Chemical and Cellular Formation of Reactive Oxygen Species from Secondary Organic Aerosols in Epithelial Lining Fluid.

INTRODUCTION: Oxidative stress mediated by reactive oxygen species (ROS) is a key process for adverse aerosol health effects. Secondary organic aerosols (SOA) account for a major fraction of particulate matter with aerodynamic diameter ≤2.5 µm (PM2.5). PM2.5 inhalation and deposition into the respiratory tract causes the formation of ROS by chemical reactions and phagocytosis of macrophages in the epithelial lining fluid (ELF), but their relative contributions are not well quantified and their link to oxidative stress remains uncertain. The specific aims of this project were (1) elucidating the chemical mechanism and quantifying the formation kinetics of ROS in the ELF by SOA; (2) quantifying the relative importance of ROS formation by chemical reactions and macrophages in the ELF. METHODS: SOA particles were generated using reaction chambers from oxidation of various precursors including isoprene, terpenes, and aromatic compounds with or without nitrogen oxides (NOx). We collected size-segregated PM at two highway sites in Anaheim, CA, and Long Beach, CA, and at an urban site in Irvine, CA, during two wildfire events. The collected particles were extracted into water or surrogate ELF that contained lung antioxidants. ROS generation was quantified using electron paramagnetic resonance (EPR) spectroscopy with a spin-trapping technique. PM oxidative potential (OP) was also quantified using the dithiothreitol assay. In addition, kinetic modeling was applied for analysis and interpretation of experimental data. Finally, we quantified cellular superoxide release by RAW264.7 macrophage cells upon exposure to quinones and isoprene SOA using a chemiluminescence assay as calibrated with an EPR spin-probing technique. We also applied cellular imaging techniques to study the cellular mechanism of superoxide release and oxidative damage on cell membranes. RESULTS: Superoxide radicals (·O2-) were formed from aqueous reactions of biogenic SOA generated by hydroxy radical (·OH) photooxidation of isoprene, ß-pinene, α-terpineol, and d-limonene. The temporal evolution of ·OH and ·O2- formation was elucidated by kinetic modeling with a cascade of aqueous reactions, including the decomposition of organic hydroperoxides (ROOH), ·OH oxidation of primary or secondary alcohols, and unimolecular decomposition of α-hydroxyperoxyl radicals. Relative yields of various types of ROS reflected the relative abundance of ROOH and alcohols contained in SOA, which generated under high NOx conditions, exhibited lower ROS yields. ROS formation by SOA was also affected by pH. Isoprene SOA had higher ·OH and organic radical yields at neutral than at acidic pH. At low pH ·O2- was the dominant species generated by all types of SOA. At neutral pH, α-terpineol SOA exhibited a substantial yield of carbon-centered organic radicals (R·), while no radical formation was observed by aromatic SOA.Organic radicals in the ELF were formed by mixtures of Fe2+ and SOA generated from photooxidation of isoprene, α-terpineol, and toluene. The molar yields of organic radicals by SOA were 5-10 times higher in ELF than in water. Fe2+ enhanced organic radical yields by a factor of 20-80. Ascorbate mediated redox cycling of iron ions and sustained organic peroxide decomposition, as supported by kinetic modeling reproducing time- and concentration-dependence of organic radical formation, as well as by additional experiments observing the formation of Fe2+ and ascorbate radicals in mixtures of ascorbate and Fe3+. ·OH and superoxide were found to be efficiently scavenged by antioxidants.Wildfire PM mainly generated ·OH and R· with minor contributions from superoxide and oxygen-centered organic radicals (RO·). PM OP was high in wildfire PM, exhibiting very weak correlation with radical forms of ROS. These results were in stark contrast with PM collected at highway and urban sites, which generated much higher amounts of radicals dominated by ·OH radicals that correlated well with OP. By combining field measurements of size-segregated chemical composition, a human respiratory tract model, and kinetic modeling, we quantified production rates and concentrations of different types of ROS in different regions of the ELF by considering particle-size-dependent respiratory deposition. While hydrogen peroxide (H2O2) and ·O2- production were governed by Fe and Cu ions, ·OH radicals were mainly generated by organic compounds and Fenton-like reactions of metal ions. We obtained mixed results for correlations between PM OP and ROS formation, providing rationale and limitations of the use of oxidative potential as an indicator for PM toxicity in epidemiological and toxicological studies.Quinones and isoprene SOA activated nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in macrophages, releasing massive amounts of superoxide via respiratory burst and overwhelming the superoxide formation by aqueous chemical reactions in the ELF. The threshold dose for macrophage activation was much smaller for quinones compared with isoprene SOA. The released ROS caused lipid peroxidation to increase cell membrane fluidity, inducing oxidative damage and stress. Further increases of doses led to the activation of antioxidant response elements, reducing the net cellular superoxide production. At very high doses and long exposure times, chemical production became comparably important or dominant if the escalation of oxidative stress led to cell death. CONCLUSIONS: The mechanistic understandings and quantitative information on ROS generation by SOA particles provided a basis for further elucidation of adverse aerosol health effects and oxidative stress by PM2.5. For a comprehensive assessment of PM toxicity and health effects via oxidative stress, it is important to consider both chemical reactions and cellular processes for the formation of ROS in the ELF. Chemical composition of PM strongly influences ROS formation; further investigations are required to study ROS formation from various PM sources. Such research will provide critical information to environmental agencies and policymakers for the development of air quality policy and regulation.

An apolipoprotein-enriched biomolecular corona switches the cellular uptake mechanism and trafficking pathway of lipid nanoparticles.

Following exposure to biological milieus (e.g. after systemic administration), nanoparticles (NPs) get covered by an outer biomolecular corona (BC) that defines many of their biological outcomes, such as the elicited immune response, biodistribution, and targeting abilities. In spite of this, the role of BC in regulating the cellular uptake and the subcellular trafficking properties of NPs has remained elusive. Here, we tackle this issue by employing multicomponent (MC) lipid NPs, human plasma (HP) and HeLa cells as models for nanoformulations, biological fluids, and target cells, respectively. By conducting confocal fluorescence microscopy experiments and image correlation analyses, we quantitatively demonstrate that the BC promotes a neat switch of the cell entry mechanism and subsequent intracellular trafficking, from macropinocytosis to clathrin-dependent endocytosis. Nano-liquid chromatography tandem mass spectrometry identifies apolipoproteins as the most abundant components of the BC tested here. Interestingly, this class of proteins target the LDL receptors, which are overexpressed in clathrin-enriched membrane domains. Our results highlight the crucial role of BC as an intrinsic trigger of specific NP-cell interactions and biological responses and set the basis for a rational exploitation of the BC for targeted delivery.

Huntington's disease cerebrospinal fluid seeds aggregation of mutant huntingtin.

Huntington's disease (HD), a progressive neurodegenerative disease, is caused by an expanded CAG triplet repeat producing a mutant huntingtin protein (mHTT) with a polyglutamine-repeat expansion. Onset of symptoms in mutant huntingtin gene-carrying individuals remains unpredictable. We report that synthetic polyglutamine oligomers and cerebrospinal fluid (CSF) from BACHD transgenic rats and from human HD subjects can seed mutant huntingtin aggregation in a cell model and its cell lysate. Our studies demonstrate that seeding requires the mutant huntingtin template and may reflect an underlying prion-like protein propagation mechanism. Light and cryo-electron microscopy show that synthetic seeds nucleate and enhance mutant huntingtin aggregation. This seeding assay distinguishes HD subjects from healthy and non-HD dementia controls without overlap (blinded samples). Ultimately, this seeding property in HD patient CSF may form the basis of a molecular biomarker assay to monitor HD and evaluate therapies that target mHTT.

Mechanistic evaluation of the transfection barriers involved in lipid-mediated gene delivery: interplay between nanostructure and composition.

Here we present a quantitative mechanism-based investigation aimed at comparing the cell uptake, intracellular trafficking, endosomal escape and final fate of lipoplexes and lipid-protamine/deoxyribonucleic acid (DNA) (LPD) nanoparticles (NPs) in living Chinese hamster ovary (CHO) cells. As a model, two lipid formulations were used for comparison. The first formulation is made of the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and the zwitterionic lipid dioleoylphosphocholine (DOPC), while the second mixture is made of the cationic 3ß-[N-(N,N-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and the zwitterionic helper lipid dioleoylphosphatidylethanolamine (DOPE). Our findings indicate that lipoplexes are efficiently taken up through fluid-phase macropinocytosis, while a less efficient uptake of LPD NPs occurs through a combination of both macropinocytosis and clathrin-dependent pathways. Inside the cell, both lipoplexes and LPD NPs are actively transported towards the cell nucleus, as quantitatively addressed by spatio-temporal image correlation spectroscopy (STICS). For each lipid formulation, LPD NPs escape from endosomes more efficiently than lipoplexes. When cells were treated with DOTAP-DOPC-containing systems the majority of the DNA was trapped in the lysosome compartment, suggesting that extensive lysosomal degradation was the rate-limiting factors in DOTAP-DOPC-mediated transfection. On the other side, escape from endosomes is large for DC-Chol-DOPE-containing systems most likely due to DOPE and cholesterol-like molecules, which are able to destabilize the endosomal membrane. The lipid-dependent and structure-dependent enhancement of transfection activity suggests that DNA is delivered to the nucleus synergistically: the process requires both the membrane-fusogenic activity of the nanocarrier envelope and the employment of lipid species with intrinsic endosomal rupture ability.

Quantitative measurement of intracellular transport of nanocarriers by spatio-temporal image correlation spectroscopy.

Spatio-temporal image correlation spectroscopy (STICS) is a powerful technique for assessing the nature of particle motion in complex systems although it has been rarely used to investigate the intracellular dynamics of nanocarriers so far. Here we introduce a method to characterize the mode of motion of nanocarriers and to quantify their transport parameters on different length scales from single-cell to subcellular level. Using this strategy we were able to study the mechanisms responsible for the intracellular transport of DOTAP-DOPC/DNA and DC-Chol-DOPE/DNA lipoplexes in CHO-K1 live cells. Measurement of both diffusion coefficients and velocity vectors (magnitude and direction) averaged over regions of the cell revealed the presence of distinct modes of motion. Lipoplexes diffused slowly on the cell surface (diffusion coefficient, D ≈ 0.003 µm2/s). In the cytosol, the lipoplexes' motion was characterized by active transport with average velocity ν ≈ 0.03 µm/s and random motion. The method permitted us to generate intracellular transport map showing several regions of concerted motion of lipoplexes.

Role of temperature-independent lipoplex-cell membrane interactions in the efficiency boost of multicomponent lipoplexes.

Multicomponent lipoplexes have recently emerged as especially promising transfection candidates, as they are from 10 to 100 times more efficient than binary complexes usually employed for gene delivery purposes. Previously, we investigated a number of chemical-physical properties of DNA-lipid complexes that were proposed to affect transfection efficiency (TE) of lipoplexes, such as nanoscale structure, size, surface potential, DNA-protection ability and DNA release from complexes upon interaction with cellular lipids. Although some minor differences between multicomponent and binary lipoplexes were found, they did not correlate clearly with efficiency. Instead, here we show that a marked difference between the cell internalization mechanism of binary and multicomponent lipoplexes does exist. Multicomponent lipoplexes significantly transfect cells at 4 °C, when endocytosis does not take place suggesting that they can enter cells via a temperature-independent mechanism. Confocal fluorescence microscopy experiments showed the existence of a correlation between endosomal escape and TE. Multicomponent lipoplexes exhibited a distinctive ability of endosomal escape and release DNA into the nucleus, whereas, poorly efficient binary lipoplexes exhibited minor, if any, endosomal rupture ability and remained confined in perinuclear late endosomes. Stopped-flow mixing measurements showed that the fusion rates of multicomponent cationic liposomes with anionic vesicles, used as model systems of cell membranes, were definitely shorter than those of binary liposomes. As either lipoplex uptake and endosomal escape involve fusion between lipoplex and cellular membranes, we suggest that a mechanism of lipoplex-cellular membrane interaction, driven by lipid mixing between cationic and anionic cellular lipids, does explain the TE boost of multicomponent lipoplexes.

Fluctuation methods to study protein aggregation in live cells: concanavalin A oligomers formation.

Prefibrillar oligomers of proteins are suspected to be the primary pathogenic agents in several neurodegenerative diseases. A key approach for elucidating the pathogenic mechanisms is to probe the existence of oligomers directly in living cells. In this work, we were able to monitor the process of aggregation of Concanavalin A in live cells. We used number and brightness analysis, two-color cross number and brightness analysis, and Raster image correlation spectroscopy to obtain the number of molecules, aggregation state, and diffusion coefficient as a function of time and cell location. We observed that binding of Concanavalin A to the membrane and the formation of small aggregates paralleled cell morphology changes, indicating progressive cell compaction and death. Upon protein aggregation, we observed increased membrane water penetration as reported by Laurdan generalized polarization imaging.

Raster image correlation spectroscopy (RICS) for measuring fast protein dynamics and concentrations with a commercial laser scanning confocal microscope.

Raster image correlation spectroscopy (RICS) is a new and novel technique for measuring molecular dynamics and concentrations from fluorescence confocal images. The RICS technique extracts information about molecular dynamics and concentrations from images of living cells taken on commercial confocal systems. Here we develop guidelines for performing the RICS analysis on an analogue commercial laser scanning confocal microscope. Guidelines for typical instrument settings, image acquisition settings and analogue detector characterization are presented. Using appropriate instrument/acquisition parameters, diffusion coefficients and concentrations can be determined, even for highly dynamic dye molecules in solution. Standard curves presented herein demonstrate the ability to detect protein concentrations as low as approximately 2 nM. Additionally, cellular measurements give accurate values for the diffusion of paxillin-enhanced-green fluorescent protein (EGFP), an adhesion adaptor molecule, in the cytosol of the cell and also show slower paxillin dynamics near adhesions where paxillin interacts with immobile adhesion components. Methods are presented to account for bright immobile structures within the cell that dominate spatial correlation functions; allowing the extraction of fast protein dynamics within and near these structures. A running average algorithm is also presented to address slow cellular movement or movement of cellular features such as adhesions. Finally, methods to determine protein concentration in the presence of immobile structures within the cell are presented. A table is presented giving guidelines for instrument and imaging setting when performing RICS on the Olympus FV300 confocal and these guidelines are a starting point for performing the analysis on other commercial confocal systems.