Combining analytical techniques to assess the translocation of diesel particles across an alveolar tissue barrier in vitro.

BACKGROUND: During inhalation, airborne particles such as particulate matter ≤ 2.5 µm (PM2.5), can deposit and accumulate on the alveolar epithelial tissue. In vivo studies have shown that fractions of PM2.5 can cross the alveolar epithelium to blood circulation, reaching secondary organs beyond the lungs. However, approaches to quantify the translocation of particles across the alveolar epithelium in vivo and in vitro are still not well established. In this study, methods to assess the translocation of standard diesel exhaust particles (DEPs) across permeable polyethylene terephthalate (PET) inserts at 0.4, 1, and 3 µm pore sizes were first optimized with transmission electron microscopy (TEM), ultraviolet-visible spectroscopy (UV-VIS), and lock-in thermography (LIT), which were then applied to study the translocation of DEPs across human alveolar epithelial type II (A549) cells. A549 cells that grew on the membrane (pore size: 3 µm) in inserts were exposed to DEPs at different concentrations from 0 to 80 µg.mL- 1 ( 0 to 44 µg.cm- 2) for 24 h. After exposure, the basal fraction was collected and then analyzed by combining qualitative (TEM) and quantitative (UV-VIS and LIT) techniques to assess the translocated fraction of the DEPs across the alveolar epithelium in vitro. RESULTS: We could detect the translocated fraction of DEPs across the PET membranes with 3 µm pore sizes and without cells by TEM analysis, and determine the percentage of translocation at approximatively 37% by UV-VIS (LOD: 1.92 µg.mL- 1) and 75% by LIT (LOD: 0.20 µg.cm- 2). In the presence of cells, the percentage of DEPs translocation across the alveolar tissue was determined around 1% at 20 and 40 µg.mL- 1 (11 and 22 µg.cm- 2), and no particles were detected at higher and lower concentrations. Interestingly, simultaneous exposure of A549 cells to DEPs and EDTA can increase the translocation of DEPs in the basal fraction. CONCLUSION: We propose a combination of analytical techniques to assess the translocation of DEPs across lung tissues. Our results reveal a low percentage of translocation of DEPs across alveolar epithelial tissue in vitro and they correspond to in vivo findings. The combination approach can be applied to any traffic-generated particles, thus enabling us to understand their involvement in public health.

The Influence of Liquid Menisci on Nanoparticle Dosimetry in Submerged Cells.

Understanding the interaction between cells and nanoparticles (NPs) is vital to understand the hazard associated with nanoparticles. This requires quantifying and interpreting dose-response relationships. Experiments with cells cultured in vitro and exposed to particle dispersions mainly rely on mathematical models that estimate the received nanoparticle dose. However, models need to consider that aqueous cell culture media wets the inner surface of hydrophilic open wells, which results in a curved liquid-air interface called the meniscus. Here the impact of the meniscus on nanoparticle dosimetry is addressed in detail. Experiments and build an advanced mathematical model, to demonstrate that the presence of the meniscus may bring about systematic errors that must be considered to advance reproducibility and harmonization is presented. The script of the model is co-published and can be adapted to any experimental setup. Finally, simple and practical solutions to this problem, such as covering the air-liquid interface with a permeable lid or soft rocking of the cell culture well plate is proposed.

Preparation of Well-Defined Fluorescent Nanoplastic Particles by Confined Impinging Jet Mixing.

Research on the origin, distribution, detection, identification, and quantification of polymer nanoparticles (NPs) in the environment and their possible impact on animal and human health is surging. For different types of studies in this field, well-defined reference materials or mimics are needed. While isolated reports on the preparation of such materials are available, a simple and broadly applicable method that allows for the production of different NP types with well-defined, tailorable characteristics is still missing. Here, we demonstrate that a confined impinging jet mixing process can be used to prepare colloidally stable NPs based on polystyrene, polyethylene, polypropylene, and poly(ethylene terephthalate) with diameters below < 100 nm. Different fluorophores were incorporated into the NPs, to allow their detection in complex environments. To demonstrate their utility and detectability, fluorescent NPs were exposed to J774A.1 macrophages and visualized using laser scanning microscopy. Furthermore, we modified the NPs in a postfabrication process and changed their shape from spherical to heterogeneous geometries, in order to mimic environmentally relevant morphologies. The methodology used here should be readily applicable to other polymers and payloads and thus a broad range of NPs that enable studies of their behavior, uptake, translocation, and biological end points in different systems.

Pitfalls in methods to study colocalization of nanoparticles in mouse macrophage lysosomes.

BACKGROUND: In the field of nanoscience there is an increasing interest to follow dynamics of nanoparticles (NP) in cells with an emphasis on endo-lysosomal pathways and long-term NP fate. During our research on this topic, we encountered several pitfalls, which can bias the experimental outcome. We address some of these pitfalls and suggest possible solutions. The accuracy of fluorescence microscopy methods has an important role in obtaining insights into NP interactions with lysosomes at the single cell level including quantification of NP uptake in a specific cell type. METHODS: Here we use J774A.1 cells as a model for professional phagocytes. We expose them to fluorescently-labelled amorphous silica NP with different sizes and quantify the colocalization of fluorescently-labelled NP with lysosomes over time. We focus on confocal laser scanning microscopy (CLSM) to obtain 3D spatial information and follow live cell imaging to study NP colocalization with lysosomes. RESULTS: We evaluate different experimental parameters that can bias the colocalization coefficients (i.e., Pearson's and Manders'), such as the interference of phenol red in the cell culture medium with the fluorescence intensity and image post-processing (effect of spatial resolution, optical slice thickness, pixel saturation and bit depth). Additionally, we determine the correlation coefficients for NP entering the lysosomes under four different experimental set-ups. First, we found out that not only Pearson's, but also Manders' correlation coefficient should be considered in lysosome-NP colocalization studies; second, there is a difference in NP colocalization when using NP of different sizes and fluorescence dyes and last, the correlation coefficients might change depending on live-cell and fixed-cell imaging set-up. CONCLUSIONS: The results summarize detailed steps and recommendations for the experimental design, staining, sample preparation and imaging to improve the reproducibility of colocalization studies between the NP and lysosomes.

Shear Stress-Responsive Polymersome Nanoreactors Inspired by the Marine Bioluminescence of Dinoflagellates.

Some marine plankton called dinoflagellates emit light in response to the movement of surrounding water, resulting in a phenomenon called milky seas or sea sparkle. The underlying concept, a shear-stress induced permeabilisation of biocatalytic reaction compartments, is transferred to polymer-based nanoreactors. Amphiphilic block copolymers that carry nucleobases in their hydrophobic block are self-assembled into polymersomes. The membrane of the vesicles can be transiently switched between an impermeable and a semipermeable state by shear forces occurring in flow or during turbulent mixing of polymersome dispersions. Nucleobase pairs in the hydrophobic leaflet separate when mechanical force is applied, exposing their hydrogen bonding motifs and therefore making the membrane less hydrophobic and more permeable for water soluble compounds. This polarity switch is used to release payload of the polymersomes on demand, and to activate biocatalytic reactions in the interior of the polymersomes.

Spatially Resolved Production of Platinum Nanoparticles in Metallosupramolecular Polymers.

Nanocomposites consisting of a polymer matrix and metallic nanoparticles can merge the functional, structural, and mechanical properties of the two components and are useful for applications that range from catalysis to soft electronics. Gaining spatial control over the nanoparticle incorporation is useful, for example to confine catalytic sites or create electrically conducting pathways. Here, we show that this is possible by the controlled disassembly of a metallosupramolecular polymer containing zerovalent platinum complexes to form nanoparticles in situ. To achieve this, a telechelic poly(ethylene-co-butylene) was end-functionalized with diphenylacetylene ligands and chain-extended through the formation of bis(η2-alkyne)Pt0 complexes. These complexes are stable at ambient conditions, but they can be dissociated upon heating or exposure to ultraviolet light, which allows producing Pt nanoparticles when and where needed and without auxiliary reagents or formation of byproducts. This approach was exploited to create objects with well-defined catalytically active areas.

Hydrodynamic Radius of Polymer-Coated Nanoparticles Measured by Taylor Dispersion: A Mathematical Model.

This theoretical work addresses the characterization of polymer-coated nanoparticles via the analysis of Taylor dispersion experiments. Our focus is on determining the apparent hydrodynamic radius and the related accuracy bias, which results from polydispersity and optical-absorption-weighted averages. To that end, we construct a statistical model addressing joint distributions of particle core size and ligand surface density, which determine the hydrodynamic radius and optical absorption of such nanoparticles. Our model predicts that a polymer shell that is thick compared with the core radius results in a smaller bias than a thin shell, and the bias may become even negative when ligand surface density is sufficiently high.

Resolution Limit of Taylor Dispersion: An Exact Theoretical Study.

Taylor dispersion is a microfluidic analytical technique with a high dynamic range and therefore is suited well to measuring the hydrodynamic radius of small molecules, proteins, supramolecular complexes, macromolecules, nanoparticles and their self-assembly. Here we calculate an unaddressed yet fundamental property: the limit of resolution, which is defined as the smallest change in the hydrodynamic radius that Taylor dispersion can resolve accurately and precisely. Using concepts of probability theory and inferential statistics, we present a comprehensive theoretical approach, addressing uniform and polydisperise particle systems, which involve either model-based or numerical analyses. We find a straightforward scaling relationship in which the resolution limit is linearly proportional to the optical-extinction-weighted average hydrodynamic radius of the particle systems.

Biocatalytically Initiated Precipitation Atom Transfer Radical Polymerization (ATRP) as a Quantitative Method for Hemoglobin Detection in Biological Fluids.

The hemoglobin content of blood is an important health indicator, and the presence of microscopic amounts of hemoglobin in places where it normally does not occur, e.g. in blood plasma or in urine, is a sign of diseases such as hemolytic anemia or urinary tract infections. Thus, methods to detect and quantify hemoglobin are important for clinical laboratories, blood banks, and for point-of-care diagnostics. The precipitation polymerization of N-isopropylacrylamide by hemoglobin-catalyzed atom transfer radical polymerization (ATRP) is used as an assay for hemoglobin quantification relying on the formation of turbidity as a simple optical read-out. Dose-response curves for pure hemoglobin and for hemoglobin in blood plasma, in urine, in erythrocytes, and in full blood are obtained. Turbidity formation increases with the concentration of hemoglobin. Concentrations of hemoglobin as low as 6.45 × 10-3 mg mL-1 in solution, 4.88 × 10-1 mg mL-1 in plasma, and 1.65 × 10-1 mg mL-1 in urine could be detected, which is below the clinically relevant concentrations in the respective body fluids. Total hemoglobin in full blood is also accurately determined. The reaction can be regarded as a polymerization-based signal amplification for the sensing of hemoglobin, as the analyte catalyzes the formation of radicals which add many monomer units into detectable polymer chains. While most established hemoglobin tests involve the use of highly toxic reagents such as potassium cyanide, the polymerization-based test uses simple and stable organic reagents. Thus, it is an environmentally friendlier alternative to established chemical assays for hemoglobin.

Versatile Macroscale Concentration Gradients of Nanoparticles in Soft Nanocomposites.

Nanocomposite materials benefit from the diverse physicochemical properties featured by nanoparticles, and the presence of nanoparticle concentration gradients can lend functions to macroscopic materials beyond the realm of classical nanocomposites. It is shown here that linearity and time-shift invariance obtained via the synergism of two independent physical phenomena-translational self-diffusion and shear-driven dispersion-may give access to an exceptionally high degree of flexibility in the design of scalable and programmable long-range concentration gradients of nanoparticles in solidifiable liquid matrices.

Long-Lived Photocharges in Supramolecular Polymers of Low-Band-Gap Chromophores.

Photoinduced charge separation in supramolecular aggregates of π-conjugated molecules is a fundamental photophysical process and a key criterion for the development of advanced organic electronics materials. Herein, the self-assembly of low-band-gap chromophores into helical one-dimensional aggregates, due to intermolecular hydrogen bonding, is reported. Chromophores confined in these supramolecular polymers show strong excitonic coupling interactions and give rise to charge-separated states with unusually long lifetimes of several hours and charge densities of up to 5 mol % after illumination with white light. Two-contact devices exhibit increased photoconductivity and can even show Ohmic behavior. These findings demonstrate that the confinement of organic semiconductors into one-dimensional aggregates results in a considerable stabilization of charge carriers for a variety of π-conjugated systems, which may have implications for the design of future organic electronic materials.

Tuning the Thickness of a Biomembrane by Stapling Diamidophospholipids with Bolalipids.

In a biological membrane, proteins require specific lipids of distinctive length and chain saturation surrounding them. The active tuning of the membrane thickness therefore opens new possibilities in the study and manipulation of membrane proteins. Here, we introduce the concept of stapling phospholipids to different degrees of interdigitation depth by mixing 1,3-diamidophospholipids with single-chain bolalipids. The mixed membranes were studied by calorimetric assays, electron microscopy, X-ray, and infrared measurements to provide a complete biophysical characterization of membrane stapling. The matching between the diamidophospholipids and the bolalipids can be so strong as to completely induce a new phase that is more stable than the gel phase of the individual components.

Rapid quantification of the malaria biomarker hemozoin by improved biocatalytically initiated precipitation atom transfer radical polymerizations.

The fight against tropical diseases such as malaria requires the development of innovative biosensing techniques. Diagnostics must be rapid and robust to ensure prompt case management and to avoid further transmission. The malaria biomarker hemozoin can catalyze atom transfer radical polymerizations (ATRP), which we exploit in a polymerization-amplified biosensing assay for hemozoin based on the precipitation polymerization of N-isopropyl acrylamide (NIPAAm). The reaction conditions are systematically investigated using synthetic hemozoin to gain fundamental understanding of the involved reactions and to greatly reduce the amplification time, while maintaining the sensitivity of the assay. The use of excess ascorbate allows oxygen to be consumed in situ but leads to the formation of reactive oxygen species and to the decomposition of the initiator 2-hydroxyethyl 2-bromoisobutyrate (HEBIB). Addition of sodium dodecyl sulfate (SDS) and pyruvate results in better differentiation between the blank and hemozoin-containing samples. Optimized reaction conditions (including reagents, pH, and temperature) reduce the amplification time from 37 ± 5 min to 3 ± 0.5 min while maintaining a low limit of detection of 1.06 ng mL-1. The short amplification time brings the precipitation polymerization assay a step closer to a point-of-care diagnostic device for malaria. Future efforts will be dedicated to the isolation of hemozoin from clinical samples.

Nanoparticles and Taylor Dispersion as a Linear Time-Invariant System.

The physical principles underpinning Taylor dispersion offer a high dynamic range to characterize the hydrodynamic radius of particles. While Taylor dispersion grants the ability to measure radius within nearly 5 orders of magnitude, the detection of particles is never instantaneous. It requires a finite sample volume, a finite detector area, and a finite detection time for measuring absorbance. First we show that these practical requirements bias the analysis when the self-diffusion coefficient of particles is high, which is typically the case of small nanoparticles. Second we show that the accuracy of the technique may be recovered by treating Taylor dispersion as a linear time-invariant system, which we prove by analyzing the Taylor dispersion spectra of two iron-oxide nanoparticles measured under identical experimental conditions. The consequence is that such treatment may be necessary whenever Taylor dispersion analysis is not optimized for a given size but dedicated to characterize broad groups of particles of varying size and material.

Precision of Taylor Dispersion.

Taylor dispersion is capable of measuring accurately the hydrodynamic radius over several orders of magnitude. Accordingly, it is now a highly competitive technique dedicated to characterizing small molecules, proteins, macromolecules, nanoparticles, and their self-assembly. Regardless, an in-depth analysis addressing the precision of the technique, being a key indicator of reproducibility, is not available. Benefiting from analytical modeling and statistical analysis, we address error propagation and present a comprehensive theoretical study of the precision of Taylor dispersion. Theory is then compared against experiment, and we find full consistency. Our results are most helpful when the design, objectives, or control of analytical quality is in focus.

The influence of substituents on gelation and stacking order of oligoaramid - based supramolecular networks.

Self-assembly has proven to be a powerful tool for functional, smart materials such as hydrogels derived from low molecular weight compounds. However, the targeted design of functional gelators remains difficult. Here, we present a set of four Y-shaped aromatic amide tetramers with varying functionalities able to undergo different non-covalent interactions. These compounds were explored towards their self-assembly behavior and hydrogel formation by experimental methods such as UV-vis spectroscopy, rheology, small angle X-ray scattering (SAXS), scanning/transmission electron, and atomic force microscopy. Additionally, we investigated the main mechanisms behind oligomer aggregation and the structure of the resulting supramolecular chains through full atomistic molecular dynamics simulations.

Wavelength-Selective Light-Responsive DASA-Functionalized Polymersome Nanoreactors.

Transient activation of biochemical reactions by visible light and subsequent return to the inactive state in the absence of light is an essential feature of the biochemical processes in photoreceptor cells. To mimic such light-responsiveness with artificial nanosystems, polymersome nanoreactors were developed that can be switched on by visible light and self-revert fast in the dark at room temperature to their inactive state. Donor-acceptor Stenhouse adducts (DASAs), with their ability to isomerize upon irradiation with visible light, were employed to change the permeability of polymersome membranes by switching polarity from a nonpolar triene-enol form to a cyclopentenone with increased polarity. To this end, amphiphilic block copolymers containing poly(pentafluorophenyl methacrylate) in their hydrophobic block were synthesized by reversible addition-fragmentation chain-transfer (RAFT) radical polymerization and functionalized either with a DASA that is based on Meldrum's acid or with a novel fast-switching pyrazolone-based DASA. These polymers were self-assembled into vesicles. Release of hydrophilic payload could be triggered by light and stopped as soon as the light was turned off. The encapsulation of enzymes yielded photoresponsive nanoreactors that catalyzed reactions only if they were irradiated with light. A mixture of polymersome nanoreactors, one that switches in green light, the other switching in red light, permitted specific control of the individual reactions of a reaction cascade in one pot by irradiation with varied wavelengths, thus enabling light-controlled wavelength-selective catalysis. The DASA-based nanoreactors demonstrate the potential of DASAs to switch permeability of membranes and could find application to switch reactions on and off, on demand, e.g., in microfluidics or in drug delivery.

Taylor Dispersion of Polydisperse Nanoclusters and Nanoparticles: Modeling, Simulation, and Analysis.

The dimensions of ultrasmall inorganic nanoparticles (US-NPs) is in the heart of the design of diagnostic and therapeutic efficacy; yet its accurate measurement is challenging for most experimental techniques. We show here how to design and analyze Taylor dispersion experiments to characterize the two most sought-after parameters describing size distributions: the number-averaged mean size and polydispersity index. To demonstrate the power of the method, we simulated and analyzed taylograms corresponding to gold US-NPs distributed normally. By using simulation and including experimental noise, we had the advantage that the true values describing size distribution were known exactly, and thus, we were able test the absolute accuracy of our analysis and its robustness against noise. Theory and computational experiments were found in very good agreement, providing a significant step in the analysis of ultrasmall inorganic nanoparticles and Taylor dispersion experiments.

Hypothesis Test of the Photon Count Distribution for Dust Discrimination in Dynamic Light Scattering.

Users of dynamic light scattering (DLS) are challenged when a sample of nanoparticles (NPs) contains dust. This is a frequently inevitable scenario and a major problem that critically affects the reproducibility and accuracy of DLS measurements. Current methods approach this problem via photon correlation spectroscopy, but remedy exists only for a few special cases. We introduce here a general criterion and a clearly defined measure to discriminate between NPs and dust particles. The experimental results show that, in contrast to photon correlation spectroscopy, hypothesis testing and the statistical moment analysis of the photon count distribution provides an accurate and precise way to characterize NPs and Brownian dynamics in the presence of dust. To demonstrate, analyses of silica, iron oxide, and gold NPs of low polydispersity are presented.

The Kinetics of ß-Hematin Crystallization Measured by Depolarized Light Scattering.

Malaria is caused by Plasmodium sp. parasites transmitted by infected female Anopheles sp. mosquitoes. The survival of the parasites in the host relies on detoxifying free heme by biocrystallization into insoluble crystals called hemozoin. This mechanism of self-preservation is targeted by a certain class of antimalarial drugs, which are screened and selected based on their capacity to inhibit the formation of hemozoin crystals. Therefore, experimental techniques capable of accurately characterizing the kinetics of crystal formation are valuable. Relying on the optical anisotropy of hemozoin, the kinetics of ß-hematin crystal formation through the statistical analysis of photon counts of dynamic depolarized light scattering (DDLS), in the absence and presence of an antimalarial drug (chloroquine, CQ), is described. It is found that CQ has an impact on both the nucleation and growth of the crystals.