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.

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.

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.

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.

Imaging lysosomal enzyme activity in live cells using self-quenched substrates.

Endocytosis, the internalization and transport of extracellular cargo, is an essential cellular process. The ultimate step in endocytosis is the intracellular degradation of extracellular cargo for use by the cell. While live cell imaging and single particle tracking have been well-utilized to study the internalization and transport of cargo, the final degradation step has required separate biochemical assays. We describe the use of self-quenched endocytic cargo to image the intracellular transport and degradation of endocytic cargo directly in live cells. We first outline the fluorescent labeling and quantification of two common endocytic cargos: a protein, bovine serum albumin, and a lipid nanoparticle, low-density lipoprotein. In vitro measurements confirm that self-quenching is a function of the number of fluorophores bound to the protein or particle and that recovery of the fluorescent signal occurs in response to enzymatic degradation. We then use confocal fluorescence microscopy and flow cytometry to demonstrate the use of self-quenched bovine serum albumin with standard fluorescence techniques. Using live cell imaging and single particle tracking, we find that the degradation of bovine serum albumin occurs in an endo-lysosomal vesicle that is positive for LAMP1.

Single particle tracking as a method to resolve differences in highly colocalized proteins.

Single particle tracking fluorescence microscopy was used to study two late endosomal proteins, Rab7 and LAMP1, that appear to be highly colocalized in static fluorescence microscopy images. Imaging these proteins simultaneously reveals that Rab7 and LAMP1 undergo periods of separation within the cell. Single particle tracking carried out during these periods of separation shows that Rab7-vesicles have greater velocities, but undergo less efficient transport than LAMP1-vesicles. This research demonstrates the use of single particle tracking as a tool to resolve functional differences in highly colocalized proteins in intact live cells.

Electrical Control of Escherichia coli Growth Measured with Simultaneous Modulation and Imaging.

Background: The use of electricity to mediate bacterial growth is unique in providing spatial control, but requires a more detailed understanding. Methods: We use two gold wires on a glass coverslip with an overlayer of agar to image Escherichia coli cells with brightfield and fluorescence microscopy while simultaneously applying a voltage. Cells outside of the wires provide a control population to measure cell growth as a function of voltage, rather than any difference in culture conditions or growth phase. Results: An applied voltage suppresses the fraction of E. coli undergoing elongation and division with recovery to control values when the voltage is removed. Depolarization is observed over the same voltage range suggesting a membrane potential-mediated response. Conclusions: Our experiments identify and use subcytotoxic voltages to measure differences in the fraction of E. coli cells elongating and dividing as a function of applied voltage. It is hoped that this research will inform the developing field of bacterial electrophysiology.

DNA-nanoparticle interactions: Formation of a DNA corona and its effects on a protein corona.

There has been much recent interest in the protein "corona," the nonspecific adsorption of proteins on the surface of nanoparticles used in biological applications. This research investigates an analogous DNA corona. We find that particles (200 nm and 1 µm) incubated with DNA form a DNA corona, with a higher concentration of DNA adsorbed on the surface of cationic nanoparticles. With protein present, a combined DNA and protein corona is formed although DNA in solution displaces protein from the nanoparticle surface. Displacement of protein from the nanoparticle surface is dependent on the concentration of DNA in solution and was also observed for planar surfaces. Overall, we expect this investigation of the DNA corona to be important for nanomedicine applications, as well as disease states, especially systemic lupus erythematosus, in which biological particles with bound DNA are important mediators of inflammation and thrombosis.

Pyrenebutyrate-mediated delivery of quantum dots across the plasma membrane of living cells.

Quantum dots have been delivered directly across the plasma membrane to the cytosol of living cells using a combination of a cationic peptide, polyarginine, and a hydrophobic counterion, pyrenebutyrate. Quantum dot delivery did not disrupt the plasma membrane and bypassed the barrier of endocytic vesicles. Cellular uptake was independent of temperature but highly dependent on the surface charge of the quantum dot and the membrane potential of the cell, suggesting a direct translocation across the membrane. This method of delivery can find immediate application for quantum dots and may be broadly applicable to other nanoparticles.