In vitro study of carbon black nanoparticles on human pulmonary artery endothelial cells: effects on calcium signaling and mitochondrial alterations.

Human exposure to manufactured nanoparticles (NPs) is a public health concern. Endothelial cells lining the inner surface of arteries could be one of the primary targets for inhaled nanoparticles. Moreover, it is well known that alteration in calcium signaling is a critical event involved in the physiopathology of cardiovascular diseases. The objective of this study was to assess the role of oxidative stress in carbon black FW2 NPs-induced alteration in calcium signaling and mitochondria in human pulmonary artery endothelial cells. To this end, cells were exposed for 4 or 24 h to FW2 NPs (1-10 µg/cm2) and the following endpoints were studied: (i) production of ROS by fluorimetry and electron paramagnetic resonance, (ii) variation in intracellular calcium concentration by confocal microscopy, and (iii) mitochondrial alteration and apoptosis by confocal microscopy and transmission electronic microscopy. Exposure to FW2 NPs concentration-dependently increases oxidative stress, evidenced by the production of superoxide anion leading to an alteration in calcium content of intracellular organelles, such as endoplasmic reticulum and mitochondria activating, in turn, intrinsic apoptosis. This study provides evidence that FW2 NPs exposure impairs calcium signaling and mitochondria triggered by oxidative stress, and, thus, could act as a cardiovascular disease risk owing to the key role of calcium homeostasis in the control of vascular tone.

Supported pulmonary surfactant bilayers on silica nanoparticles: formulation, stability and impact on lung epithelial cells.

Studies have shown that following exposure to particulate matter, ultrafine fractions (<100 nm) may deposit along the respiratory tract down to the alveolar region. To assess the effects of nanoparticles on the lungs, it is essential to address the question of their biophysicochemical interaction with the different pulmonary environments, including the lung lining fluids and the epithelia. Here we examine one of these interactive scenarios and study the role of supported lipid bilayers (SLB) in the effect of 40 nm fluorescent silica particles on living cells. We first study the particle phase behavior in the presence of Curosurf®, a pulmonary surfactant substitute used in replacement therapies. It is found that Curosurf® vesicles interact strongly with the nanoparticles, but do not spontaneously form SLBs. To achieve this goal, we use sonication to reshape the vesicular membranes and induce lipid fusion around the particles. Centrifugal sedimentation and electron microscopy are carried out to determine the optimum coating conditions and layer thickness. We then explore the impact of surfactant SLBs on the cytotoxic potential and interactions towards a malignant epithelial cell line. All in vitro assays indicate that SLBs mitigate the particle toxicity and internalization rates. In the cytoplasm, the particle localization is also strongly coating dependent. It is concluded that SLBs profoundly affect cellular interactions and functions in vitro and could represent an alternative strategy for particle coating. The current data also shed some light on the potential mechanisms pertaining to the particle or pathogen transport through the air-blood barrier.

Robust raspberry-like metallo-dielectric nanoclusters of critical sizes as SERS substrates.

Raspberry-like nano-objects made of large plasmonic satellites (>10 nm) covering a central dielectric particle have many potential applications as photonic materials, superlenses and (bio-) sensors, but their synthesis remains challenging. Herein, we show how to build stable and robust raspberry-like nano-systems with close-packed satellites, by combining monodisperse silica particles (80 or 100 nm diameter) and oppositely charged noble metal nanoparticles (Au or Ag) with well-defined sizes (10-50 nm). The spectral characteristics of their associated plasmonic resonances (wavelength, linewidth, extinction cross-section) and the electromagnetic coupling between satellites were observed using the spatial modulation spectroscopy technique and interpreted through a numerical model. The composite nano-objects exhibit numerous hot spots at satellite junctions, resulting in excellent surface-enhanced Raman scattering (SERS) performance. The SERS efficiency of the raspberry-like clusters is highly dependent on their structure.

Templated growth of gold satellites on dimpled silica cores.

We synthesize robust clusters of gold satellites positioned with tetrahedral symmetry on the surface of a patchy silica core by adsorption and growth of gold on the patches. First we conduct emulsion polymerization of styrene in the presence of 52 nm silica seeds whose surface has been modified with methacryloxymethyltriethoxysilane (MMS). We derive four-dimple particles from the resulting silica/polystyrene tetrapods. Polystyrene chains are covalently bound to the silica surface within the dimples due to the MMS grafts and they may be thiolated to induce adsorption of 12 nm gold particles. Using chloroauric acid, ascorbic acid and sodium citrate at room temperature, we grow gold from these 12 nm seeds without detachment from or deformation of the dimpled silica surface. We obtain gold satellites of tunable diameter up to 140 nm.

Polyelectrolyte assisted charge titration spectrometry: Applications to latex and oxide nanoparticles.

The electrostatic charge density of particles is of paramount importance for the control of the dispersion stability. Conventional methods use potentiometric, conductometric or turbidity titration but require large amount of samples. Here we report a simple and cost-effective method called polyelectrolyte assisted charge titration spectrometry or PACTS. The technique takes advantage of the propensity of oppositely charged polymers and particles to assemble upon mixing, leading to aggregation or phase separation. The mixed dispersions exhibit a maximum in light scattering as a function of the volumetric ratio X, and the peak position XMax is linked to the particle charge density according to σ∼D0XMax where D0 is the particle diameter. The PACTS is successfully applied to organic latex, aluminum and silicon oxide particles of positive or negative charge using poly(diallyldimethylammonium chloride) and poly(sodium 4-styrenesulfonate). The protocol is also optimized with respect to important parameters such as pH and concentration, and to the polyelectrolyte molecular weight. The advantages of the PACTS technique are that it requires minute amounts of sample and that it is suitable to a broad variety of charged nano-objects.

Extracellular vesicles from blood plasma: determination of their morphology, size, phenotype and concentration.

BACKGROUND: Plasma and other body fluids contain membranous extracellular vesicles (EVs), which are considered to derive from activated or apoptotic cells. EVs participate in physiological and pathological processes and have potential applications in diagnostics or therapeutics. Knowledge on EVs is, however, limited, mainly due to their sub-micrometer size and to intrinsic limitations in methods applied for their characterization. OBJECTIVES: Our aim was to provide a comprehensive description of EVs from plasma of healthy subjects. METHODS: Cryo-transmission electron microscopy combined with receptor-specific gold labeling was used to reveal the morphology, size and phenotype of EVs. An original approach based on sedimentation on electron microscopy grids was developed for enumerating EVs. A correlation was performed between conventional flow cytometry and electron microscopy results. RESULTS: We show that platelet-free plasma samples contain spherical EVs, 30 nm to 1 µm in diameter, tubular EVs, 1-5 µm long, and membrane fragments, 1-8 µm large. We show that only a minority of EVs expose the procoagulant lipid phosphatidylserine, in contrast to the classical theory of EV formation. In addition, the concentrations of the main EV sub-populations are determined after sedimentation on EM grids. Finally, we show that conventional flow cytometry, the main method of EV characterization, detects only about 1% of them. CONCLUSION: This study brings novel insights on EVs from normal plasma and provides a reference for further studies of EVs in disease situations.

Synthesis of size-monodisperse spherical Ag@SiO2 nanoparticles and 3-D assembly assisted by microfluidics.

This article reports a one-step approach for the fabrication of highly uniform, spherical Ag particles with tailored dimensions ranging from 10 to 30 nm. Coated with silica shell, the high uniformity of the particles allows their spontaneous assembly into millimeter-long extended 3-D arrays with transverse dimensions of tens of micrometers, using a microfluidic evaporation-based process.

Magnetic resonance imaging tracking of human adipose derived stromal cells within three-dimensional scaffolds for bone tissue engineering.

For bone tissue engineering, human Adipose Derived Stem Cells (hADSCs) are proposed to be associated with a scaffold for promoting bone regeneration. After implantation, cellularised scaffolds require a non-invasive method for monitoring their fate in vivo. The purpose of this study was to use Magnetic Resonance Imaging (MRI)-based tracking of these cells, labelled with magnetic agents for in vivo longitudinal assessment. hADSCs were isolated from adipose tissue and labelled with USPIO-rhodamine (Ultrasmall SuperParamagnetic Iron Oxide). USPIO internalisation, absence of toxicity towards hADSCs, and osteogenic differentiation of the labelled cells were evaluated in standard culture conditions. Labelled cells were then seeded within a 3D porous polysaccharide-based scaffold and imaged in vitro using fluorescence microscopy and MRI. Cellularised scaffolds were implanted subcutaneously in nude mice and MRI analyses were performed from 1 to 28 d after implantation. In vitro, no effect of USPIO labelling on cell viability and osteogenic differentiation was found. USPIO were efficiently internalised by hADSCs and generated a high T2* contrast. In vivo MRI revealed that hADSCs remain detectable until 28 d after implantation and could migrate from the scaffold and colonise the area around it. These data suggested that this scaffold might behave as a cell carrier capable of both holding a cell fraction and delivering cells to the site of implantation. In addition, the present findings evidenced that MRI is a reliable technique to validate cell-seeding procedures in 3D porous scaffolds, and to assess the fate of hADSCs transplanted in vivo.

Silica encapsulated manganese perovskite nanoparticles for magnetically induced hyperthermia without the risk of overheating.

Nanoparticles of manganese perovskite of the composition La(0.75)Sr(0.25)MnO(3) uniformly coated with silica were prepared by encapsulation of the magnetic cores (mean crystallite size 24 nm) using tetraethoxysilane followed by fractionation. The resulting hybrid particles form a stable suspension in an aqueous environment at physiological pH and possess a narrow hydrodynamic size distribution. Both calorimetric heating experiments and direct measurements of hysteresis loops in the alternating field revealed high specific power losses, further enhanced by the encapsulation procedure in the case of the coated particles. The corresponding results are discussed on the basis of complex characterization of the particles and especially detailed magnetic measurements. Moreover, the Curie temperature (335 K) of the selected magnetic cores resolves the risk of local overheating during hyperthermia treatment.

Use of nanopatterned surfaces to enhance immunoreaction efficiency.

In this work, we compare the immunoreaction efficiency between uniformly functionalized surface and chemically nanopatterned surfaces when applied as platforms for antigen/antibody interactions with and without the use of protein A as orienting protein. On the nanopatterned platform, the immunoreaction efficiency is higher than all the other cases with no protein A pretreatment of the surface, providing evidence of the capability of the adhesive/antiadhesive nanopatterned surface to immobilize the molecules in a reactive state, increasing their possibility to form complexes.