the differentiation of the cells, and at antimicrobial concentrations of Ag-NPs resulted in a minimal decrease in viability. CONCLUSION: The biocompatibility of Ag-NPs with both undifferentiated and differentiated hASC establishes their suitability for incorporation into tissue-engineered graft scaffolds, for the prevention of bacterial contamination upon implantation.

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

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-1ß, 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.

Leveraging nanoscale plasmonic modes to achieve reproducible enhancement of light.

The strongly enhanced and localized optical fields that occur within the gaps between metallic nanostructures can be leveraged for a wide range of functionality in nanophotonic and optical metamaterial applications. Here, we introduce a means of precise control over these nanoscale gaps through the application of a molecular spacer layer that is self-assembled onto a gold film, upon which gold nanoparticles (NPs) are deposited electrostatically. Simulations using a three-dimensional finite element model and measurements from single NPs confirm that the gaps formed by this process, between the NP and the gold film, are highly reproducible transducers of surface-enhanced resonant Raman scattering. With a spacer layer of roughly 1.6 nm, all NPs exhibit a strong Raman signal that decays rapidly as the spacer layer is increased.

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]-1ß, 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.

Evaluation of silver nanoparticle toxicity in skin in vivo and keratinocytes in vitro.

INTRODUCTION: Products using the antimicrobial properties of silver nanoparticles (Ag-nps) may be found in health and consumer products that routinely contact skin. OBJECTIVES: This study was designed to assess the potential cytotoxicity of Ag-nps in human epidermal keratinocytes (HEKs) and their inflammatory and penetrating potential into porcine skin in vivo. MATERIALS AND METHODS: We used eight different Ag-nps in this study [unwashed/uncoated (20, 50, and 80 nm particle diameter), washed/uncoated (20, 50, and 80 nm), and carbon-coated (25 and 35 nm)]. Skin was dosed topically for 14 consecutive days. HEK viability was assessed by MTT, alamarBlue (aB), and CellTiter 96 AQueous One (96AQ). Release of the proinflammatory mediators interleukin (IL)-1beta, IL-6, IL-8, IL-10, and tumor necrosis factor-alpha (TNF-alpha) were measured. RESULTS: The effect of the unwashed Ag-nps on HEK viability after a 24-hr exposure indicated a significant dose-dependent decrease (p < 0.05) at 0.34 microg/mL with aB and 96AQ and at 1.7 microg/mL with MTT. However, both the washed Ag-nps and carbon-coated Ag-nps showed no significant decrease in viability at any concentration assessed by any of the three assays. For each of the unwashed Ag-nps, we noted a significant increase (p < 0.05) in IL-1beta, IL-6, IL-8, and TNF-alpha concentrations. We observed localization of all Ag-nps in cytoplasmic vacuoles of HEKs. Macroscopic observations showed no gross irritation in porcine skin, whereas microscopic and ultrastructural observations showed areas of focal inflammation and localization of Ag-nps on the surface and in the upper stratum corneum layers of the skin. CONCLUSION: This study provides a better understanding Ag-nps safety in vitro as well as in vivo and a basis for occupational and risk assessment. Ag-nps are nontoxic when dosed in washed Ag-nps solutions or carbon coated.

Interactions of aluminum nanoparticles with human epidermal keratinocytes.

Aluminum nanoparticles (Al NP) have been used in applications as diverse as drug delivery, material surface coatings and an ingredient for solid rocket fuel in military explosives and artillery. Although Al NP are used in many civilian and military applications, the health and safety implications of these nanosize particles are not known. To understand the interactions and biological activity of Al NP in human cells, cultured human neonatal epidermal keratinocytes (HEK) were exposed for 24 h to 50 and 80 nm Al NP in concentrations from 4.0 to 0.0004 mg ml(-1) to assess the cytotoxicity and inflammatory potential. UV-Vis measurements and nanoparticle controls revealed that the Al NP interact with the assay dyes. Viability did not decrease in HEK exposed to both the 50 and the 80 nm Al NP at all treatment concentrations with MTT, CellTiter 96 AQueous One (96 AQ) and alamar Blue (aB) viability assays. The 96 AQ and aB assays interact with the Al NP less than MTT, and proved to be the best assays to use with these Al NP. TEM depicted Al NP localized within the cytoplasmic vacuoles of the cells. Cytokine data was variable, indicating possible nanoparticle interactions with the cytokine assays. These studies illustrate the difficulties involved in assessing the biological safety of nanomaterials such as Al NP due to media- and temperature-dependent particle agglomeration and nanoparticle interactions with biomarkers of cytotoxicity.

Nanoparticle characteristics affecting environmental fate and transport through soil.

Nanoparticles are being used in broad range of applications; therefore, these materials probably will enter the environment during their life cycle. The objective of the present study is to identify changes in properties of nanoparticles released into the environment with a case study on aluminum nanoparticles. Aluminum nanoparticles commonly are used in energetic formulations and may be released into the environment during their handling and use. To evaluate the transport of aluminum nanoparticles, it is necessary not only to understand the properties of the aluminum in its initial state but also to determine how the nanoparticle properties will change when exposed to relevant environmental conditions. Transport measurements were conducted with a soil-column system that delivers a constant upflow of a suspension of nanoparticles to a soil column and monitors the concentration, size, agglomeration state, and charge of the particles in the eluent. The type of solution and surface functionalization had a marked effect on the charge, stability, and agglomeration state of the nanoparticles, which in turn impacted transport through the receiving matrix. Transport also is dependent on the size of the nanoparticles, although it is the agglomerate size, not the primary size, that is correlated with transportability. Electrostatically induced binding events of positively charged aluminum nanoparticles to the soil matrix were greater than those for negatively charged aluminum nanoparticles. Many factors influence the transport of nanoparticles in the environment, but size, charge, and agglomeration rate of nanoparticles in the transport medium are predictive of nanoparticle mobility in soil.

Base pair mismatch recognition using plasmon resonant particle labels.

We demonstrate the use of silver plasmon resonant particles (PRPs), as reporter labels, in a microarray-based DNA hybridization assay in which we screen for a known polymorphic site in the breast cancer gene BRCA1. PRPs (40-100 nm in diameter) image as diffraction-limited points of colored light in a standard microscope equipped with dark-field illumination, and can be individually identified and discriminated against background scatter. Rather than overall intensity, the number of PRPs counted in a CCD image by a software algorithm serves as the signal in these assays. In a typical PRP hybridization assay, we achieve a detection sensitivity that is approximately 60 x greater than that achieved by using fluorescent labels. We conclude that single particle counting is robust, generally applicable to a wide variety of assay platforms, and can be integrated into low-cost and quantitative detection systems for single nucleotide polymorphism analysis.