Christian Mühlfeld

Quantitative Evaluation of Cellular Uptake and Trafficking of Plain and Polyethylene Glycol-Coated Gold Nanoparticles

This study addresses the cellular uptake and intracellular trafficking of 15-nm gold nanoparticles (NPs), either plain (i.e., stabilized with citrate) or coated with polyethylene glycol (PEG), exposed to human alveolar epithelial cells (A549) at the air-liquid interface for 1, 4, and 24 h. Quantitative analysis by stereology on transmission electron microscopy images reveals a significant, nonrandom intracellular distribution for both NP types. No particles are observed in the nucleus, mitochondria, endoplasmic reticulum, or golgi. The cytosol is not a preferred cellular compartment for both NP types, although significantly more PEG-coated than citrate-stabilized NPs are present there. The preferred particle localizations are vesicles of different sizes (<150, 150-1000, >1000 nm). This is observed for both NP types and indicates a predominant uptake by endocytosis. Subsequent inhibition of caveolin- and clathrin-mediated endocytosis by methyl-beta-cyclodextrin (MbetaCD) results in a significant reduction of intracellular NPs. The inhibition, however, is more pronounced for PEG-coated than citrate-stabilized NPs. The latter are mostly found in larger vesicles; therefore, they are potentially taken up by macropinocytosis, which is not inhibited by MbetaCD. With prolonged exposure times, both NPs are preferentially localized in larger-sized intracellular vesicles such as lysosomes, thus indicating intracellular particle trafficking. This quantitative evaluation reveals that NP surface coatings modulate endocytotic uptake pathways and cellular NP trafficking. Other nonendocytotic entry mechanisms are found to be involved as well, as indicated by localization of a minority of PEG-coated NPs in the cytosol.

Translocation of particles and inflammatory responses after exposure to fine particles and nanoparticles in an epithelial airway model

BackgroundExperimental studies provide evidence that inhaled nanoparticles may translocate over the airspace epithelium and cause increased cellular inflammation. Little is known, however, about the dependence of particle size or material on translocation characteristics, inflammatory response and intracellular localization.ResultsUsing a triple cell co-culture model of the human airway wall composed of epithelial cells, macrophages and dendritic cells we quantified the entering of fine (1 μm) and nano-sized (0.078 μm) polystyrene particles by laser scanning microscopy. The number distribution of particles within the cell types was significantly different between fine and nano-sized particles suggesting different translocation characteristics. Analysis of the intracellular localization of gold (0.025 μm) and titanium dioxide (0.02–0.03 μm) nanoparticles by energy filtering transmission electron microscopy showed differences in intracellular localization depending on particle composition. Titanium dioxide nanoparticles were detected as single particles without membranes as well as in membrane-bound agglomerations. Gold nanoparticles were found inside the cells as free particles only. The potential of the different particle types (different sizes and different materials) to induce a cellular response was determined by measurements of the tumour necrosis factor-α in the supernatants. We measured a 2–3 fold increase of tumour necrosis factor-α in the supernatants after applying 1 μm polystyrene particles, gold nanoparticles, but not with polystyrene and titanium dioxide nanoparticles.ConclusionQuantitative laser scanning microscopy provided evidence that the translocation and entering characteristics of particles are size-dependent. Energy filtering transmission electron microscopy showed that the intracellular localization of nanoparticles depends on the particle material. Both particle size and material affect the cellular responses to particle exposure as measured by the generation of tumour necrosis factor-α.

Interactions of nanoparticles with pulmonary structures and cellular responses

Combustion-derived and synthetic nano-sized particles (NSP) have gained considerable interest among pulmonary researchers and clinicians for two main reasons. 1) Inhalation exposure to combustion-derived NSP was associated with increased pulmonary and cardiovascular morbidity and mortality as suggested by epidemiological studies. Experimental evidence has provided a mechanistic picture of the adverse health effects associated with inhalation of combustion-derived and synthetic NSP. 2) The toxicological potential of NSP contrasts with the potential application of synthetic NSP in technological as well as medicinal settings, with the latter including the use of NSP as diagnostics or therapeutics. To shed light on this paradox, this article aims to highlight recent findings about the interaction of inhaled NSP with the structures of the respiratory tract including surfactant, alveolar macrophages, and epithelial cells. Cellular responses to NSP exposure include the generation of reactive oxygen species and the induction of an inflammatory response. Furthermore, this review places special emphasis on methodological differences between experimental studies and the caveats associated with the dose metrics and points out ways to overcome inherent methodological problems.

Silk protein fibroin from Antheraea mylitta for cardiac tissue engineering

The human heart cannot regenerate after an injury. Lost cardiomyocytes are replaced by scar tissue resulting in reduced cardiac function causing high morbidity and mortality. One possible solution to this problem is cardiac tissue engineering. Here, we have investigated the suitability of non-mulberry silk protein fibroin from Indian tropical tasar Antheraea mylitta as a scaffold for engineering a cardiac patch in vitro. We have tested cell adhesion, cellular metabolic activity, response to extracellular stimuli, cell-to-cell communication and contractility of 3-days postnatal rat cardiomyocytes on silk fibroin. Our data demonstrate that A. mylitta silk fibroin exhibits similar properties as fibronectin, a component of the natural matrix for cardiomyocytes. Comparison to mulberry Bombyx mori silk protein fibroin shows that A. mylitta silk fibroin is superior probably due to its RGD domains. 3D scaffolds can efficiently be loaded with cardiomyocytes resulting in contractile patches. In conclusion, our findings demonstrate that A. mylitta silk fibroin 3D scaffolds are suitable for the engineering of cardiac patches.

Effects and uptake of gold nanoparticles deposited at the air–liquid interface of a human epithelial airway model

The impact of nanoparticles (NPs) in medicine and biology has increased rapidly in recent years. Gold NPs have advantageous properties such as chemical stability, high electron density and affinity to biomolecules, making them very promising candidates as drug carriers and diagnostic tools. However, diverse studies on the toxicity of gold NPs have reported contradictory results. To address this issue, a triple cell co-culture model simulating the alveolar lung epithelium was used and exposed at the air-liquid interface. The cell cultures were exposed to characterized aerosols with 15 nm gold particles (61 ng Au/cm2 and 561 ng Au/cm2 deposition) and incubated for 4 h and 24 h. Experiments were repeated six times. The mRNA induction of pro-inflammatory (TNFalpha, IL-8, iNOS) and oxidative stress markers (HO-1, SOD2) was measured, as well as protein induction of pro- and anti-inflammatory cytokines (IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, GM-CSF, TNFalpha, INFgamma). A pre-stimulation with lipopolysaccharide (LPS) was performed to further study the effects of particles under inflammatory conditions. Particle deposition and particle uptake by cells were analyzed by transmission electron microscopy and design-based stereology. A homogeneous deposition was revealed, and particles were found to enter all cell types. No mRNA induction due to particles was observed for all markers. The cell culture system was sensitive to LPS but gold particles did not cause any synergistic or suppressive effects. With this experimental setup, reflecting the physiological conditions more precisely, no adverse effects from gold NPs were observed. However, chronic studies under in vivo conditions are needed to entirely exclude adverse effects.

Cholinergic chemosensory cells in the trachea regulate breathing

In the epithelium of the lower airways, a cell type of unknown function has been termed “brush cell” because of a distinctive ultrastructural feature, an apical tuft of microvilli. Morphologically similar cells in the nose have been identified as solitary chemosensory cells responding to taste stimuli and triggering trigeminal reflexes. Here we show that brush cells of the mouse trachea express the receptors (Tas2R105, Tas2R108), the downstream signaling molecules (α-gustducin, phospholipase Cβ2) of bitter taste transduction, the synthesis and packaging machinery for acetylcholine, and are addressed by vagal sensory nerve fibers carrying nicotinic acetylcholine receptors. Tracheal application of an nAChR agonist caused a reduction in breathing frequency. Similarly, cycloheximide, a Tas2R108 agonist, evoked a drop in respiratory rate, being sensitive to nicotinic receptor blockade and epithelium removal. This identifies brush cells as cholinergic sensors of the chemical composition of the lower airway luminal microenvironment that are directly linked to the regulation of respiration.

In vitro models of the human epithelial airway barrier to study the toxic potential of particulate matter

Background: Several epidemiological studies show that inhalation of particulate matter may cause increased pulmonary morbidity and mortality. Of particular interest are the ultrafine particles that are particularly toxic. In addition more and more nanoparticles are released into the environment; however, the potential health effects of these nanoparticles are yet unknown. Objectives: To avoid particle toxicity studies with animals many cell culture models have been developed during the past years. Methods: This review focuses on the most commonly used in vitro epithelial airway and alveolar models to study particle–cell interactions and particle toxicity and highlights advantages and disadvantages of the different models. Results/conclusion: There are many lung cell culture models but none of these models seems to be perfect. However, they might be a great tool to perform basic research or toxicity tests. The focus here is on 3D and co-culture models, which seem to be more realistic than monocultures.

Cardioprotection and lifespan extension by the natural polyamine spermidine

Aging is associated with an increased risk of cardiovascular disease and death. Here we show that oral supplementation of the natural polyamine spermidine extends the lifespan of mice and exerts cardioprotective effects, reducing cardiac hypertrophy and preserving diastolic function in old mice. Spermidine feeding enhanced cardiac autophagy, mitophagy and mitochondrial respiration, and it also improved the mechano-elastical properties of cardiomyocytes in vivo, coinciding with increased titin phosphorylation and suppressed subclinical inflammation. Spermidine feeding failed to provide cardioprotection in mice that lack the autophagy-related protein Atg5 in cardiomyocytes. In Dahl salt-sensitive rats that were fed a high-salt diet, a model for hypertension-induced congestive heart failure, spermidine feeding reduced systemic blood pressure, increased titin phosphorylation and prevented cardiac hypertrophy and a decline in diastolic function, thus delaying the progression to heart failure. In humans, high levels of dietary spermidine, as assessed from food questionnaires, correlated with reduced blood pressure and a lower incidence of cardiovascular disease. Our results suggest a new and feasible strategy for protection against cardiovascular disease.

Increased Airway Smooth Muscle Mass in Children with Asthma, Cystic Fibrosis, and Non-Cystic Fibrosis Bronchiectasis

RATIONALE Structural alterations to airway smooth muscle (ASM) are a feature of asthma and cystic fibrosis (CF) in adults. OBJECTIVES We investigated whether increase in ASM mass is already present in children with chronic inflammatory lung disease. METHODS Fiberoptic bronchoscopy was performed in 78 children (median age [IQR], 11.3 [8.5-13.8] yr): 24 with asthma, 27 with CF, 16 with non-CF bronchiectasis (BX), and 11 control children without lower respiratory tract disease. Endobronchial biopsy ASM content and myocyte number and size were quantified using stereology. MEASUREMENTS AND MAIN RESULTS The median (IQR) volume fraction of subepithelial tissue occupied by ASM was increased in the children with asthma (0.27 [0.12-0.49]; P < 0.0001), CF (0.12 [0.06-0.21]; P < 0.01), and BX (0.16 [0.04-0.21]; P < 0.01) compared with control subjects (0.04 [0.02-0.05]). ASM content was related to bronchodilator responsiveness in the asthmatic group (r = 0.66, P < 0.01). Median (IQR) myocyte number (cells per mm(2) of reticular basement membrane) was 8,204 (5,270-11,749; P < 0.05) in children with asthma, 4,504 (2,838-8,962; not significant) in children with CF, 4,971 (3,476-10,057; not significant) in children with BX, and 1,944 (1,596-6,318) in control subjects. Mean (SD) myocyte size (mum(3)) was 3,344 (801; P < 0.01) in children with asthma, 3,264 (809; P < 0.01) in children with CF, 3,177 (873; P < 0.05) in children with BX, and 1,927 (386) in control subjects. In all disease groups, the volume fraction of ASM in subepithelial tissue was related to myocyte number (asthma: r = 0.84, P < 0.001; CF: r = 0.81, P < 0.01; BX: r = 0.95, P < 0.001), but not to myocyte size. CONCLUSIONS Increases in ASM (both number and size) occur in children with chronic inflammatory lung diseases that include CF, asthma, and BX.

A review of recent methods for efficiently quantifying immunogold and other nanoparticles using TEM sections through cells, tissues and organs

Detecting, localising and counting ultrasmall particles and nanoparticles in sub- and supra-cellular compartments are of considerable current interest in basic and applied research in biomedicine, bioscience and environmental science. For particles with sufficient contrast (e.g. colloidal gold, ferritin, heavy metal-based nanoparticles), visualization requires the high resolutions achievable by transmission electron microscopy (TEM). Moreover, if particles can be counted, their spatial distributions can be subjected to statistical evaluation. Whatever the level of structural organisation, particle distributions can be compared between different compartments within a given structure (cell, tissue and organ) or between different sets of structures (in, say, control and experimental groups). Here, a portfolio of stereology-based methods for drawing such comparisons is presented. We recognise two main scenarios: (1) section surface localisation, in which particles, exemplified by antibody-conjugated colloidal gold particles or quantum dots, are distributed at the section surface during post-embedding immunolabelling, and (2) section volume localisation (or full section penetration), in which particles are contained within the cell or tissue prior to TEM fixation and embedding procedures. Whatever the study aim or hypothesis, the methods for quantifying particles rely on the same basic principles: (i) unbiased selection of specimens by multistage random sampling, (ii) unbiased estimation of particle number and compartment size using stereological test probes (points, lines, areas and volumes), and (iii) statistical testing of an appropriate null hypothesis. To compare different groups of cells or organs, a simple and efficient approach is to compare the observed distributions of raw particle counts by a combined contingency table and chi-squared analysis. Compartmental chi-squared values making substantial contributions to total chi-squared values help identify where the main differences between distributions reside. Distributions between compartments in, say, a given cell type, can be compared using a relative labelling index (RLI) or relative deposition index (RDI) combined with a chi-squared analysis to test whether or not particles preferentially locate in certain compartments. This approach is ideally suited to analysing particles located in volume-occupying compartments (organelles or tissue spaces) or surface-occupying compartments (membranes) and expected distributions can be generated by the stereological devices of point, intersection and particle counting. Labelling efficiencies (number of gold particles per antigen molecule) in immunocytochemical studies can be determined if suitable calibration methods (e.g. biochemical assays of golds per membrane surface or per cell) are available. In addition to relative quantification for between-group and between-compartment comparisons, stereological methods also permit absolute quantification, e.g. total volumes, surfaces and numbers of structures per cell. Here, the utility, limitations and recent applications of these methods are reviewed.