Characterization of Thermoplastic Polyurethane (TPU) and Ag-Carbon Black TPU Nanocomposite for Potential Application in Additive Manufacturing.

Electromechanical, adhesion, and viscoelastic properties of polymers and polymer nanocomposites (PNCs) are of interest for additive manufacturing (AM) and flexible electronics. Development/optimization of inks for AM is complex, expensive, and substrate/interface dependent. This study investigates properties of free standing films of a thermoplastic polyurethane (TPU) polymer and an Ag⁻carbon black (Ag-CB) TPU PNC in a lightly loaded low strain compression contact as a rough measure of their suitability for AM. The TPU exhibited high hysteresis and a large viscoelastic response, and sufficient dwell time was needed for polymer chain relaxation and measurable adhesion. A new discovery is that large enough contact area is needed to allow longer time constant polymer ordering in the contact that led to higher adhesion and better performance/reliability. This has previously unknown implications for interface size relative to polymer chain length in AM design. The standard linear model was found to be a good fit for the viscoelastic behavior of the TPU. The PNC exhibited no adhesion (new result), low electrical resistance, and relatively small viscoelastic response. This implies potential for AM electrical trace as well as switch applications.

Assessment of Carbon- and Metal-Based Nanoparticle DNA Damage with Microfluidic Electrophoretic Separation Technology.

In this study, we examined the feasibility of extracting DNA from whole cell lysates exposed to nanoparticles using two different methodologies for evaluation of fragmentation with microfluidic electrophoretic separation. Human lung macrophages were exposed to five different carbon- and metal-based nanoparticles at two different time points (2 h, 24 h) and two different doses (5 µg/ml, 100 µg/ml). The primary difference in the banding patterns after 2 h of nanoparticle exposure is more DNA fragmentation at the higher NP concentration when examining cells exposed to nanoparticles of the same composition. However, higher doses of carbon and silver nanoparticles at both short and long dosing periods can contribute to erroneous or incomplete data with this technique. Also comparing DNA isolation methodologies, we recommend the centrifugation extraction technique, which provides more consistent banding patterns in the control samples compared to the spooling technique. Here we demonstrate that multi-walled carbon nanotubes, 15 nm silver nanoparticles and the positive control cadmium oxide cause similar DNA fragmentation at the short time point of 2 h with the centrifugation extraction technique. Therefore, the results of these studies contribute to elucidating the relationship between nanoparticle physicochemical properties and DNA fragmentation results while providing the pros and cons of altering the DNA isolation methodology. Overall, this technique provides a high throughput way to analyze subcellular alterations in DNA profiles of cells exposed to nanomaterials to aid in understanding the consequences of exposure and mechanistic effects. Future studies in microfluidic electrophoretic separation technologies should be investigated to determine the utility of protein or other assays applicable to cellular systems exposed to nanoparticles.

Organophosphate vapor detection on gold electrodes using peptide nanotubes.

Peptide nanotubes (PNTs) encapsulating horseradish peroxidase and surface coated with acetylcholinesterase (AChE) were attached to gold screen printed electrodes to construct a novel gas phase organophosphate (OP) biosensor. When the sensor with the AChE enzyme is put in contact with acetylthiocholine (ATCh), the ATCh is hydrolyzed to produce thiocholine, which is then oxidized by horseradish peroxidase (HRP). Direct electron transfer between HRP and electrode is achieved through PNTs. The signal produced by the electron transfer is measured with cyclic voltammetry (CV). The presence of an OP compound inhibits this signal by binding with the AChE enzyme. In this study, gas phase malathion was used as a model OP due to the fact that it displays the identical binding mechanism with acetylcholinesterase (AChE) as its more potent counterparts such as sarin and VX, but has low toxicity, making it more practical and safer to handle. The CV signal was proportionally inhibited by malathion vapor concentrations as low as 12 ppbv. Depending on the method used in their preparation, the electrodes maintained their activity for up to 45 days. This research demonstrates the potential of applying nano-modified biosensors for the detection of low levels of OP vapor, an important development in countering weaponized organophosphate nerve agents and detecting commercially-used OP pesticides.

Magnesium cluster film synthesis by helium nanodroplets.

Atomic and molecular clusters are a unique class of substances with properties that differ greatly from those of the bulk or single atoms due to changes in surface to volume ratio and finite size effects. Here, we demonstrate the ability to create cluster matter films using helium droplet mediated cluster assembly and deposition, a recently developed methodology that condenses atoms or molecules within liquid helium droplets and then gently deposits them onto a surface. In this work, we examine magnesium nanocluster films, which exhibit growth behavior comparable to low-energy cluster beam methods, and demonstrate physical properties and morphology dependent on helium droplet size.

Assessment of cytotoxicity of carbon nanoparticles using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cell viability assay.

Ever since the discovery of carbon nanotubes, there has been an increasing interest in technologies that rely upon these incredibly small particles for their unique properties. However, assessment of their biological consequences has been riddled with assay limitations. Here, we describe application of a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium cell viability assay to study cytotoxicity of various carbon-based nanomaterials on cells and discuss some pitfalls of this method.

Toxicity testing of nanomaterials.

The large-scale production and consumer exposure to a variety of nanotechnology innovations has stirred interest concerning the health consequences of human exposure to nanomaterials. In order to investigate these questions, in vitro systems are used to rapidly and inexpensively predict the effects of nanomaterials at the cellular level. Recent advances in the toxicity testing of nanomaterials are beginning to shed light on the characteristics, uptake and mechanisms of their toxicity in a variety of cell types. Once the nanomaterials have been satisfactorily characterized, the evaluation of their interactions with cells can be studied with microscopy and biochemical assays. The combination of viability testing, observation of morphology and the generation of oxidative stress provide clues to the mechanisms of nanomaterial toxicity. The results of these studies are used to better understand how the size, chemical composition, shape and functionalization may contribute to their toxicity. This chapter will introduce the reader to the impact of nanomaterials in the workplace and marketplace with an emphasis on carbon-based and metal-based nanomaterials, which are most commonly encountered. While most purified carbon nanomaterials were nontoxic to many cell lines, many metal nanoparticles (e.g., silver or manganese) were more toxic. Other side- effects of nanoparticle interactions with cells can also occur, such as increased branching and dopamine depletion. Further investigation into the characteristics, uptake and mechanisms of nanomaterial toxicity will continue to elucidate this fascinating and rapidly growing area of science.

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-1ß, 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-1ß 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.

Fundamental examination of nanoparticle heating kinetics upon near infrared (NIR) irradiation.

Near infrared (NIR) light, which spans wavelengths from ~700-1100 nm holds particular promise in bionanotechnology-enabled applications because both NIR light and nanoparticles (NPs) have the potential for remote activation leading to exquisite localization and targeting scenarios. In this study, aqueous solutions of carbon and metal-based NPs (carbon black, single-walled carbon nanotubes, silver nanoparticles and copper nanoparticles) were exposed to continuous NIR laser (λ = 1064 nm) irradiation at powers of 2.2W and 4.5W. The differential heating of bulk aqueous suspension of NPs with varying physicochemical properties revealed maximum temperatures of 67 °C with visible evidence of condensation and bubble formation. The basis of the NP heating is due to the strong intrinsic optical absorbance in the NIR spectral window and the transduction of this NIR photon energy into thermal energy. In this regard, UV-vis measurements can accurately predict NP heating kinetics prior to NIR irradiation. Further, a uniform thermodynamic heating model demonstrates close agreement with the experimental data for the low NIR-absorbing NPs. However, the uniform thermodynamic heating model used in this study does not accurately portray the energy release upon localized NP heating because of bubble formation for the highly absorbing NPs. Therefore, this study reveals the differential heating kinetics of NPs excited with NIR with implications in the development of novel NIR-NP-based systems.

Surface charge of gold nanoparticles mediates mechanism of toxicity.

Recently gold nanoparticles (Au NPs) have shown promising biological and military applications due to their unique electronic and optical properties. However, little is known about their biocompatibility in the event that they come into contact with a biological system. In the present study, we have investigated whether modulating the surface charge of 1.5 nm Au NPs induced changes in cellular morphology, mitochondrial function, mitochondrial membrane potential (MMP), intracellular calcium levels, DNA damage-related gene expression, and of p53 and caspase-3 expression levels after exposure in a human keratinocyte cell line (HaCaT). The evaluation of three different Au NPs (positively charged, neutral, and negatively charged) showed that cell morphology was disrupted by all three NPs and that they demonstrated a dose-dependent toxicity; the charged Au NPs displayed toxicity as low as 10 µg ml(-1) and the neutral at 25 µg ml(-1). Furthermore, there was significant mitochondrial stress (decreases in MMP and intracellular Ca2+ levels) following exposure to the charged Au NPs, but not the neutral Au NPs. In addition to the differences observed in the MMP and Ca2+ levels, up or down regulation of DNA damage related gene expression suggested a differential cell death mechanism based on whether or not the Au NPs were charged or neutral. Additionally, increased nuclear localization of p53 and caspase-3 expression was observed in cells exposed to the charged Au NPs, while the neutral Au NPs caused an increase in both nuclear and cytoplasmic p53 expression. In conclusion, these results indicate that surface charge is a major determinant of how Au NPs impact cellular processes, with the charged NPs inducing cell death through apoptosis and neutral NPs leading to necrosis.

Temporal and mechanistic tracking of cellular uptake dynamics with novel surface fluorophore-bound nanodiamonds.

Nanoparticles (NPs) offer promise for a multitude of biological applications including cellular probes at the bio-interface for targeted delivery of anticancer substances, Raman and fluorescent-based imaging and directed cell growth. Nanodiamonds (NDs), in particular, have several advantages compared to other carbon-based nanomaterials - including a rich surface chemistry useful for chemical conjugation, high biocompatibility with little reactive oxygen species (ROS) generation, physical and chemical stability that affords sterilization, high surface area to volume ratio, transparency and a high index of refraction. The visualization of ND internalization into cells is possible via photoluminescence, which is produced by direct dye conjugation or high energy irradiation that creates nitrogen vacancy centers. Here, we explore the kinetics and mechanisms involved in the intracellular uptake and localization of novel, highly-stable, fluorophore-conjugated NDs. Examination in a neuronal cell line (N2A) shows ND localization to early endosomes and lysosomes with eventual release into the cytoplasm. The addition of endocytosis and exocytosis inhibitors allows for diminished uptake and increased accumulation, respectively, which further corroborates cellular behavior in response to NDs. Ultimately, the ability of the NDs to travel throughout cellular compartments of varying pH without degradation of the surface-conjugated fluorophore or alteration of cell viability over extended periods of time is promising for their use in biomedical applications as stable, biocompatible, fluorescent probes.

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

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.

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.

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]-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.

Interaction of silver nanoparticles with Tacaribe virus.

BACKGROUND: Silver 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. RESULTS: This 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. CONCLUSIONS: Silver 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.

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 (approximately 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 approximately 1 week.

Can silver nanoparticles be useful as potential biological labels?

Silver (Ag) nanoparticles have unique plasmon-resonant optical scattering properties that are finding use in nanomedical applications such as signal enhancers, optical sensors, and biomarkers. In this study, we examined the chemical and biological properties of Ag nanoparticles of similar sizes, but that differed primarily in their surface chemistry (hydrocarbon versus polysaccharide), in neuroblastoma cells for their potential use as biological labels. We observed strong optical labeling of the cells in a high illumination light microscopy system after 24 h of incubation due to the excitation of plasmon resonance by both types of Ag nanoparticle. Surface binding of both types of Ag nanoparticle to the plasma membrane of the cells was verified with scanning electron microscopy as well as the internalization and localization of the Ag nanoparticles into intracellular vacuoles in thin cell sections with transmission electron microscopy. However, the induction of reactive oxygen species (ROS), degradation of mitochondrial membrane integrity, disruption of the actin cytoskeleton, and reduction in proliferation after stimulation with nerve growth factor were found after incubation with Ag nanoparticles at concentrations of 25 µg ml(-1) or greater, with a more pronounced effect produced by the hydrocarbon-based Ag nanoparticles in most cases. Therefore, the use of Ag nanoparticles as potential biological labels, even if the surface is chemically modified with a biocompatible material, should be approached with caution.

Characterization of nanomaterial dispersion in solution prior to in vitro exposure using dynamic light scattering technique.

The need to characterize nanoparticles in solution before assessing the in vitro toxicity is a high priority. Particle size, size distribution, particle morphology, particle composition, surface area, surface chemistry, and particle reactivity in solution are important factors which need to be defined to accurately assess nanoparticle toxicity. Currently, there are no well-defined techniques for characterization of wet nanomaterials in aqueous or biological solutions. Previously reported nanoparticle characterization techniques in aqueous or biological solutions have consisted of the use of ultra-high illumination light microscopy and disc centrifuge sedimentation; however, these techniques are limited by the measurement size range. The current study focuses on characterizing a wide range of nanomaterials using dynamic light scattering (DLS) and transmission electron microscopy, including metals, metal oxides, and carbon-based materials, in water and cell culture media, with and without serum. Cell viability and cell morphology studies were conducted in conjunction with DLS experiments to evaluate toxicological effects from observed agglomeration changes in the presence or absence of serum in cell culture media. Observations of material-specific surface properties were also recorded. It was also necessary to characterize the impact of sonication, which is implemented to aid in particle dispersion and solution mixture. Additionally, a stock solution of nanomaterials used for toxicology studies was analyzed for changes in agglomeration and zeta potential of the material over time. In summary, our results demonstrate that many metal and metal oxide nanomaterials agglomerate in solution and that depending upon the solution particle agglomeration is either agitated or mitigated. Corresponding toxicity data revealed that the addition of serum to cell culture media can, in some cases, have a significant effect on particle toxicity possibly due to changes in agglomeration or surface chemistry. It was also observed that sonication slightly reduces agglomeration and has minimal effect on particle surface charge. Finally, the stock solution experienced significant changes in particle agglomeration and surface charge over time.

Cellular interaction of different forms of aluminum nanoparticles in rat alveolar macrophages.

Nanomaterials, with dimensions in the 1-100 nm range, possess numerous potential benefits to society. However, there is little characterization of their effects on biological systems, either within the environment or on human health. The present study examines cellular interaction of aluminum oxide and aluminum nanomaterials, including their effect on cell viability and cell phagocytosis, with reference to particle size and the particle's chemical composition. Experiments were performed to characterize initial in vitro cellular effects of rat alveolar macrophages (NR8383) after exposure to aluminum oxide nanoparticles (Al2O3-NP at 30 and 40 nm) and aluminum metal nanoparticles containing a 2-3 nm oxide coat (Al-NP at 50, 80, and 120 nm). Characterization of the nanomaterials, both as received and in situ, was performed using transmission electron microscopy (TEM), dynamic light scattering (DLS), laser Doppler velocimetry (LDV), and/or CytoViva150 Ultra Resolution Imaging (URI)). Particles showed significant agglomeration in cell exposure media using DLS and the URI as compared to primary particle size in TEM. Cell viability assay results indicate a marginal effect on macrophage viability after exposure to Al2O3-NP at doses of 100 microg/mL for 24 h continuous exposure. Al-NP produced significantly reduced viability after 24 h of continuous exposure with doses from 100 to 250 microg/mL. Cell phagocytotic ability was significantly hindered by exposure to 50, 80, or 120 nm Al-NP at 25 microg/mL for 24 h, but the same concentration (25 microg/mL) had no significant effect on the cellular viability. However, no significant effect on phagocytosis was observed with Al2O3-NP. In summary, these results show that Al-NP exhibit greater toxicity and more significantly diminish the phagocytotic ability of macrophages after 24 h of exposure when compared to Al2O3-NP.

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.