Publications

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Biomolecular interactions and responses of human epithelial and macrophage cells to engineered nanomaterials

Bachand, George B.; Brozik, Susan M.; Bachand, Marlene B.; Aaron, Jesse S.; Timlin, Jerilyn A.; Achyuthan, Komandoor A.; Kotula, Paul G.

Engineered nanomaterials (ENMs) are increasingly being used in commercial products, particularly in the biomedical, cosmetic, and clothing industries. For example, pants and shirts are routinely manufactured with silver nanoparticles to render them 'wrinkle-free.' Despite the growing applications, the associated environmental health and safety (EHS) impacts are completely unknown. The significance of this problem became pervasive within the general public when Prince Charles authored an article in 2004 warning of the potential social, ethical, health, and environmental issues connected to nanotechnology. The EHS concerns, however, continued to receive relatively little consideration from federal agencies as compared with large investments in basic nanoscience R&D. The mounting literature regarding the toxicology of ENMs (e.g., the ability of inhaled nanoparticles to cross the blood-brain barrier; Kwon et al., 2008, J. Occup. Health 50, 1) has spurred a recent realization within the NNI and other federal agencies that the EHS impacts related to nanotechnology must be addressed now. In our study we proposed to address critical aspects of this problem by developing primary correlations between nanoparticle properties and their effects on cell health and toxicity. A critical challenge embodied within this problem arises from the ability to synthesize nanoparticles with a wide array of physical properties (e.g., size, shape, composition, surface chemistry, etc.), which in turn creates an immense, multidimensional problem in assessing toxicological effects. In this work we first investigated varying sizes of quantum dots (Qdots) and their ability to cross cell membranes based on their aspect ratio utilizing hyperspectral confocal fluorescence microscopy. We then studied toxicity of epithelial cell lines that were exposed to different sized gold and silver nanoparticles using advanced imaging techniques, biochemical analyses, and optical and mass spectrometry methods. Finally we evaluated a new assay to measure transglutaminase (TG) activity; a potential marker for cell toxicity.

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TYPE SAND Report YEAR 2011
OSTIDOI

Advanced optical imaging reveals the dependence of particle geometry on interactions between CdSe quantum dots and immune cells

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Aaron, Jesse S.; Greene, Adrienne C.; Kotula, Paul G.; Bachand, George B.; Timlin, Jerilyn A.

The biocompatibility and possible toxicological consequences of engineered nanomaterials, including quantum dots (QDs) due to their unique suitability for biomedical applications, remain intense areas of interest. We utilized advanced imaging approaches to characterize the interactions of CdSe QDs of various sizes and shapes with live immune cells. Particle diffusion and partitioning within the plasma membrane, cellular uptake kinetics, and sorting of particles into lysosomes were all independantly characterized. Using high-speed total internal reflectance fluorescence (TIRF) microscopy, we show that QDs with an average aspect ratio of 2.0 (i.e., rod-shaped) diffuse nearly an order of magnitude slower in the plasma membrane than more spherical particles with aspect ratios of 1.2 and 1.6, respectively. Moreover, more rod-shaped QDs were shown to be internalized into the cell 2-3 fold more slowly. Hyperspectral confocal fluorescence microscopy demonstrates that QDs tend to partition within the cell membrane into regions containing a single particle type. Furthermore, data examining QD sorting mechanisms indicate that endocytosis and lysosomal sorting increases with particle size. Together, these observations suggest that both size and aspect ratio of a nanoparticle are important characteristics that significantly impact interactions with the plasma membrane, uptake into the cell, and localization within intracellular vesicles. Thus, rather than simply characterizing nanoparticle uptake into cells, we show that utilization of advanced imaging approaches permits a more nuanced and complete examination of the multiple aspects of cell-nanoparticle interactions that can ultimately aid understanding possible mechanisms of toxicity, resulting in safer nanomaterial designs. Using hyperspectral confocal fluorescence (HCF) microscopy, it is shown that quantum dots of various sizes and shapes partition themselves into distinct regions within the cell membrane of RBL-2H3 rat mast cells. HCF microscopy allows for deconvolving the signal from multiple, overlapping fluorophores in the sample in order to reveal precise concentrations and distributions of nanoparticles in the cell. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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TYPE Journal Article YEAR 2011
ScopusOSTI

Biomolecular transport and separation in nanotubular networks

Sasaki, Darryl Y.; Wang, Julia W.; Hayden, Carl C.; Stachowiak, Jeanne C.; Branda, Steven B.; Bachand, George B.; Meagher, Robert M.; Stevens, Mark J.; Robinson, David R.; Zendejas, Frank Z.

Cell membranes are dynamic substrates that achieve a diverse array of functions through multi-scale reconfigurations. We explore the morphological changes that occur upon protein interaction to model membrane systems that induce deformation of their planar structure to yield nanotube assemblies. In the two examples shown in this report we will describe the use of membrane adhesion and particle trajectory to form lipid nanotubes via mechanical stretching, and protein adsorption onto domains and the induction of membrane curvature through steric pressure. Through this work the relationship between membrane bending rigidity, protein affinity, and line tension of phase separated structures were examined and their relationship in biological membranes explored.

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TYPE SAND Report YEAR 2010
OSTIDOI

Toxicological studies of semiconductor quantum dots on immune cells

Bachand, George B.; Greene, Adrienne C.; Poschet, Jens F.

Nanoengineered materials hold a vast promise of enabling revolutionary technologies, but also pose an emerging and potentially serious threat to human and environmental health. While there is increasing knowledge concerning the risks posed by engineered nanomaterials, significant inconsistencies exist within the current data based on the high degree of variability in the materials (e.g., synthesis method, coatings, etc) and biological test systems (e.g., cell lines, whole organism, etc). In this project, we evaluated the uptake and response of two immune cell lines (RAW macrophage and RBL mast cells) to nanocrystal quantum dots (Qdots) with different sizes and surface chemistries, and at different concentrations. The basic experimental design followed a 2 x 2 x 3 factorial model: two Qdot sizes (Qdot 520 and 620), two surface chemistries (amine 'NH{sub 2}' and carboxylic acid 'COOH'), and three concentrations (0, 1 nM, and 1 {micro}M). Based on this design, the following Qdots from Evident Technologies were used for all experiments: Qdot 520-COOH, Qdot 520-NH{sub 2}, Qdot 620-COOH, and Qdot 620-NH{sub 2}. Fluorescence and confocal imaging demonstrated that Qdot 620-COOH and Qdot 620-NH{sub 2} nanoparticles had a greater level of internalization and cell membrane association in RAW and RBL cells, respectively. From these data, a two-way interaction between Qdot size and concentration was observed in relation to the level of cellular uptake in RAW cells, and association with RBL cell membranes. Toxicity of both RBL and RAW cells was also significantly dependent on the interaction of Qdot size and concentration; the 1 {micro}M concentrations of the larger, Qdot 620 nanoparticles induced a greater toxic effect on both cell lines. The RBL data also demonstrate that Qdot exposure can induce significant toxicity independent of cellular uptake. A significant increase in TNF-{alpha} and decrease in IL-10 release was observed in RAW cells, and suggested that Qdot exposure induced a pro-inflammatory response. In contrast, significant decreases in both TNF-{alpha} and IL-4 releases were observed in RBL cells, which is indicative of a suppressed inflammatory response. The changes in cytokine release observed in RAW and RBL cells were primarily dependent on Qdot concentration and independent of size and surface chemistry. Changes in the activity of superoxide dismutase were observed in RAW, but not RBL cells, suggesting that RAW cells were experiencing oxidative stress induced by Qdot exposure. Overall, our results demonstrate that the uptake/association and biomolecular response of macrophage and mast cells is primarily driven by an interaction between Qdot size and concentration. Based on these findings, a more detailed understanding of how size directly impacts cellular interactions and response will be critical to developing predictive models of Qdot toxicity.

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TYPE SAND Report YEAR 2008
OSTIDOI

In vivo collection of rare proteins using kinesin-based "nano-harvesters"

Bachand, George B.; Greene, Adrienne C.

In this project, we have developed a novel platform for capturing, transport, and separating target analytes using the work harnessed from biomolecular transport systems. Nanoharvesters were constructed by co-organizing kinesin motor proteins and antibodies on a nanocrystal quantum dot (nQD) scaffold. Attachment of kinesin and antibodies to the nQD was achieved through biotin-streptavidin non-covalent bonds. Assembly of the nanoharvesters was characterized using a modified enzyme-linked immunosorbent assay (ELISA) that confirmed attachment of both proteins. Nanoharvesters selective against tumor necrosis factor-{alpha} (TNF-{alpha}) and nuclear transcription factor-{kappa}B (NF-{kappa}B) were capable of detecting target antigens at <100 ng/mL in ELISAs. A motility-based assay was subsequently developed using an antibody-sandwich approach in which the target antigen (TNF-{alpha}) formed a sandwich with the red-emitting nanoharvester and green-emitting detection nQD. In this format, successful sandwich formation resulted in a yellow emission associated with surface-bound microtubules. Step-wise analysis of sandwich formation suggested that the motility function of the kinesin motors was not adversely affected by either antigen capture or the subsequent binding of the detection nQDs. TNF-{alpha} was detected as low as {approx}1.5 ng/mL TNF-{alpha}, with 5.2% of the nanoharvesters successfully capturing the target analyte and detection nQDs. Overall, these results demonstrate the ability to capture target protein analytes in vitro using the kinesin-based nanoharvesters in nanofluidic environments. This system has direct relevance for lab-on-a-chip applications where pressure-driven or electrokinetic movement of fluids is impractical, and offers potential application for in vivo capture of rare proteins within the cytoplasmic domain of live cells.

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TYPE SAND Report YEAR 2008
OSTIDOI
Results 101–125 of 156
Results 101–125 of 156