Galya Orr

Interlaboratory evaluation of in vitro cytotoxicity and inflammatory responses to engineered nanomaterials: the NIEHS Nano GO Consortium.

Background: Differences in interlaboratory research protocols contribute to the conflicting data in the literature regarding engineered nanomaterial (ENM) bioactivity. Objectives: Grantees of a National Institute of Health Sciences (NIEHS)-funded consortium program performed two phases of in vitro testing with selected ENMs in an effort to identify and minimize sources of variability. Methods: Consortium program participants (CPPs) conducted ENM bioactivity evaluations on zinc oxide (ZnO), three forms of titanium dioxide (TiO2), and three forms of multiwalled carbon nanotubes (MWCNTs). In addition, CPPs performed bioassays using three mammalian cell lines (BEAS-2B, RLE-6TN, and THP-1) selected in order to cover two different species (rat and human), two different lung epithelial cells (alveolar type II and bronchial epithelial cells), and two different cell types (epithelial cells and macrophages). CPPs also measured cytotoxicity in all cell types while measuring inflammasome activation [interleukin-1β (IL-1β) release] using only THP-1 cells. Results: The overall in vitro toxicity profiles of ENM were as follows: ZnO was cytotoxic to all cell types at ≥ 50 μg/mL, but did not induce IL-1β. TiO2 was not cytotoxic except for the nanobelt form, which was cytotoxic and induced significant IL-1β production in THP-1 cells. MWCNTs did not produce cytotoxicity, but stimulated lower levels of IL-1β production in THP-1 cells, with the original MWCNT producing the most IL-1β. Conclusions: The results provide justification for the inclusion of mechanism-linked bioactivity assays along with traditional cytotoxicity assays for in vitro screening. In addition, the results suggest that conducting studies with multiple relevant cell types to avoid false-negative outcomes is critical for accurate evaluation of ENM bioactivity.

Probing Conformational Changes of Gramicidin Ion Channels by Single-Molecule Patch-Clamp Fluorescence Microscopy

Complex conformational changes influence and regulate the dynamics of ion channels. Such conformational changes are stochastic and often inhomogeneous, which makes it extremely difficult, if not impossible, to characterize them by ensemble-averaged experiments or by single-channel recordings of the electric current that report the open-closed events but do not specifically probe the associated conformational changes. Here, we report our studies on ion channel conformational changes using a new approach, patch-clamp fluorescence microscopy, which simultaneously combines single-molecule fluorescence spectroscopy and single-channel current recordings to probe the open-closed transitions and the conformational dynamics of individual ion channels. We demonstrate patch-clamp fluorescence microscopy by measuring gramicidin ion channel conformational changes in a lipid bilayer formed at a patch-clamp micropipette tip under a buffer solution. By measuring single-pair fluorescence resonance energy transfer and fluorescence self-quenching from dye-labeled gramicidin channels, we observed that the efficiency of single-pair fluorescence resonance energy transfer and self-quenching is widely distributed, which reflects a broad distribution of conformations. Our results strongly suggest a hitherto undetectable correlation between the multiple conformational states of the gramicidin channel and its closed and open states in a lipid bilayer.

Enzyme-Directed Assembly of Nanoparticles in Tumors Monitored by in Vivo Whole Animal Imaging and ex Vivo Super-Resolution Fluorescence Imaging

Matrix metalloproteinase enzymes, overexpressed in HT-1080 human fibrocarcinoma tumors, were used to guide the accumulation and retention of an enzyme-responsive nanoparticle in a xenograft mouse model. The nanoparticles were prepared as micelles from amphiphilic block copolymers bearing a simple hydrophobic block and a hydrophilic peptide brush. The polymers were end-labeled with Alexa Fluor 647 dyes leading to the formation of labeled micelles upon dialysis of the polymers from DMSO/DMF to aqueous buffer. This dye-labeling strategy allowed the presence of the retained material to be visualized via whole animal imaging in vivo and in ex vivo organ analysis following intratumoral injection into HT-1080 xenograft tumors. We propose that the material is retained by virtue of an enzyme-induced accumulation process whereby particles change morphology from 20 nm spherical micelles to micrometer-scale aggregates, kinetically trapping them within the tumor. This hypothesis is tested here via an unprecedented super-resolution fluorescence analysis of ex vivo tissue slices confirming a particle size increase occurs concomitantly with extended retention of responsive particles compared to unresponsive controls.

Aerosolized ZnO Nanoparticles Induce Toxicity in Alveolar Type II Epithelial Cells at the Air-Liquid Interface

The majority of in vitro studies characterizing the impact of engineered nanoparticles (NPs) on cells that line the respiratory tract were conducted in cells exposed to NPs in suspension. This approach introduces processes that are unlikely to occur during inhaled NP exposures in vivo, such as the shedding of toxic doses of dissolved ions. ZnO NPs are used extensively and pose significant sources for human exposure. Exposures to airborne ZnO NPs can induce adverse effects, but the relevance of the dissolved Zn(2+) to the observed effects in vivo is still unclear. Our goal was to mimic in vivo exposures to airborne NPs and decipher the contribution of the intact NP from the contribution of the dissolved ions to airborne ZnO NP toxicity. We established the exposure of alveolar type II epithelial cells to aerosolized NPs at the air-liquid interface (ALI) and compared the impact of aerosolized ZnO NPs and NPs in suspension at the same cellular doses, measured as the number of particles per cell. By evaluating membrane integrity and cell viability 6 and 24 h post-exposure, we found that aerosolized NPs induced toxicity at the ALI at doses that were in the same order of magnitude as doses required to induce toxicity in submersed cultures. In addition, distinct patterns of oxidative stress were observed in the two exposure systems. These observations unravel the ability of airborne ZnO NPs to induce toxicity without the contribution of dissolved Zn(2+) and suggest distinct mechanisms at the ALI and in submersed cultures.

Submicrometer and nanoscale inorganic particles exploit the actin machinery to be propelled along microvilli-like structures into alveolar cells.

The growing commerce in micro- and nanotechnology is expected to increase human exposure to submicrometer and nanoscale particles, including certain forms of amorphous silica. When inhaled, these particles are likely to reach the alveoli, where alveolar type II epithelial cells that are distinguished by apical microvilli are found. These cells play critical roles in the function of the alveoli and participate in the immune response to amorphous silica and other particles by releasing chemokines. The cellular interactions of the particles, which drive the cellular responses, are still unclear. Adverse effects of nanoparticles have been attributed, in part, to the unique properties of materials at the nanoscale. However, little is known about the cellular interactions of individual or small nanoparticle aggregates, mostly because of their tendency to agglomerate under experimental conditions. Here we investigate the interaction and internalization pathway of individual precipitated amorphous silica particles with specific surface properties and size, by following one particle at a time. We find that both 100 and 500 nm particles can take advantage of the actin turnover machinery within filopodia and microvilli-like structures to advance their way into alveolar type II epithelial cells. This pathway is strictly dependent on the positive surface charge of the particle and on the integrity of the actin filaments, unraveling the coupling of the particle with the intracellular environment across the cell membrane. The retrograde pathway brings a new mechanism by which positive surface charge supports particle recruitment, and potential subsequent toxicity, by polarized epithelial cells bearing microvilli.

Cellular recognition and trafficking of amorphous silica nanoparticles by macrophage scavenger receptor A

Abstract The cellular uptake of engineered nanoparticles (ENPs) is known to involve active transport mechanisms, yet the biological molecules involved are poorly understood. We demonstrate that the uptake of amorphous silica ENPs by macrophage cells, and the secretion of proinflammatory cytokines, is strongly inhibited by silencing expression of scavenger receptor A (SR-A). Conversely, ENP uptake is augmented by introducing SR-A expression into human cells that are normally non-phagocytic. Confocal microscopy analyses show that the majority of single or small clusters of silica ENPs co-localize with SR-A and are internalized through a pathway characteristic of clathrin-dependent endocytosis. In contrast, larger silica ENP agglomerates (>500 nm) are poorly co-localized with the receptor, suggesting that the physical agglomeration state of an ENP influences its cellular trafficking. As SR-A is expressed in macrophages throughout the reticulo-endothelial system, this pathway is likely an important determinant of the biological response to ENPs.

Biological Responses to Engineered Nanomaterials: Needs for the Next Decade.

The interaction of nanomaterials with biomolecules, cells, and organisms is an enormously vital area of current research, with applications in nanoenabled diagnostics, imaging agents, therapeutics, and contaminant removal technologies. Yet the potential for adverse biological and environmental impacts of nanomaterial exposure is considerable and needs to be addressed to ensure sustainable development of nanomaterials. In this Outlook four research needs for the next decade are outlined: (i) measurement of the chemical nature of nanomaterials in dynamic, complex aqueous environments; (ii) real-time measurements of nanomaterial–biological interactions with chemical specificity; (iii) delineation of molecular modes of action for nanomaterial effects on living systems as functions of nanomaterial properties; and (iv) an integrated systems approach that includes computation and simulation across orders of magnitude in time and space.

Three Human Cell Types Respond to Multi-Walled Carbon Nanotubes and Titanium Dioxide Nanobelts with Cell-Specific Transcriptomic and Proteomic Expression Patterns.

Abstract The growing use of engineered nanoparticles (NPs) in commercial and medical applications raises the urgent need for tools that can predict NP toxicity. Global transcriptome and proteome analyses were conducted on three human cell types, exposed to two high aspect ratio NP types, to identify patterns of expression that might indicate high versus low NP toxicity. Three cell types representing the most common routes of human exposure to NPs, including macrophage-like (THP-1), small airway epithelial and intestinal (Caco-2/HT29-MTX) cells, were exposed to TiO2 nanobelts (TiO2-NB; high toxicity) and multi-walled carbon nanotubes (MWCNT; low toxicity) at low (10 µg/mL) and high (100 µg/mL) concentrations for 1 and 24 h. Unique patterns of gene and protein expressions were identified for each cell type, with no differentially expressed (p < 0.05, 1.5-fold change) genes or proteins overlapping across all three cell types. While unique to each cell type, the early response was primarily independent of NP type, showing similar expression patterns in response to both TiO2-NB and MWCNT. The early response might, therefore, indicate a general response to insult. In contrast, the 24 h response was unique to each NP type. The most significantly (p < 0.05) enriched biological processes in THP-1 cells indicated TiO2-NB regulation of pathways associated with inflammation, apoptosis, cell cycle arrest, DNA replication stress and genomic instability, while MWCNT-regulated pathways indicated increased cell proliferation, DNA repair and anti-apoptosis. These two distinct sets of biological pathways might, therefore, underlie cellular responses to high and low NP toxicity, respectively.

Comprehensive metabolomic, lipidomic and microscopic profiling of Yarrowia lipolytica during lipid accumulation identifies targets for increased lipogenesis

Yarrowia lipolytica is an oleaginous ascomycete yeast that accumulates large amounts of lipids and has potential as a biofuel producing organism. Despite a growing scientific literature focused on lipid production by Y. lipolytica, there remain significant knowledge gaps regarding the key biological processes involved. We applied a combination of metabolomic and lipidomic profiling approaches as well as microscopic techniques to identify and characterize the key pathways involved in de novo lipid accumulation from glucose in batch cultured, wild-type Y. lipolytica. We found that lipids accumulated rapidly and peaked at 48 hours during the five day experiment, concurrent with a shift in amino acid metabolism. We also report that exhaustion of extracellular sugars coincided with thickening of the cell wall, suggesting that genes involved in cell wall biogenesis may be a useful target for improving the efficiency of lipid producing yeast strains.

Lipopolysaccharide Density and Structure Govern the Extent and Distance of Nanoparticle Interaction with Actual and Model Bacterial Outer Membranes

Design of nanomedicines and nanoparticle-based antimicrobial and antifouling formulations and assessment of the potential implications of nanoparticle release into the environment requires understanding nanoparticle interaction with bacterial surfaces. Here we demonstrate the electrostatically driven association of functionalized nanoparticles with lipopolysaccharides of Gram-negative bacterial outer membranes and find that lipopolysaccharide structure influences the extent and location of binding relative to the outer leaflet-solution interface. By manipulating the lipopolysaccharide content in Shewanella oneidensis outer membranes, we observed the electrostatically driven interaction of cationic gold nanoparticles with the lipopolysaccharide-containing leaflet. We probed this interaction by quartz crystal microbalance with dissipation monitoring (QCM-D) and second harmonic generation (SHG) using solid-supported lipopolysaccharide-containing bilayers. The association of cationic nanoparticles increased with lipopolysaccharide content, while no association of anionic nanoparticles was observed. The harmonic-dependence of QCM-D measurements suggested that a population of the cationic nanoparticles was held at a distance from the outer leaflet-solution interface of bilayers containing smooth lipopolysaccharides (those bearing a long O-polysaccharide). Additionally, smooth lipopolysaccharides held the bulk of the associated cationic particles outside of the interfacial zone probed by SHG. Our results demonstrate that positively charged nanoparticles are more likely to interact with Gram-negative bacteria than are negatively charged particles, and this interaction occurs primarily through lipopolysaccharides.