Fluorescent-magnetic hybrid nanoparticles induce a dose-dependent increase in proinflammatory response in lung cells in vitro correlated with intracellular localization

Iron-platinum nanoparticles embedded in a poly(methacrylic acid) (PMA) polymer shell and fluorescently labeled with the dye ATTO 590 (FePt-PMA-ATTO-2%) are investigated in terms of their intracellular localization in lung cells and potential to induce a proinflammatory response dependent on concentration and incubation time. A gold core coated with the same polymer shell (Au-PMA-ATTO-2%) is also included. Using laser scanning and electron microscopy techniques, it is shown that the FePt-PMA-ATTO-2% particles penetrate all three types of cell investigated but to a higher extent in macrophages and dendritic cells than epithelial cells. In both cell types of the defense system but not in epithelial cells, a particle-dose-dependent increase of the cytokine tumor necrosis factor alpha (TNFalpha) is found. By comparing the different nanoparticles and the mere polymer shell, it is shown that the cores combined with the shells are responsible for the induction of proinflammatory effects and not the shells alone. It is concluded that the uptake behavior and the proinflammatory response upon particle exposure are dependent on the time, cell type, and cell culture.

The use of light- and electron microscopy for studies on the cell- and molecular biology of parasites and parasitic diseases

Lightmicroscopical (LM) and electron microscopi cal (EM) techniques, have had a major influence on the development and direction of cell biology, and particularly also on the investigation of complex host-parasite relationships. Earlier, microscopy has been rather descriptive, but new technical and scientific advances have changed the situation. Microscopy has now become analytical, quantitative and three-dimensional, with greater emphasis on analysis of live cells with fluorescent markers. The new or improved techniques that have become available include immunocytochemistry using immunogold labeling techniques or fluorescent probes, cryopreservation and cryosectioning, in situ hybridization, fluorescent reporters for subcellular localization, micro-analytical methods for elemental distribution, confocal laser scanning microscopy, scanning tunneling microscopy and live-imaging. Taken together, these tools are providing both researchers and students with a novel and multidimensional view of the intricate biological processes during parasite development in the host.

Visualization and quantitative analysis of nanoparticles in the respiratory tract by transmission electron microscopy

ABSTRACT: Nanotechnology in its widest sense seeks to exploit the special biophysical and chemical properties of materials at the nanoscale. While the potential technological, diagnostic or therapeutic applications are promising there is a growing body of evidence that the special technological features of nanoparticulate material are associated with biological effects formerly not attributed to the same materials at a larger particle scale. Therefore, studies that address the potential hazards of nanoparticles on biological systems including human health are required. Due to its large surface area the lung is one of the major sites of interaction with inhaled nanoparticles. One of the great challenges of studying particle-lung interactions is the microscopic visualization of nanoparticles within tissues or single cells both in vivo and in vitro. Once a certain type of nanoparticle can be identified unambiguously using microscopic methods it is desirable to quantify the particle distribution within a cell, an organ or the whole organism. Transmission electron microscopy provides an ideal tool to perform qualitative and quantitative analyses of particle-related structural changes of the respiratory tract, to reveal the localization of nanoparticles within tissues and cells and to investigate the 3D nature of nanoparticle-lung interactions.This article provides information on the applicability, advantages and disadvantages of electron microscopic preparation techniques and several advanced transmission electron microscopic methods including conventional, immuno and energy-filtered electron microscopy as well as electron tomography for the visualization of both model nanoparticles (e.g. polystyrene) and technologically relevant nanoparticles (e.g. titanium dioxide). Furthermore, we highlight possibilities to combine light and electron microscopic techniques in a correlative approach. Finally, we demonstrate a formal quantitative, i.e. stereological approach to analyze the distributions of nanoparticles in tissues and cells.This comprehensive article aims to provide a basis for scientists in nanoparticle research to integrate electron microscopic analyses into their study design and to select the appropriate microscopic strategy.

Cellular uptake and localization of inhaled gold nanoparticles in lungs of mice with chronic obstructive pulmonary disease

BACKGROUND: Inhalative nanocarriers for local or systemic therapy are promising. Gold nanoparticles (AuNP) have been widely considered as candidate material. Knowledge about their interaction with the lungs is required, foremost their uptake by surface macrophages and epithelial cells.Diseased lungs are of specific interest, since these are the main recipients of inhalation therapy. We, therefore, used Scnn1b-transgenic (Tg) mice as a model of chronic obstructive pulmonary disease (COPD) and compared uptake and localization of inhaled AuNP in surface macrophages and lung tissue to wild-type (Wt) mice. METHODS: Scnn1b-Tg and Wt mice inhaled a 21-nm AuNP aerosol for 2 h. Immediately (0 h) or 24 h thereafter, bronchoalveolar lavage (BAL) macrophages and whole lungs were prepared for stereological analysis of AuNP by electron microscopy. RESULTS: AuNP were mainly found as singlets or small agglomerates of <= 100 nm diameter, at the epithelial surface and within lung-surface structures. Macrophages contained also large AuNP agglomerates (> 100 nm). At 0 h after aerosol inhalation, 69.2+/-4.9% AuNP were luminal, i.e. attached to the epithelial surface and 24.0+/-5.9% in macrophages in Scnn1b-Tg mice. In Wt mice, 35.3+/-32.2% AuNP were on the epithelium and 58.3+/-41.4% in macrophages. The percentage of luminal AuNP decreased from 0 h to 24 h in both groups. At 24 h, 15.5+/-4.8% AuNP were luminal, 21.4+/-14.2% within epithelial cells and 63.0+/-18.9% in macrophages in Scnn1b-Tg mice. In Wt mice, 9.5+/-5.0% AuNP were luminal, 2.2+/-1.6% within epithelial cells and 82.8+/-0.2% in macrophages. BAL-macrophage analysis revealed enhanced AuNP uptake in Wt animals at 0 h and in Scnn1b-Tg mice at 24 h, confirming less efficient macrophage uptake and delayed clearance of AuNP in Scnn1b-Tg mice. CONCLUSIONS: Inhaled AuNP rapidly bound to the alveolar epithelium in both Wt and Scnn1b-Tg mice. Scnn1b-Tg mice showed less efficient AuNP uptake by surface macrophages and concomitant higher particle internalization by alveolar type I epithelial cells compared to Wt mice. This likely promotes AuNP depth translocation in Scnn1b-Tg mice, including enhanced epithelial targeting. These results suggest AuNP nanocarrier delivery as successful strategy for therapeutic targeting of alveolar epithelial cells and macrophages in COPD.

Granular layer in the periplasmic space of gram-positive bacteria and fine structures of Enterococcus gallinarum and Streptococcus gordonii septa revealed by cryo-electron microscopy of vitreous sections.

High-resolution structural information on optimally preserved bacterial cells can be obtained with cryo-electron microscopy of vitreous sections. With the help of this technique, the existence of a periplasmic space between the plasma membrane and the thick peptidoglycan layer of the gram-positive bacteria Bacillus subtilis and Staphylococcus aureus was recently shown. This raises questions about the mode of polymerization of peptidoglycan. In the present study, we report the structure of the cell envelope of three gram-positive bacteria (B. subtilis, Streptococcus gordonii, and Enterococcus gallinarum). In the three cases, a previously undescribed granular layer adjacent to the plasma membrane is found in the periplasmic space. In order to better understand how nascent peptidoglycan is incorporated into the mature peptidoglycan, we investigated cellular regions known to represent the sites of cell wall production. Each of these sites possesses a specific structure. We propose a hypothetic model of peptidoglycan polymerization that accommodates these differences: peptidoglycan precursors could be exported from the cytoplasm to the periplasmic space, where they could diffuse until they would interact with the interface between the granular layer and the thick peptidoglycan layer. They could then polymerize with mature peptidoglycan. We report cytoplasmic structures at the E. gallinarum septum that could be interpreted as cytoskeletal elements driving cell division (FtsZ ring). Although immunoelectron microscopy and fluorescence microscopy studies have demonstrated the septal and cytoplasmic localization of FtsZ, direct visualization of in situ FtsZ filaments has not been obtained in any electron microscopy study of fixed and dehydrated bacteria.

Solvation-Driven Charge Transfer and Localization in Metal Complexes

In any physicochemical process in liquids, the dynamical response of the solvent to the solutes out of equilibrium plays a crucial role in the rates and products: the solvent molecules react to the changes in volume and electron density of the solutes to minimize the free energy of the solution, thus modulating the activation barriers and stabilizing (or destabilizing) intermediate states. In charge transfer (CT) processes in polar solvents, the response of the solvent always assists the formation of charge separation states by stabilizing the energy of the localized charges. A deep understanding of the solvation mechanisms and time scales is therefore essential for a correct description of any photochemical process in dense phase and for designing molecular devices based on photosensitizers with CT excited states. In the last two decades, with the advent of ultrafast time-resolved spectroscopies, microscopic models describing the relevant case of polar solvation (where both the solvent and the solute molecules have a permanent electric dipole and the mutual interaction is mainly dipole−dipole) have dramatically progressed. Regardless of the details of each model, they all assume that the effect of the electrostatic fields of the solvent molecules on the internal electronic dynamics of the solute are perturbative and that the solvent−solute coupling is mainly an electrostatic interaction between the constant permanent dipoles of the solute and the solvent molecules. This well-established picture has proven to quantitatively rationalize spectroscopic effects of environmental and electric dynamics (time-resolved Stokes shifts, inhomogeneous broadening, etc.). However, recent computational and experimental studies, including ours, have shown that further improvement is required. Indeed, in the last years we investigated several molecular complexes exhibiting photoexcited CT states, and we found that the current description of the formation and stabilization of CT states in an important group of molecules such as transition metal complexes is inaccurate. In particular, we proved that the solvent molecules are not just spectators of intramolecular electron density redistribution but significantly modulate it. Our results solicit further development of quantum mechanics computational methods to treat the solute and (at least) the closest solvent molecules including the nonperturbative treatment of the effects of local electrostatics and direct solvent−solute interactions to describe the dynamical changes of the solute excited states during the solvent response.