DNA corona on nanoparticles leads to an enhanced immunostimulatory effect with implications for autoimmune diseases.

Autoimmune and inflammatory diseases are highly complex, limiting treatment and the development of new therapies. Recent work has shown that cell-free DNA bound to biological microparticles is linked to systemic lupus erythematosus, a prototypic autoimmune disease. However, the heterogeneity and technical challenges associated with the study of biological particles have hindered a mechanistic understanding of their role. Our goal was to develop a well-controlled DNA-particle model system to understand how DNA-particle complexes affect cells. We first characterized the adsorption of DNA on the surface of polystyrene nanoparticles (200 nm and 2 µm) using transmission electron microscopy, dynamic light scattering, and colorimetric DNA concentration assays. We found that DNA adsorbed on the surface of nanoparticles was resistant to degradation by DNase 1. Macrophage cells incubated with the DNA-nanoparticle complexes had increased production of pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6). We probed two intracellular DNA sensing pathways, toll-like receptor 9 (TLR9) and cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING), to determine how cells sense the DNA-nanoparticle complexes. We found that the cGAS-STING pathway is the primary route for the interaction between DNA-nanoparticles and macrophages. These studies provide a molecular and cellular-level understanding of DNA-nanoparticle-macrophage interactions. In addition, this work provides the mechanistic information necessary for future in vivo experiments to elucidate the role of DNA-particle interactions in autoimmune diseases, providing a unique experimental framework to develop novel therapeutic approaches.

Palmitate-mediated disruption of the endoplasmic reticulum decreases intracellular vesicle motility.

Essential cellular processes such as metabolism, protein synthesis, and autophagy require the intracellular transport of membrane-bound vesicles. The importance of the cytoskeleton and associated molecular motors for transport is well documented. Recent research has suggested that the endoplasmic reticulum (ER) may also play a role in vesicle transport through a tethering of vesicles to the ER. We use single-particle tracking fluorescence microscopy and a Bayesian change-point algorithm to characterize vesicle motility in response to the disruption of the ER, actin, and microtubules. This high-throughput change-point algorithm allows us to efficiently analyze thousands of trajectory segments. We find that palmitate-mediated disruption of the ER leads to a significant decrease in vesicle motility. A comparison with the disruption of actin and microtubules shows that disruption of the ER has a significant impact on vesicle motility, greater than the disruption of actin. Vesicle motility was dependent on cellular region, with greater motility in the cell periphery than the perinuclear region, possibly due to regional differences in actin and the ER. Overall, these results suggest that the ER is an important factor in vesicle transport.

Transport of lysosomes decreases in the perinuclear region: Insights from changepoint analysis.

Lysosomes are membrane-bound organelles that serve as the endpoint for endocytosis, phagocytosis, and autophagy, degrading the molecules, pathogens, and organelles localized within them. These cellular functions require intracellular transport. We use fluorescence microscopy to characterize the motion of lysosomes as a function of intracellular region, perinuclear or periphery, and lysosome diameter. Single-particle tracking data are complemented by changepoint identification and analysis of a mathematical model for state switching. We first classify lysosomal motion as motile or stationary. We then study how lysosome location and diameter affects the proportion of time spent in each state and quantify the speed during motile periods. We find that the proportion of time spent stationary is strongly region dependent, with significantly decreased motility in the perinuclear region. Increased lysosome diameter only slightly decreases speed. Overall, these results demonstrate the importance of decomposing particle trajectories into qualitatively different behaviors before conducting population-wide statistical analysis. Our results suggest that intracellular region is an important factor to consider in studies of intracellular transport.

Concentration and composition of the protein corona as a function of incubation time and serum concentration: an automated approach to the protein corona.

Nanoparticles in contact with proteins form a "corona" of proteins adsorbed on the nanoparticle surface. Subsequent biological responses are then mediated by the adsorbed proteins rather than the bare nanoparticles. The use of nanoparticles as nanomedicines and biosensors would be greatly improved if researchers were able to predict which specific proteins will adsorb on a nanoparticle surface. We use a recently developed automated workflow with a liquid handling robot and low-cost proteomics to determine the concentration and composition of the protein corona formed on carboxylate-modified iron oxide nanoparticles (200 nm) as a function of incubation time and serum concentration. We measure the concentration of the resulting protein corona with a colorimetric assay and the composition of the corona with proteomics, reporting both abundance and enrichment relative to the fetal bovine serum (FBS) proteins used to form the corona. Incubation time was found to be an important parameter for corona concentration and composition at high (100% FBS) incubation concentrations, with only a slight effect at low (10%) FBS concentrations. In addition to these findings, we describe two methodological advances to help reduce the cost associated with protein corona experiments. We have automated the digest step necessary for proteomics and measured the variability between triplicate samples at each stage of the proteomics experiments. Overall, these results demonstrate the importance of understanding the multiple parameters that influence corona formation, provide new tools for corona characterization, and advance bioanalytical research in nanomaterials.

Intracellular Generation of Superoxide by TiO2 Nanoparticles Decreases Histone Deacetylase 9 (HDAC9), an Epigenetic Modifier.

Titanium dioxide (TiO2) nanoparticles are used on a massive scale in commercial and industrial products. Of specific concern is how the inhalation of these nanoparticles in a manufacturing setting may affect human health. We examine the cellular response to TiO2 nanoparticles using a combination of cell-free spectroscopic assays, fluorescence microscopy, Western blotting, and TiO2 nanoparticle surface modifications. These experiments show that TiO2 nanoparticles generate superoxide, both in solution and in cells, and this intracellular superoxide decreases expression of histone deacetylase 9 (HDAC9), an epigenetic modifier. We use protein coronas formed from superoxide dismutase (SOD) and catalase, enzymes that scavenge reactive oxygen species (ROS), to probe the relationship between TiO2 nanoparticles, ROS, and the subsequent cellular response. These protein coronas provide nanoparticle-localized scavengers that demonstrate that the nanoparticles are the source of the intracellular superoxide. Importantly, the use of a SOD corona or surface passivated TiO2 nanoparticles prevents the decrease of HDAC9. These experiments elucidate the underlying mechanism of TiO2 nanoparticle-mediated cellular responses including oxidative stress and changes in gene expression. They also provide the first demonstration of a protein corona as a tool for probing cellular responses to nanoparticles. Overall, this research shows that low, nontoxic concentrations of TiO2 nanoparticles alter an enzyme responsible for epigenetic modifications, which points to concerns regarding long-term exposures in manufacturing settings.

Microscopy and Cell Biology: New Methods and New Questions.

Understanding the cellular basis of human health and disease requires the spatial resolution of microscopy and the molecular-level details provided by spectroscopy. This review highlights imaging methods at the intersection of microscopy and spectroscopy with applications in cell biology. Imaging methods are divided into three broad categories: fluorescence microscopy, label-free approaches, and imaging tools that can be applied to multiple imaging modalities. Just as these imaging methods allow researchers to address new biological questions, progress in biological sciences will drive the development of new imaging methods. We highlight four topics in cell biology that illustrate the need for new imaging tools: nanoparticle-cell interactions, intracellular redox chemistry, neuroscience, and the increasing use of spheroids and organoids. Overall, our goal is to provide a brief overview of individual imaging methods and highlight recent advances in the use of microscopy for cell biology.

Food-Grade TiO2 Particles Generate Intracellular Superoxide and Alter Epigenetic Modifiers in Human Lung Cells.

Titanium dioxide (TiO2) particles are a common ingredient in food, providing the bright white color for many candies, gums, and frostings. While ingestion of these materials has been examined previously, few studies have examined the effect of these particles on lung cells. Inhalation is an important exposure pathway for workers processing these foods and, more recently, home users who purchase these particles directly. We examine the response of lung cells to food-grade TiO2 particles using a combination of fluorescence microscopy and RT-PCR. These experiments show that TiO2 particles generate intracellular reactive oxygen species, specifically superoxide, and alter expression of two epigenetic modifiers, histone deacetylase 9 (HDAC9) and HDAC10. We use a protein corona formed from superoxide dismutase (SOD), an enzyme that scavenges superoxide, to probe the relationship between TiO2 particles and superoxide generation. These experiments show that low, non-cytotoxic, concentrations of food-grade TiO2 particles lead to cellular responses, including altering two enzymes responsible for epigenetic modifications. This production of superoxide and change in epigenetic modifiers could affect human health following inhalation. We expect this research will motivate future in vivo experiments examining the pulmonary response to food-grade TiO2 particles.

Automation and low-cost proteomics for characterization of the protein corona: experimental methods for big data.

Nanoparticles used in biological settings are exposed to proteins that adsorb on the surface forming a protein corona. These adsorbed proteins dictate the subsequent cellular response. A major challenge has been predicting what proteins will adsorb on a given nanoparticle surface. Instead, each new nanoparticle and nanoparticle modification must be tested experimentally to determine what proteins adsorb on the surface. We propose that any future predictive ability will depend on large datasets of protein-nanoparticle interactions. As a first step towards this goal, we have developed an automated workflow using a liquid handling robot to form and isolate protein coronas. As this workflow depends on magnetic separation steps, we test the ability to embed magnetic nanoparticles within a protein nanoparticle. These experiments demonstrate that magnetic separation could be used for any type of nanoparticle in which a magnetic core can be embedded. Higher-throughput corona characterization will also require lower-cost approaches to proteomics. We report a comparison of fast, low-cost, and standard, slower, higher-cost liquid chromatography coupled with mass spectrometry to identify the protein corona. These methods will provide a step forward in the acquisition of the large datasets necessary to predict nanoparticle-protein interactions.

A protein corona primer for physical chemists.

Nanoparticles present in any biological environment are exposed to extracellular proteins. These proteins adsorb on the surface of the nanoparticle forming a "protein corona." These proteins control the interaction of nanoparticles with cells. The interaction of proteins with the nanoparticle surface is governed by physical chemistry. Understanding this process requires spectroscopy, microscopy, and computational tools that are familiar to physical chemists. This perspective provides an overview of the protein corona along with two future directions: first, the need for new computational approaches, including machine learning, to predict corona formation and second, the extension of protein corona studies to more complex environments ranging from lung fluids to waste water treatment.

Protein Corona in Response to Flow: Effect on Protein Concentration and Structure.

Nanoparticles used in cellular applications encounter free serum proteins that adsorb onto the surface of the nanoparticle, forming a protein corona. This protein layer controls the interaction of nanoparticles with cells. For nanomedicine applications, it is important to consider how intravenous injection and the subsequent shear flow will affect the protein corona. Our goal was to determine if shear flow changed the composition of the protein corona and if these changes affected cellular binding. Colorimetric assays of protein concentration and gel electrophoresis demonstrate that polystyrene nanoparticles subjected to flow have a greater concentration of serum proteins adsorbed on the surface, especially plasminogen. Plasminogen, in the absence of nanoparticles, undergoes changes in structure in response to flow, characterized by fluorescence and circular dichroism spectroscopy. The protein-nanoparticle complexes formed from fetal bovine serum after flow had decreased cellular binding, as measured with flow cytometry. In addition to the relevance for nanomedicine, these results also highlight the technical challenges of protein corona studies. The composition of the protein corona was highly dependent on the initial mixing step: rocking, vortexing, or flow. Overall, these results reaffirm the importance of the protein corona in nanoparticle-cell interactions and point toward the challenges of predicting corona composition based on nanoparticle properties.

Controlling the Resting Membrane Potential of Cells with Conducting Polymer Microwires.

All cells have a resting membrane potential resulting from an ion gradient across the plasma membrane. The resting membrane potential of cells is tightly coupled to regeneration and differentiation. The ability to control this parameter provides the opportunity for both biomedical advances and the probing of fundamental bioelectric pathways. The use of poly(3,4-ethylenedioxythiophene): polystyrene sulfonate (PEDOT:PSS) conducting polymer microwires to depolarize cells is tested using E. coli cells loaded with a fluorescent dye that is pumped out of the cells in response to depolarization; a more positive membrane potential. Fluorescence imaging of the cells in response to a conducting-polymer-microwire applied voltage confirms depolarization and shows that the rate of depolarization is a function of the applied voltage and frequency. Microwire activity does not damage the cells, demonstrated with a propidium iodide assay of membrane integrity. The conducting polymer microwires do not penetrate the cell, or even come into contact with the cell; they only need to generate a minimum electric field, controlled by the placement of the wires. It is expected that these microwires will provide a new, noninvasive, cellular-scale tool for the control of resting membrane potential with high spatial precision.

Conducting polymer nanowires for control of local protein concentration in solution.

Interfacing devices with cells and tissues requires new nanoscale tools that are both flexible and electrically active. We demonstrate the use of PEDOT:PSS conducting polymer nanowires for the local control of protein concentration in water and biological media. We use fluorescence microscopy to compare the localization of serum albumin in response to electric fields generated by narrow (760 nm) and wide (1.5 µm) nanowires. We show that proteins in deionized water can be manipulated over a surprisingly large micron length scale and that this distance is a function of nanowire diameter. In addition, white noise can be introduced during the electrochemical synthesis of the nanowire to induce branches into the nanowire allowing a single device to control multiple nanowires. An analysis of growth speed and current density suggests that branching is due to the Mullins-Sekerka instability, ultimately controlled by the roughness of the nanowire surface. These small, flexible, conductive, and biologically compatible PEDOT:PSS nanowires provide a new tool for the electrical control of biological systems.

RNA-induced silencing complex (RISC) Proteins PACT, TRBP, and Dicer are SRA binding nuclear receptor coregulators.

The cytoplasmic RNA-induced silencing complex (RISC) contains dsRNA binding proteins, including protein kinase RNA activator (PACT), transactivation response RNA binding protein (TRBP), and Dicer, that process pre-microRNAs into mature microRNAs (miRNAs) that target specific mRNA species for regulation. There is increasing evidence for important functional interactions between the miRNA and nuclear receptor (NR) signaling networks, with recent data showing that estrogen, acting through the estrogen receptor, can modulate initial aspects of nuclear miRNA processing. Here, we show that the cytoplasmic RISC proteins PACT, TRBP, and Dicer are steroid receptor RNA activator (SRA) binding NR coregulators that target steroid-responsive promoters and regulate NR activity and downstream gene expression. Furthermore, each of the RISC proteins, together with Argonaute 2, associates with SRA and specific pre-microRNAs in both the nucleus and cytoplasm, providing evidence for links between NR-mediated transcription and some of the factors involved in miRNA processing.

Nanoparticle-cell interactions: molecular structure of the protein corona and cellular outcomes.

The use of nanoparticles (NPs) in biology and medicine requires a molecular-level understanding of how NPs interact with cells in a physiological environment. A critical difference between well-controlled in vitro experiments and in vivo applications is the presence of a complex mixture of extracellular proteins. It has been established that extracellular serum proteins present in blood will adsorb onto the surface of NPs, forming a "protein corona". Our goal was to understand how this protein layer affected cellular-level events, including NP binding, internalization, and transport. A combination of microscopy, which provides spatial resolution, and spectroscopy, which provides molecular information, is necessary to probe protein-NP-cell interactions. Initial experiments used a model system composed of polystyrene NPs functionalized with either amine or carboxylate groups to provide a cationic or anionic surface, respectively. Serum proteins adsorb onto the surface of both cationic and anionic NPs, forming a net anionic protein-NP complex. Although these protein-NP complexes have similar diameters and effective surface charges, they show the exact opposite behavior in terms of cellular binding. In the presence of bovine serum albumin (BSA), the cellular binding of BSA-NP complexes formed from cationic NPs is enhanced, whereas the cellular binding of BSA-NP complexes formed from anionic NPs is inhibited. These trends are independent of NP diameter or cell type. Similar results were obtained for anionic quantum dots and colloidal gold nanospheres. Using competition assays, we determined that BSA-NP complexes formed from anionic NPs bind to albumin receptors on the cell surface. BSA-NP complexes formed from cationic NPs are redirected to scavenger receptors. The observation that similar NPs with identical protein corona compositions bind to different cellular receptors suggested that a difference in the structure of the adsorbed protein may be responsible for the differences in cellular binding of the protein-NP complexes. Circular dichroism spectroscopy, isothermal titration calorimetry, and fluorescence spectroscopy show that the structure of BSA is altered following incubation with cationic NPs, but not anionic NPs. Single-particle-tracking fluorescence microscopy was used to follow the cellular internalization and transport of protein-NP complexes. The single particle-tracking experiments show that the protein corona remains bound to the NP throughout endocytic uptake and transport. The interaction of protein-NP complexes with cells is a challenging question, as the adsorbed protein corona controls the interaction of the NP with the cell; however, the NP itself alters the structure of the adsorbed protein. A combination of microscopy and spectroscopy is necessary to understand this complex interaction, enabling the rational design of NPs for biological and medical applications.

Tumor-targeted TNFα stabilizes tumor vessels and enhances active immunotherapy.

Solid tumors are intrinsically resistant to immune rejection. Abnormal tumor vasculature can act as a barrier for immune cell migration into tumors. We tested whether targeting IFNγ and/or TNFα into pancreatic neuroendocrine tumors can alleviate immune suppression. We found that intratumoral IFNγ causes rapid vessel loss, which does not support anti-tumor immunity. In contrast, low-dose TNFα enhances T-cell infiltration and overall survival, an effect that is exclusively mediated by CD8(+) effector cells. Intriguingly, lymphocyte influx does not correlate with increased vessel leakiness. Instead, low-dose TNFα stabilizes the vascular network and improves vessel perfusion. Inflammatory vessel remodeling is, at least in part, mediated by tumor-resident macrophages that are reprogrammed to secrete immune and angiogenic modulators. Moreover, inflammatory vessel remodeling with low-dose TNFα substantially improves antitumor vaccination or adoptive T-cell therapy. Thus, low-dose TNFα promotes both vessel remodeling and antitumor immune responses and acts as a potent adjuvant for active immunotherapy.

Cellular and In Vivo Response to Industrial, Food Grade, and Photocatalytic TiO2 Nanoparticles.

We encounter titanium dioxide nanoparticles (TiO2 NPs) throughout our daily lives in the form of food coloring, cosmetics, and industrial materials. They are used on a massive industrial scale, with over 1 million metric tons in the global market. For the workers who process these materials, inhalation is a major concern. The goal of our current research is to provide a direct comparison of the three major types of TiO2 NPs (P25, E171, R101) in terms of surface characterization, cellular response, and in vivo response following introduction into the lungs of mice. In both cellular and in vivo experiments, we observe a pro-inflammatory response to the P25 TiO2 NPs that is not observed in the E171 or R101 TiO2 NPs at mass-matched concentrations. Cellular experiments measured a cytokine, TNF-α, as a marker of a pro-inflammatory response. In vivo experiments in mice measured the number of immune cells and four pro-inflammatory cytokines (IL-6, MIP-2, IP-10, and MCP-1) present in bronchoalveolar lavage fluid. A detailed physical and chemical characterization of the TiO2 NPs shows that the P25 TiO2 NPs are distinguished by smaller primary particles suggesting that samples matched by mass contain a larger number of P25 TiO2 NPs. Cellular dose-response measurements with the P25, E171, and R101 TiO2 NPs support this hypothesis showing increased TNF-α release by macrophages as a function of TiO2 NP dose. Overall, this direct comparison of the three major types of TiO2 NPs shows that the number of particles in a dose, which is dependent on the particle diameter, is a key parameter in TiO2 NP-induced inflammation.

House Dust Mite Proteins Adsorb on Multiwalled Carbon Nanotubes Forming an Allergen Corona That Intensifies Allergic Lung Disease in Mice.

The increasing use of multiwalled carbon nanotubes (MWCNTs) could increase the risk of allergic lung disease in occupational or consumer settings. We previously reported that MWCNTs exacerbated allergic lung disease in mice induced by extract from house dust mites (HDM), a common cause of asthma in humans. Because MWCNTs avidly bind biomolecules to form protein coronas that can modify immunotoxicity, we hypothesized that exacerbation of allergic lung disease in mice caused by coexposure to MWCNTs and HDM extract was due to the formation of an allergen corona. In a first set of experiments, male and female C57BL/6J mice were coexposed to MWCNTs and HDM extract over 3 weeks compared to MWCNTs or HDM extract alone. In a second set of experiments, mice were exposed to pristine MWCNTs or MWCNTs with an HDM allergen corona (HDM-MWCNTs). HDM-MWCNTs were formed by incubating MWCNTs with HDM extract, where ∼7% of proteins adsorbed to MWCNTs, including Der p 1 and Der p 2. At necropsy, bronchoalveolar lavage fluid was collected from lungs to assess lactate dehydrogenase, total protein and inflammatory cells, while lung tissue was used for histopathology, qPCR, and Western blotting. Compared to MWCNTs or HDM extract alone, coexposure to MWCNTs and HDM extract or exposure to HDM-MWCNTs increased pathological outcomes associated with allergic lung disease (eosinophilia, fibrosis, mucous cell metaplasia), increased mRNAs associated with fibrosis (Col1A1, Arg1) and enhanced STAT6 phosphorylation in lung tissue. These findings indicated that exacerbation of HDM-induced allergic lung disease by MWCNTs is due to an allergen corona.

House Dust Mite Extract Forms a Der p 2 Corona on Multi-Walled Carbon Nanotubes: Implications for Allergic Airway Disease.

Multi-walled carbons nanotubes (MWCNTs) are used in materials for the construction, automotive, and aerospace industries. Workers and consumers are exposed to these materials via inhalation. Existing recommended exposure limits are based on MWCNT exposures that do not take into account more realistic co-exposures. Our goal was to understand how a common allergen, house dust mites, interacts with pristine MWCNTs and lung fluid proteins. We used gel electrophoresis, western blotting, and proteomics to characterize the composition of the allergen corona formed from house dust mite extract on the surface of MWCNTs. We found that the corona is dominated by der p 2, a protein associated with human allergic responses to house dust mites. Der p 2 remains adsorbed on the surface of the MWCNTs following subsequent exposures to lung fluid proteins. The high concentration of der p 2, localized on surface of MWCNTs, has important implications for house dust mite-induced allergies and asthma. This research provides a detailed characterization of the complex house dust mite-lung fluid protein coronas for future cellular and in vivo studies. These studies will help to address the molecular and biochemical mechanisms underlying the exacerbation of allergic lung disease by nanomaterials.

Targeting Lyn tyrosine kinase through protein fusions encompassing motifs of Cbp (Csk-binding protein) and the SOCS box of SOCS1.

The tyrosine kinase Lyn is involved in oncogenic signalling in several leukaemias and solid tumours, and we have previously identified a pathway centred on Cbp [Csk (C-terminal Src kinase)-binding protein] that mediates both enzymatic inactivation, as well as proteasomal degradation of Lyn via phosphorylation-dependent recruitment of Csk (responsible for phosphorylating the inhibitory C-terminal tyrosine of Lyn) and SOCS1 (suppressor of cytokine signalling 1; an E3 ubiquitin ligase). In the present study we show that fusing specific functional motifs of Cbp and domains of SOCS1 together generates a novel molecule capable of directing the proteasomal degradation of Lyn. We have characterized the binding of pY (phospho-tyrosine) motifs of Cbp to SFK (Src-family kinase) SH2 (Src homology 2) domains, identifying those with high affinity and specificity for the SH2 domain of Lyn and that are preferred substrates of active Lyn. We then fused them to the SB (SOCS box) of SOCS1 to facilitate interaction with the ubiquitination-promoting elongin B/C complex. As an eGFP (enhanced green fluorescent protein) fusion, these proteins can direct the polyubiquitination and proteasomal degradation of active Lyn. Expressing this fusion protein in DU145 cancer cells (but not LNCaP or MCF-7 cells), that require Lyn signalling for survival, promotes loss of Lyn, loss of caspase 3, appearance of an apoptotic morphology and failure to survive/expand. These findings show how functional domains of Cbp and SOCS1 can be fused together to generate molecules capable of inhibiting the growth of cancer cells that express high levels of active Lyn.

Interaction of TiO2 nanoparticles with lung fluid proteins and the resulting macrophage inflammatory response.

Inhalation is a major exposure route to nanoparticles. Following inhalation, nanoparticles first interact with the lung lining fluid, a complex mixture of proteins, lipids, and mucins. We measure the concentration and composition of lung fluid proteins adsorbed on the surface of titanium dioxide (TiO2) nanoparticles. Using proteomics, we find that lung fluid results in a unique protein corona on the surface of the TiO2 nanoparticles. We then measure the expression of three cytokines (interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-α), and macrophage inflammatory protein 2 (MIP-2)) associated with lung inflammation. We find that the corona formed from lung fluid leads to elevated expression of these cytokines in comparison to bare TiO2 nanoparticles or coronas formed from serum or albumin. These experiments show that understanding the concentration and composition of the protein corona is essential for understanding the pulmonary response associated with human exposure to nanoparticles.