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Record Count: 14
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header (Title, Principal Investigator, Institution, City, ST, Award Code, or
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DESCRIPTION (provided by applicant): The proposed work will develop nanoscale tools for characterizing the mammalian cell; it will ultimately lead to new tools for drug discovery, diagnosis of disease, and studying fundamental cell biology. Its justification is that study of biological entities fundamentally involves the study of nanoscale components of the cell: subcellular organelles, pathogens, macromolecules. Nanoscale tools are required to examine and analyze these components at the subcellular scale. The research will create nanometer-scale components (rods, particles, and surfaces) using "biology-friendly" nanotechnology (soft lithography and self-assembled monolayers), and use them to examine mammalian cells. It will use nanoscience-based approaches to:1) create 2D and 3D microenvironments with controlled shapes, molecular composition, and mechanical characteristics for studies of cells; 2) create electrically, optically, and mechanically functional nanosystems that permit selective stimulation of cells, and allow read-out of cellular electrical, chemical and mechanical responses with subcellular resolution; 3) leverage systems that exhibit quantum phenomena unique to nanosystems (e.g., superparamagnetism, superluminosity) to generate new physics and chemistry relevant to biology, and use this understanding of physical science to afford fundamentally new classes of information about cell structure and function; 4) develop methods to multiplex nanoscale technologies to measure functions and characteristics of single cells in parallel, with high statistical reliability; 5) demonstrate the relevance and application of these tools using important biological problems. The work will combine to generate a "nanotool cellular workbench"; it has four specific aims: 1) To create novel multifunctional nanometer-scale structures, particles, components, and surfaces, and analytical systems that use these entities, 2) To use this "work bench" of nanotools to understand how individual cells sense mechanical cues and integrate them with chemical and electrical signals in 2D and 3D microenvironments, 3) To create nanoscale control interfaces that rapidly actuate changes in cellular signal transduction and read-out biochemical responses, and 4) To combine these nanotechnologies with microfluidic systems to create prototypes for integrated cellular biochip-based medical devices.
DESCRIPTION (provided by applicant):
Data for performing a preliminary risk assessment of manufactured nanomaterials are just beginning to emerge. However, early studies of nanomaterial toxicity in aqueous media have tended to be more observational than mechanistic, and have often focused on a single, advanced stage of toxicity that could yield contradictory results. Moreover, the ability to generalize findings to other nanomaterials is limited by the lack of a rational basis for categorizing nanomaterials. Elucidating the mechanisms of toxicity for a given nanomaterial will provide a basis for classifying materials for regulatory purposes, postulating dose-response curves, screening potential risks, and prescribing strategies for risk management. The primary objective of this work is to elucidate the mechanism(s) by which manufactured nanoparticles may induce toxicity in vitro and in vivo. Specifically, this study will consider fullerene-based materials, comparing them with, (i) reference standards (TiO2 and carbon black); (ii) ultrafine particles obtained from an urban airshed (well characterized by in vitro toxicology studies); We will explore a methodology for rapidly screening potentially toxic nanoparticles based on their propensity to generate ROS. The principal hypothesis is that certain classes of nanoparticles such as fullerenes induce ROS production, cellular oxidative stress and cytotoxicity. Fullerenes are selected based on the relatively novel properties (e.g. strength.arid electron affinity) that make them attractive for commercialization. The investigators propose that oxidative stress induced by fullerene derivatives occurs in several stages (tiers), beginning with the induction of phase II antioxidant defenses at the lowest tier of oxidative stress (tier 1), followed by pro-inflammatory (tier 2) and mitochondrion-mediated cytotoxic effects (tier 3) as the level of oxidative stress increases. Particle size, shape, surface area, charge, and chemical composition are important physical variables that could determine their ROS-generating or scavenging properties. Rapid physicochemical determination of ROS production might provide a paradigm to assess the possible toxicity of nanomaterials that act via these mechanisms.
Specific Aim 1 will characterize commercial nanoparticles and their derivatives in terms of particle size, shape, surface area, charge, aqueous solubility, propensity to aggregate, and their ability to catalyze or quench ROS production in vitro. Materials will also be characterized in model solutions containing naturally occurring organic matter, proteins and ions at levels similar to those present in natural waters. Aim 2 will determine whether various fullerenes can generate a hierarchical oxidative stress response in macrophages, bronchial epithelial cells, endothelial cells, neural cells and hepatocytes. This will be accomplished by comparing the effects of fullerenes and reference nanoparticles on, (i) phase II enzyme expression and activation of the heme oxygenase 1 (HO-1) promoter (tier 1); (ii) cytokine and chernokine expression as well as assays for MAP kinase activation (tier 2); (iii) mitochondria! perturbation and induction of cellular apoptosis (tier 3). These biological responses will be compared to the physicochemieal properties of nanomaterials elucidated in Aim 1. Aim 3 will perform in vivo imaging of the oxidative stress-sensitive HO-1 promoter linked to a luciferase reporter in transgenic mice. Organs and tissues showing increased luciferase activity will be investigated for histological evidence of inflammation and cytotoxicity. Aim 4 will compare the biologic responses elicited by each of the nano-scale particles with their ability to generate ROS abiotically, and test the hypothesis that ROS generation can be used to screen toxicity.
By focusing on mechanisms of toxicity rather than outcomes alone, this work will provide the basis for classifying nanomaterials for regulatory purposes. Based on preliminary results presented in this proposal, we anticipate that ROS generation in solution and under UV radiation will be good predictors of nanoparticle toxicity and that ROS measurements can be adapted to screen nanomaterials. A broader assessment of nanomaterial toxicity in the context of the hierarchical oxidative stress response is likely to yield a more sensitive paradigm for toxicity testing, perhaps resolving inconsistencies reported in the literature.
DESCRIPTION (provided by applicant)
Recent advances in nanotechnology have produced a new class of fluorescent nanoparticles, semiconductor quantum dots (QDs). These nanometer-sized crystals have unique photochemical and photophysical properties that are not available from either isolated molecules or bulk solids, and consequently have enabled new opportunities in many areas including optoelectronics, anti-counterfeiting inks, and photovoltaics. More recently, the research interest in QDs has shifted toward the life sciences, where material scientists, chemists and biologists are working together to develop these quantum-confined nanocrystals as fluorescent probes for biomedical imaging. Compared with organic dyes and fluorescent proteins, semiconductor QDs offer several unique advantages, such as size- and composition-tunable emission from visible to infrared wavelengths, large absorption coefficients across a wide spectral range, and very high levels of brightness and photostability. High-quality QDs are generally made from group II-VI and -V elements in the chemical periodic table including Cd and Hg, which are toxic heavy metals. Our preliminary results indicate that polymer-protected QDs remain intact in live cells and animals for up to 2-4 months and are non-toxic. But their long term degradation, metabolism, and clearance are still unknown, which will ultimately determine the suitability of QDs for biomedical applications. Furthermore, because QDs intended for applications other than biomedicine will not necessarily be designed with biocompatibility in mind, concerns have been raised regarding potential occupational and environmental exposures to QDs. In this context, we propose to systematically investigate the toxicity of QDs of different chemical compositions. We will focus on QDs that have practical applications in photonics, optics, solar cells, sensors and biomedical imaging. We hypothesize that the colloidal stability and nanomaterial surface properties such as surface ligands and functional groups will affect in vitro and in vivo behavior and toxicity of QDs. In order to address this hypothesis, this application will focus on innovations of nanoparticle synthesis and surface engineering, in vitro toxicity to multiple murine and human cell types, and in vivo toxicity in genetically modified mice. Information gathered from these studies will be valuable in helping to predict the role of various constituent metals on the toxicity of QDs, as well as in the design of cores and coatings that will optimize the desirable properties of these materials while minimizing their adverse health impacts. Such information will be useful in risk assessments of these materials, and thus help to inform regulatory policy making.
We propose to develop artificial membranes containing nanoscale pores whose mechanical, electrical, and chemical properties are controlled at a nanoscale with organic molecules. The pores will be fabricated in 30-50 nm silicon nitride membranes, and as an integral component of Pt/glass nanopore electrodes. The goal of the proposed work is to covalently modify the inner walls of 5-50 nm wide nanopores with organic molecules whose size and shape are sensitive to external stimuli, such as pH, solvent polarity, pX (where X is an ion or a small molecule), external electric field, light, etc., thus producing responsive nanopores. Specific aims of the project are: (1) to develop the preparation of the nanopores in silicon nitride and in glass to the specified size; (2) to design the molecules for the responsive nanopores, and to optimize
the chemistry needed for their attachment to the nanopore walls; (3) to study the stimulus-response behavior of the attached molecules and their interactions within the confines of the nanopores; and (4) to study the transport dynamics within the resulting responsive nanopores. The pores have long-range applications in stochastic biosensor devices, in separations investigations of biomolecules, and in controlled drug release devices.
The proposed work is a highly innovative, design-driven effort that will lead to the creation and use of nanoscale devices with a host of biological and medical applications. This effort brings together such disciplines as organic synthesis, surface chemistry, analytical chemistry, material science, and electrical engineering.
Crisp Terms/Key Words: acidity /alkalinity, biological transport, biomaterial evaluation, biomaterial development /preparation, biomaterial interface interaction, electric field, lighting, ion, molecular polarity, nitrite, scanning electron microscopy, glass, silicon, stimulus /response, transmission electron microscopy, artificial membrane, atomic force microscopy, nanotechnology, small molecule
DESCRIPTION (provided by applicant):
Carbon nanoparticles (CNPs) are finding increased use in commercial, diagnostic, clinical and other applications. CNP production is anticipated to greatly increase and there is evidence of CNP formation by anthropogenic activity. However, evidence of their potential toxicity is also increasing and respiratory exposure is considered one route of exposure. A number of studies have now demonstrated that manufactured CNP (single walled carbon nanotubes (SWNT), multi-walled carbon nanotubes (MWNT) and to a lesser extent C60 carbon spheres (C60CS)) all cause pulmonary toxicity ranging from inflammation, granuloma formation, fibrosis and airway changes. Macrophages are considered potential targets of CNP in the Jung that may contribute to pathologic outcomes. In vitro toxicity of these particles has been demonstrated with a suggested rank order of potency most often SWNT> MWNT> C60CS. Although reports of nanoparticle toxicity are increasing, the results are often difficult to correlate since the sources (and compositions) and methods of suspension are different for CNP, and often only one particle type is examined. Furthermore, the mechanism(s) by which these uniquely hydrophobic particles are acting is unknown. Based on the unusual physical properties and size of the carbon nanoparticles, as well as our preliminary results with SWNT, MWNT and C60CS, we postulate that CNP are causing membrane disruption leading to changes in macrophage function contributing to lung inflammation. Furthermore, the different sizes of the CNPs may contribute to the rank order of potency and effects. In order to test these hypotheses and gain new information on mechanisms of action we propose the following goals.
1) Compare and evaluate how much variability in biological activity of CNP stems from differences in sources and methods of suspension.
2) Demonstrate that CNP cause alteration of membrane properties and function.
3) Evaluate the effects of CNP on macrophage function related to membrane activity.
The results of these studies will provide important new information regarding the roles that different CNP sources, suspension (disaggregation) protocols and CNP types have on membrane disruption. In addition, the studies will provide information on the mechanism of action in membranes how that translates to alterations of macrophage function.
DESCRIPTION (provided by applicant): Although the toxic nature of many chemicals, such as carcinogenic species, which have found their way into the environment is well-known, the mechanisms by which these chemicals exert their toxic effects at the cellular and molecular level are only now beginning to be understood. There is a strong need for developing a molecular tool that is capable of monitoring biochemical processes or detecting specific biomarkers in a living cell following chemical exposure. Minimally invasive analysis of cellular signaling pathways inside single intact cells is becoming increasingly important fundamentally because cells in a population respond asynchronously to external stimuli. The objective of this research is to conduct fundamental research and develop nanobiosensors for probing chemical exposure and health effects of individual living cells. The proposed technique could provide unprecedented insights into intact cell function, allowing studies of molecular functions in the context of the functional cell architecture. This proposed research will contribute to future advancements in biology and medicine, which is directly relevant to the mission of National Institute of Environmental Health Sciences (NIEHS). The specific aims of the project are: 1) Develop a nanosensor for monitoring the carcinogenic compound, benzo[a]pyrene (BaP) and the related biomarker of exposure, benzopyrene tetrol (BPT), 2) Develop dual-target sensing technology for the nanobiosensor to achieve simultaneous detection of BaP and BPT using phase-resolved detection, and 3) Investigation of exposure and metabolism pathways in a single living cell exposed to BaP. The nanoprobes to be developed in this project will open new horizons to a host of applications in biotechnology, molecular biology and environmental health research, and the study of in situ intracellular signaling processes, and investigations of the chemical transport and toxicity.
DESCRIPTION (provided by applicant)
We propose a quantitative structure activity relationship (QSAR) approach to investigate the specific physical and chemical surface properties that influence nanoparticle biocompatibility. Amorphous silica is chosen as an experimental particle due to its widespread use in consumer products, and because it is readily synthesized in a wide range of defined sizes and surface chemistries. Our recent work shows that the bioactivity of amorphous silica is greatly enhanced in particles <50 nm. We hypothesize that this is due to changes in the silanol site surface chemistry as particle diameter is decreased.
Our approach involves three aims: 1) A panel of nanoparticle-induced secreted proteins will be identified using advanced mass spectrometry-based proteomic analysis of conditioned medium from macrophages exposed to amorphous silica, coupled with our existing gene microarray data. Bioinformatic pathway analysis will be performed to select ~20 pathway biomarkers, which will be used to modify an antibody sandwich-based protein ELISA microarray platform for multiplexed response analyses. 2) A series of silicabased particles where size and surface chemistry is selectively altered with functional groups will be prepared and characterized for size, charge, aggregation state, dissolution products and silanol types. X-ray absorption near-edge spectroscopy will be used to identify surface silicon isoforms in particles adsorbed to micelles mimicking cell membranes. The biological responses of each particle will be assessed in multiwell cellular assays with macrophages, using the ELISA microarray platform to provide quantitative measures of dose-response for ~20 different pathway markers. QSAR analyses will be performed with the measured physicochemical parameters and biological response data to identify relationships that correlate most strongly. 3) Particles selected from QSAR analysis will be further tested in mice exposed by intratracheal instillation, and biological responses will be determined by histopathology along with ELISA microarray analysis of bronchial lavage fluid. Comparison of QSAR results obtained in aims 2 and 3 will determine how predictive the in vitro assay is, as well as highlight particle characteristics that are most important for dictating biocompatibility in vivo. The results will determine properties of nano-scale amorphous silica that determine its biocompatibility, and reveal general principals relevant to other types of nanomaterial. In addition, the approach and biomarkers developed from this work will provide a screening platform that can be deployed to a variety of nanomaterials in the future.
DESCRIPTION (provided by applicant):
The objective of this study is to determine the long-term cardiovascular effects of inhaled nanoparticles. The hypothesize that long-term inhalation of nanoparticles can enhance the development and progression of vascular dysfunction leading to atherosclerosis in a sensitive animal model, and that the vascular dysfunction process is mediated by oxidative stress through the disruption of nitric oxide (NO) regulation. Preliminary studies have demonstrated that 6 months of 6-hr weekday exposures of mice lacking apolipoprotein E (ApoE-/-) to fine ambient concentrated particles enhanced atherosclerosis, and altered vasoconstrictor responses to phenylephrine and serotonin challenge in the thoracic aorta. These changes were accompanied by marked increases in macrophage infiltration, the inducible isoform of nitric oxide synthase (iNOS), increased generation of reactive oxygen species (ROS) and greater immunostaining for the protein nitration product 3-nitrotyrosine. Since nanoparticles have been shown to be able to penetrate into the systemic circulation after inhalation, and are capable of affecting endothelial cell function, it is likely that manufactured nanoparticles could produce cardiovascular effects similar to those seen in preliminary studies. While a tremendous amount of research has addressed the greater pulmonary toxicity associated with ultrafine particles (< 100nm) compared to fine or coarse sized particles, little research has examined the cardiovascular effects of ultrafine or nanoparticles (< 50nm). Furthermore, the few studies that have investigated the cardiovascular effects of inhaled particles were short-term exposure in.nature. To date, there is no study that has investigated the chronic cardiovascular effects of inhaled nanoparticles.
Three different nanoparticles will be used to test the hypothesis, nickel, titanium, and carbon. Nanoparticles (count median diameter = 20 nm) will be produced using a spark generator with pure electrodes of nickel, titanium, and carbon. ApoE (-/-) mice will be exposed to 0 (filtered air control), 25, 50, or 100 ng/m3 nanoparticles for 6 hr/d, 5 d/wk, for up to 6 months. The development and progression of atherosclerosis will be assessed using non-invasive ultrasound biomicroscopy (UBM) as well as serial morphometric measurements. Serial endpoints such as vasoconstriction response, macrophage infiltration, NOS expression level (both inducible and endothelial isoforms), ROS, and 3-nitrotyrosine production will also be investigated as the major mechanisms involved in vascular dysfunction leading to nanoparticle enhanced atherosclerosis.
It is expected that nanoparticle toxicity will be influenced by a variety of exposure conditions including concentration, duration of exposure, and composition. This study will allow us to begin to understand the long-term exposure effects of nanoparticles on the cardiovascular system. The data obtained in the proposed animal studies can readily be used for extrapolation to occupational and ambient settings. In summary, the results from this proposal address a number of the research needs identified in this solicitation, including toxicity and exposure assessment. Ultimately, a systems approach will be developed to the understanding of nanomaterials toxicology sufficiently complete to allow predictions about health effects.
DESCRIPTION (provided by applicant)
The ability to several manufactured nanomaterials to induce oxidative stress has been suggested to be the most appropriate means of assessing the potential toxicity of manufactured nanomaterials. Since oxidative stress is a common pathogenic mechanism in numerous diseases, including various neurodegenerative diseases, it is possible that the various nanomaterials may contribute to the disease process. We have shown that the redox state (dynamic balance between reduced and oxidized components) of neurons (in vitro and in vivo) can be spatially resolved by subcellular compartment. Neurotoxicants can preferentially oxidize cytoplasmic, mitochondrial, or nuclear redox components, such as thioredoxin or GSH. We hypothesize that the overall toxicity of nanomaterials will correspond to their ability to induce oxidative stress in distinct subcellular compartments and that these measures will provide a superior means of assessing their potential toxicity. We propose a series of in vitro and in vivo experiments aimed at determining the subcellular redox state of a cellular population known to be especially vulnerable to oxidative injury, namely, the dopamine neurons in the substantia nigra pars compacta. Aim 1. To determine the ability of the manufactured nanomaterials fullerene (C60), fullerol (C60(OH)22-24), manganese oxide (MnO2), titanium dioxide (TiO2), magnetic iron oxide (FeO4), and nanoscale zero valent iron (n-ZVI) to preferentially oxidize sucellular redox components. In this aim, we will examine the ability of suspended nanoparticles to induce oxidative stress in cell cultures of neuronal origin in the absence and presence of an oxidative challenge (6-OHDA). In addition, we will assess the physico-chemical properties of the nanomaterials prior to and after exposure to the cellular model. Aim 2. To determine the ability of the manufactured nanomaterials "manganese oxide (MnO2), titanium dioxide (TiO2), magnetic iron oxide (FeO4), and nanoscale zero valent iron (n-ZVI) to induce oxidative stress in dopaminergic brain regions. This aim will examine the ability of nanomaterials to alter subcellular redox state and induce oxidative damage in dopaminergic brain regions and determine the physico-chemical state of the nanomaterials prior to administration and in the brain tissue of exposed animals. Aim 3. To determine the ability of bioavailable antioxidants to attenuate the oxidative stress induced by manufactured nanomaterials. In this aim N-acetyl cysteine and alpha tocopherol will be tested for their ability to attenuate oxidative stress in in vitro and in vivo settings. Completion of these aims will provide novel information on the ability of nanomaterials to induce oxidative stress with subcellular spatial resolution and determine if their physico-chemical state is altered after exposure to the biological system.
DESCRIPTION (provided by applicant): Exposure to toxic metals like cadmium (Cd), lead (Pb), mercury (Hg), chromate (CrO42-), arsenite (As) (III), and arsenate (AsO43-) are known to induce various diseases that are detrimental to human health. One of NIEHS emphasis areas is the development of "Chelation chemistry that can serve as the foundation for therapies to ameliorate aberrant metal accumulations and the effects of toxic exposures." In response to PA-06-181, this Exploratory/Developmental R21 proposal will develop and evaluate novel biocompatible chelating materials that will lead to a breakthrough in the field of chelation chemistry, specifically for heavy metals in humans after environmental exposure. This work addresses a key mission of NIEHS since the institute "has primary responsibility with respect to toxic metal exposure from environmental sources." Complete chelation therapies encompass (1) chelating the metal ions in the gastrointestinal fluids in order to limit systemic absorption of ingested materials and (2) chelating the metal ions in blood that have been absorbed systemically from all routes of exposure (oral, dermal and inhalation). Since the 1940s, in vivo toxic metal immobilization has involved the use of ethylenediamine-tetraacetate (EDTA) or dimercaptosuccinic acid (DMSA) following metal exposures. However, these chelation agents still have many disadvantages and low efficacy. They are also not effective in removing Cd and toxic anions such as chromate and arsenate. We hypothesize that functionalized silica (SAMMS) and magnetic nanoparticles, both proven in our numerous preliminary data and publications to be highly efficient and stable sorbents for removal of toxic metals in environmental cleanups, can also be used effectively in biological matrices for metal decorporation in humans. They will be better than the currently FDA-approved EDTA and DMSA in terms of higher affinity, specificity and capacity. In addition, we hypothesize that these new chelating materials will exhibit a faster removal rate, less toxicity, and overall their use will result in lower costs of treatment. To test the hypothesis, SAMMS will be evaluated for toxic metal removal from gastrointestinal (GI) tract while magnetic nanoparticles will be evaluated for extracorporeal chelation of toxic metals from whole blood. Differing organic groups will be carefully designed to have a high affinity for the target metals. Assessment of chelating performance, material stability, protein fouling, and cell uptake of the nanomaterials will be done in vitro using relevant physicochemical forms and concentrations of the metals appropriate to acute and chronic human exposure of the toxic metals. Refinement of both materials to increase their stability as well as minimize protein fouling and cell uptake will also be performed. The results will form a strong foundation for our continued effort with in vivo studies using animal models in a future R01 project. Exposure to toxic metals like cadmium (Cd), lead (Pb), mercury (Hg), chromate (CrO42-), arsenite (As(III)), and arsenate (AsO43-) are known to induce various diseases that are detrimental to human health. One of NIEHS emphasis areas is the development of "Chelation chemistry that can serve as the foundation for therapies to ameliorate aberrant metal accumulations and the effects of toxic exposures." In response to PA-06-181, this exploratory R21 proposal will develop and evaluate novel biocompatible nanomaterials that will lead to a breakthrough in the field of chelation therapies of the above heavy metals by substantially outperforming the current FDA-approved chelating agents.
DESCRIPTION (provided by applicant): We will engage a multidisciplinary team from the University of California Lead Campus Program for Nanotoxicology in studying how the physicochemical characteristics of metal oxide (NP) influence biocompatibility and toxicity in vivo and in vitro. The long-term goal is to develop a rapid screening procedure that classifies NP into potentially safe or dangerous categories. Outline: Titanium dioxide (TiO2), zinc oxide (ZnO) and Ceria (CeO2/Ce2O3) metal oxide NP were chosen based on high volume of production, potential for airborne spread and ability to induce airway inflammation through the generation of reactive oxygen species (ROS). We are particularly interested in how a variation in the physicochemical characteristics of engineered NP influences their biocompatibility or toxicity in portal-of- entry (e.g., macrophages, epithelial, endothelial cells) cellular targets in the lung. In particular, we would like to determine whether our predictive hierarchical oxidative stress model, which is comprised of compensatory as well as injurious cellular responses, could be used for generating a high throughput screening procedure in tissue culture cells that can be used to predict the toxic potential of NP in vivo. To achieve this goal, we will determine how controlled design of the physicochemical characteristics (chemical composition, particle size, state of agglomeration, encapsulation, surface charge) of metal oxide influence ROS production in cells. Metal oxide NP will be synthesized by flame pyrollysis, followed by studying the NP characteristics under dry and wet conditions. We will determine how the variation in the design properties influences: (i) induction of incremental levels of oxidative stress that culminate in adaptive, pro-inflammatory and cytotoxic responses (Aim 1). (2) cellular uptake, subcellular localization and mitochondrial targeting as a prelude to cellular toxicity or biological adaptation (Aim 2); These in vitro cellular studies will involve the use of flow cytometry, real-time PCR, Western blotting, ELISA assays electron microscopy, and confocal microscopy. Once toxicity profiling has been accomplished, the different readouts will be combined into a high-throughput epifluorescence screening procedure that compares several particle types and design modifications simultaneously (Aim 3). Finally, we will determine how the variation in NP physicochemical characteristics influences the induction of airway inflammation in a murine intratracheal instillation model (Aim 4). We will also attempt to relate the vitro oxidant stress effects to the induction of in vivo oxidative stress by using a transgenic mouse that expresses the heme oxygenase 1 promoter linked to a luciferase reporter. We will determine whether the knockout of a key antioxidant defense regulator (Nrf2) renders these animals more susceptible to NP-induced oxidative stress injury.
PUBLIC HEALTH RELEVANCE: This application will develop a novel testing strategy to screen for the safety of a large number of new nanomaterials that are coming onto the market. We will use the design of metal oxide nanoparticle as a screening tool to develop our toxicity screening method that will assess how these particles lead to cell death by different uptake mechanisms and ability to produce toxic oxygen radicals.
DESCRIPTION (provided by applicant): Nanotechnology is an enabling platform that will provide a broad range of novel applications and improved technologies for biomedical science due to the unique physical and chemical properties inherent to nanomaterials. Pertinent to the development of promising biomedical nanotechnologies, and to the safety of nanomaterials in general, is a thorough understanding of nanomaterial-biological interactions. Yet, the principal characteristics that may be predictive of nanomaterial interactions with biological systems have not been elucidated because of the current lack of data, the enormous diversity of nanomaterials, and the lack of coordinated efforts to share findings and translate data into knowledge. The embryonic zebrafish model is a dynamic in vivo system that offers the power of whole-animal investigations with the convenience of cell culture to rapidly evaluate interactions between engineered nanomaterials and biological systems. Investigations using this model system can reveal subtle interactions at multiple levels of biological organization, i.e. molecular, cellular, systems, organismal. Our approach couples the many advantages of the embryonic zebrafish assay with an ideal nanoparticle platform in order to systematically assess the relative influence of various physiochemical parameters on overall biological responses to nanomaterial exposure. High-purity, ligand-functionalized gold nanoparticles (AuNPs) synthesized in aqueous environments can be precisely engineered such that individual aspects of the material can be evaluated independently. It is well understood that data from this emerging field will be extremely diverse including a multitude of widely varying nanomaterials that are being/or will be tested in a broad array of animal systems and in vitro assays. Knowledge of nanomaterial-biological interactions will likely only be arrived at upon inclusion and consideration of the entire body of data produced from global efforts in this research area. To address these needs in the nascent field of nanobiotechnology, our group has developed a collaborative knowledgebase of Nanomaterial-Biological Interactions (NB). The NBI knowledgebase serves as a repository for annotated data on nanomaterial characterization, synthesis methods, and nanomaterial-biological interactions define at multiple levels of biological organization. Relevant computational, analytic and data mining tools will be incorporated into NBI to the framework for species, route, dose and scenario extrapolations and for identification of key data required to predict the biological interactions of nanomaterials. PUBLIC HEALTH RELEVANCE: New nanomaterials are rapidly being developed for a wide range of biomedical applications (e.g. high-performance diagnostic probes, site-selective therapeutics, prosthetics, regenerative medicine, imaging, etc.), so it is surprising that so little is known about how or why nanomaterials interact with biological systems and even less is known about how to design them to exhibit a desired effect in whole animals. The immediate need to gain comprehensive information on biological-nanomaterial interactions requires systematic, collaborative scientific investigation to define nanomaterial-biological interactions and describe how specific properties of nanomaterials govern biological responses. Timely evaluation and dissemination of information on nanomaterial-biological interactions will provide much needed data, improve public trust of the nanotechnology industry, and provide nanomaterial designers in academia and industry with information to direct the development of high-performance, safe nanomaterials and resulting biomedical technologies.
DESCRIPTION (provided by applicant)
Adverse human health effects due to occupational and environmental exposure to nanomaterials are a major concern and a potential threat to their successful commercialization and biomedical applications. Realization of their commercial potential will require a better understanding of the interactions of nanomaterials with biological systems and the development of new strategies to manage human health risk. Manufactured carbon nanomaterials are highly variable with respect to chemical and physical properties, state of aggregation, and purity. Toxicological screening is urgently needed to identify potentially hazardous nanomaterials; however, their wide variability and unique properties complicate interpretation of traditional in vitro and in vivo toxicity assays. An interdisciplinary research team at Brown University including a materials scientist, a toxicologic pathologist, and a molecular biologist has developed a panel of novel nanomaterials and innovative approaches for nanotoxicology assays. This panel of model nanomaterials will be expanded to include selected commercial materials subjected to rigorous characterization of lexicologically relevant materials properties. Novel synthesis and characterization methods will be used to carry out systematic studies (Specific Aims 1 and 2) that reveal the chemical (surface state, metals bioavailability, biopersistence), structural (size, shape, elasticity), and superstructural (aggregate size and shape) basis of carbon nanomaterial toxicity. This team will develop and validate a unique platform for cellular assays in 3- dimensional culture using formation of granulomas, persistent macrophage activation, and fibrosis as pathologic endpoints (Specific Aim 3). This platform will incorporate post-exposure characterization of nanomaterials in parallel with an acellular assay to assess biopersistence and aggregation state in simulated intracellular environments (Specific Aim 4). A cytokine expression profile will be developed to predict toxicity of carbon nanomaterials relative to standard reference materials (Specific Aim 5). It is anticipated that this validated toxicologic screening assay will provide an alternative to chronic rodent inhalation assays at lower cost and reduced burden of animal testing. Identification of specific chemical and physical properties of nanomaterials responsible for cellular toxicity will enable development of manufacturing methods and post processing steps to eliminate intrinsic toxicity.
DESCRIPTION (provided by applicant)
Carbon nanotubes exhibit exceptional structural properties and conductivity, and are being incorporated into diverse manufacturing processes, yet little is known about the risks that these novel molecules pose to human health. Emerging observations indicate that pulmonary exposure to carbon nanotubes induce a fibrotic response in the lungs that is related at least in part due to their size and shape. Preliminary experiments from our group indicate that pulmonary exposure to multiwalled carbon nanotubes (MWCNT) alters hemostasis, vascular reactivity, and myocardial injury resulting from acute coronary occulusion and reperfusion. Furthermore, comparison to other nano-scale particles, including elemental carbon (Printex 90), ceramic nanoparticles and ambient particulate matter, indicates that cardiotoxicity of nano-scale particles is related more to the particle composition and surface characteristics than to its shape.
In this project, we propose to test the hypothesis that respirable MWCNT are cardiotoxic, and how MWCNTtissue interaction impacts their biological activity. MWCNT of uniform length (10 to 20 |am) and well-defined surface characteristics will be generated through a collaboration with the Institute of Regenerative Medicine at Winston-Salem Health Sciences Center and NanoTechLabs, Inc. Investigators at the East Carolina University will deliver the nanotubes by acute inhalation to mice, verify the pulmonary distribution and define the acute and chronic effects of particle exposure on hemostasis, vascular reactivity, and myocardial response to ischemia. The modifications to be compared represent alteration to the pristine MWCNT and surface functionalization paradigms that are common in nanotechnology and will include a) nitrogen-doping, b) surface carboxylation, and c) surface amination.
The effects of these modifications will be tested in each of the following Specific Aims:
Specific Aim 1. Evaluate the fate of inhaled MWCNT,
Specific Aim 2. Demonstrate that aspiration of multiwalled carbon nanotubes alters hemostasis and myocardial response to ischemia and reperfusion.
Specific Aim 3. Test the hypothesis that inhalation of MWCNT alters vascular reactivity via impared adenosine signaling.
Specific Aim 4. Assess the role of endothiela nitric oxide, oxidative stress and vascular function in MWCNT cardiotoxicity.
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