Nano Cell Interactions
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Scientific Unit
Interference of silica nanoparticles with the traditional Limulus amebocyte lysate gel clot assay
(2014)
Endotoxin contaminations of engineered nanomaterials can be responsible for observed biological responses, especially for misleading results in in vitro test systems, as well as in vivo studies. Therefore, endotoxin testing of nanomaterials is necessary to benchmark their influence on cells. Here, we tested the traditional Limulus amebocyte lysate gel clot assay for the detection of endotoxins in nanoparticle suspensions with a focus on possible interference of the particles with the test system. We systematically investigated the effects of nanomaterials made of, or covered by, the same material. Different types of bare or PEGylated silica nanoparticles, as well as iron oxide-silica core shell nanoparticles, were tested. Detailed inhibition/enhancement controls revealed enhanced activity in the Limulus coagulation cascade for all particles with bare silica surface. In comparison, PEGylation led to a lower degree of enhancement. These results indicate that the protein-particle interactions are the basis for the observed inhibition and enhancement effects. The enhancement activity of a particle type was positively related to the calculated particle surface area. For most silica particles tested, a dilution of the sample within the maximum valid dilution was sufficient to overcome non-valid enhancement, enabling semi-quantification of the endotoxin contamination.
Moderne Nanomaterialien nehmen eine immer größere Bedeutung in industriell gefertigten Produkten ein. Der Mensch kommt deshalb über verschiedene Expositionswege, die z.T. auch den medizinischen Einsatz von Nanopartikeln in diagnostischen oder therapeutischen Anwendungen beinhalten, in Kontakt. Die Interaktion von Nanomaterialien mit biologischen Systemen des menschlichen Körpers wird von den physikalisch-chemischen Eigenschaften der Partikel bestimmt. Die Art der Interaktion mit einzelnen Körperzellen bzw. deren Bestandteilen entscheidet, ob positive oder negative Effekte auf die Gesundheit des Menschen entstehen.
In dieser Arbeit wurden kolloidale Silikananopartikel, die aus Tetraethylorthosilicat nach der Synthese von Hartlen (2008) hergestellt wurden, auf ihre Fähigkeit untersucht, mit den Signaltransduktionswegen epithelialer Zellen zu interagieren. Im Versuchssystem mit Rattenlungenepithelzellen (RLE 6TN) wurden drei verschiedene Größen kolloidaler Silikananopartikel (Si15 – 15 nm; Si25 – 25 nm; Si80 – 80 nm) untersucht und mit Kontrollnanopartikeln verglichen. Zusätzlich zur Zytotoxizität der Partikel wurden vor allem Effekte auf die Zellproliferation und Signaltransduktion betrachtet. Neben den molekularen Mechanismen der Partikeleffekte wurden insbesondere die Aufnahmemechanismen der Silikananopartikel und die subzelluläre Lokalisation der Partikel in den Zellen untersucht.
Die kleinsten verwendeten Silikapartikel (Si15) zeigten einen bislang nicht beschriebenen inhibitorischen Effekt auf die durch den epidermalen Wachstumsfaktor- (EGF-)vermittelte Zellproliferation. Dieser Effekt war nicht durch die Induktion von Zelltod auf der Ebene von Apoptose oder Nekrose begründet. Vielmehr zeigte sich eine spezifische Interaktion von Silikananopartikeln mit der EGFR-abhängigen proliferativen Signaltransduktion. Si15 Nanopartikel führten zu einer Reduktion der Phosphorylierung des EGFRs am Tyrosinrest 1173. Die mit der Aktivierung des Rezeptors einhergehende Internalisierung und die Aktivierung von intrazellulären Signalkinasen, insbesondere Proteinkinase B (Akt), wurden durch die Nanopartikel signifikant verringert. Die molekularen Mechanismen der antiproliferativen Wirkung der Nanopartikel wurden auf der Ebene der Interaktion mit dem natürlichen Liganden EGF identifiziert. So konnte in zellfreien Untersuchungen mit fluoreszenzmarkiertem EGF gezeigt werden, dass dieser an Silikananopartikel bindet. Konfokalmikroskopische Untersuchungen zeigten im zellulären System eine Kolokalisation der Partikel und EGF in vesikulären Strukturen. Weitere Analysen mit den Endozytose-Inhibitoren Chlorpromazin und Filipin III haben gezeigt, dass die Silikananopartikel (SiNP) in Abwesenheit von EGF Clathrin-abhängig von den Zellen internalisiert werden. Durch Färbungen mit vesikulären Markern wurden die SiNP in frühen und späten Endosomen lokalisiert, sodass von einem Transport der Partikel zu den Lysosomen ausgegangen werden kann.
The effects of engineered nanomaterials on human health are intensively studied in order to facilitate their safe application. However, relatively little is known how me-chanical strain (stretching), as induced in alveolar epithelial cells by breathing dynam-ics, modifies biological responses to nanoparticles. In this study, A549 cells as a model for human type II alveolar epithelial cells were exposed to 25 nm amorphous colloidal silica nanoparticles (Si25) under dynamic or static culture conditions. Gene array data, qPCR, and ELISA revealed that stretching, in order to mimic breathing, can amplify the inflammatory responses to nanoparticle exposure. Treatment of cells with either stretching or nanoparticles alone led to minor changes in gene expression or cytokine secretion. The amplifying effect from stretching was not influenced by nanoparticle size or an intensified stretching, but by varying fetal bovine sera for medium supple-mentation. The type of fetal bovine serum used as medium supplement determined the occurrence of the amplifying effect, affecting both baseline cytokine production, as well as cellular response to nanoparticles. Gene expression alterations induced by combined exposure to nanoparticles plus stretching showed a high similarity to those known to be induced by TNF and mediated by NFkB. However, translocation of NFkB-p65, NF-κB2-p100/p52, and NF-κB1-p105/p50 subunits upon Si25, stretch, or a combined treatment could not be observed. Confocal microscopy revealed that stretching did not lead to an increased internalization of nanoparticles in this simplified lung model, indicating that the observed response amplification was not caused secondary to an elevated intracellular nanoparticle accumulation. This study suggests that mechanical strain, which constantly affects lung epithe-lial cells in vivo, significantly determines cell response and should therefore be imple-mented in all in vitro models for pulmonary toxicity tests.
The partners of the research project NanoS-QM (Quality- and Description Standards for Nanosafety Research Data) identified and invited relevant experts from research institutions, federal agencies, and industry to evaluate the traceability of the results generated with the existing standards and quality criteria. During the discussion it emerged that numerous studies seem to be of insufficient quality for regulatory purposes or exhibit weaknesses with regard to data completeness. Deficiencies in study design could be avoided by more comprehensive use of appropriate standards, many of which already exist. The use of Electronic Laboratory Notebooks (ELNs) that allow for early collection of metadata and enrichment of datasets could be one solution to enable data re-use and simplify quality control. Generally, earlier provision and curation of data and metadata indicating their quality and completeness (e.g. guidelines, standards, standard operating procedures (SOPs) that were used) would improve their findability, accessibility, interoperability, and reusability (FAIR) in the nanosafety research field.
Ziel des Vorhabens war es, die gesundheitlichen Auswirkungen von Nanopartikeln (NP) anhand mechanistischer in vitro, in vivo und in sillco-Untersuchungen zu identifizieren und systematisch zu bewerten. Die Arbeitshypothese bestand darin Partikeleigenschaften wie die Oberflächenmodifikation deren Verteilung, Lokalisation und Auswirkungen sowohl auf zellulärer Ebene als auch im gesamten Organismus zu bestimmen. Untersuchungsgegenstand des Vorhabens waren NP mit einem Kern-Schale¬Aufbau und AnwendungsPotential im Bereich der medizinischen Diagnostik, vor allem der Magnetresonanztomographie und der Röntgendiagnostik. Ziel war es, Partikel mit einem Kern aus Eisenoxid oder Bariumsulfat so zu modifizieren, dass sie mittels unterschiedlicher bildgebender Verfahren im Magen-Darm-Trakt nachweisbar waren.[...]
Abstract Due to their importance for the outcome of the inflammatory response, the motile myeloid cells are a focus of novel treatment options. The interplay of selectins and their ligands with leukocytes and endothelial cells, which mediate endothelial attachment and transmigration of immune cells, can be modulated by selectin-binding structures. Here, a library of selectin-targeting ligands coupled to either gold, silver, iron oxide nanospheres, or quantum dots of 5–10 nm in size is used to systematically study their impact on immune cell motility. The multivalent presentation of the carbohydrate mimetics results in very low sub-nanomolar binding to L-selectin. Using human primary monocytes, granulocytes, lymphocytes, and macrophages, it is shown that the ligands exhibit only minor effects on uptake, whereas the motility of leukocytes is critically affected as observed in migration assays evaluated by flow cytometry. The carbohydrate mimetic ring structure, sulfation, in particular, and the degree of ligand presentation, are constituents which cohere in this process. Specific carbohydrate ligands can thus selectively regulate leukocyte subsets. These data form the basis for advanced immunotherapy which inhibits the amplification of inflammation by restricting leukocyte influx to injured tissue sites. Furthermore, the targeting ligands may complement existing treatment options for inflammatory diseases.
Bright fluorescent silica-nanoparticle probes for high-resolution STED and confocal microscopy
(2017)
In recent years, fluorescent nanomaterials have gained high relevance in biological applications as probes for various fluorescence-based spectroscopy and imaging techniques. Among these materials, dye-doped silica nanoparticles have demonstrated a high potential to overcome the limitations presented by conventional organic dyes such as high photobleaching, low stability and limited fluorescence intensity. In the present work we describe an effective approach for the preparation of fluorescent silica nanoparticles in the size range between 15 and 80 nm based on L-arginine-controlled hydrolysis of tetraethoxysilane in a biphasic cyclohexane–water system. Commercially available far-red fluorescent dyes (Atto647N, Abberior STAR 635, Dy-647, Dy-648 and Dy-649) were embedded covalently into the particle matrix, which was achieved by aminosilane coupling. The physical particle attributes (particle size, dispersion, degree of agglomeration and stability) and the fluorescence properties of the obtained particles were compared to particles from commonly known synthesis methods. As a result, the spectroscopic characteristics of the presented monodisperse dye-doped silica nanoparticles were similar to those of the free uncoupled dyes, but indicate a much higher photostability and brightness. As revealed by dynamic light scattering and ζ-potential measurements, all particle suspensions were stable in water and cell culture medium. In addition, uptake studies on A549 cells were performed, using confocal and stimulated emission depletion (STED) microscopy. Our approach allows for a step-by-step formation of dye-doped silica nanoparticles in the form of dye-incorporated spheres, which can be used as versatile fluorescent probes in confocal and STED imaging.
The epidermal growth factor receptor (EGFR) is an abundant membrane protein, which is essential for regulating many cellular processes including cell proliferation. In our earlier studies, we observed an activation of the EGFR and subsequent signaling events after the exposure of epithelial cells to carbon nanoparticles. In the current study, we describe molecular mechanisms that allow for discriminating carbon nanoparticle-specific from ligand-dependent receptor activation. Caveolin-1 is a key player that co-localizes with the EGFR upon receptor activation by carbon nanoparticles. This specific process mediated by nanoparticle-induced reactive oxygen species and the accumulation of ceramides in the plasma membrane is not triggered when cells are exposed to non-nano carbon particles or the physiological ligand EGF. The role of caveolae formation was demonstrated by the induction of higher order structures of caveolin-1 and by the inhibition of caveolae formation. Using an in vivo model with genetically modified mice lacking caveolin-1, it was possible to demonstrate that carbon nanoparticles in vivo trigger EGFR downstream signaling cascades via caveolin-1. The identified molecular mechanisms are, therefore, of toxicological relevance for inhaled nanoparticles. However, nanoparticles that are intentionally applied to humans might cause side effects depending on this phenomenon
In this study, a novel approach for preparation of green fluorescent protein (GFP)-doped silica nanoparticles with a narrow size distribution is presented. GFP was chosen as a model protein due to its autofluorescence. Protein-doped nanoparticles have a high application potential in the field of intracellular protein delivery. In addition, fluorescently labelled particles can be used for bioimaging. The size of these protein-doped nanoparticles was adjusted from 15 to 35 nm using a multistep synthesis process, comprising the particle core synthesis followed by shell regrowth steps. GFP was selectively incorporated into the silica matrix of either the core or the shell or both by a one-pot reaction. The obtained nanoparticles were characterised by determination of particle size, hydrodynamic diameter, ζ-potential, fluorescence and quantum yield. The measurements showed that the fluorescence of GFP was maintained during particle synthesis. Cellular uptake experiments demonstrated that the GFP-doped nanoparticles can be used as stable and effective fluorescent probes. The study reveals the potential of the chosen approach for incorporation of functional biological macromolecules into silica nanoparticles, which opens novel application fields like intracellular protein delivery.
Background: Quantum dots (QDs) have great potential as fluorescent labels but cytotoxicity relating to extra- and intracellular degradation in biological systems has to be addressed prior to biomedical applications. In this study, human intestinal cells (Caco-2) grown on transwell membranes were used to study penetration depth, intracellular localization, translocation and cytotoxicity of CdSe/ZnS QDs with amino and carboxyl surface modifications. The focus of this study was to compare the penetration depth of QDs in differentiated vs undifferentiated cells using confocal microscopy and image processing. Results: Caco-2 cells were exposed to QDs with amino (NH2) and carboxyl (COOH) surface groups for 3 days using a concentration of 45 µg cadmium ml−1. Image analysis of confocal/multiphoton microscopy z-stacks revealed no penetration of QDs into the cell lumen of differentiated Caco-2 cells. Interestingly, translocation of cadmium ions onto the basolateral side of differentiated monolayers was observed using high resolution inductively coupled plasma mass spectrometry (ICP-MS). Membrane damage was neither detected after short nor long term incubation in Caco-2 cells. On the other hand, intracellular localization of QDs after exposure to undifferentiated cells was observed and QDs were partially located within lysosomes.Conclusions: In differentiated Caco-2 monolayers, representing a model for small intestinal enterocytes, no penetration of amino and carboxyl functionalized CdSe/ZnS QDs into the cell lumen was detected using microscopy analysis and image processing. In contrast, translocation of cadmium ions onto the basolateral side could be detected using ICP-MS. However, even after long term incubation, the integrity of the cell monolayer was not impaired and no cytotoxic effects could be detected. In undifferentiated Caco-2 cells, both QD modifications could be found in the cell lumen. Only to some extend, QDs were localized in endosomes or lysosomes in these cells. The results indicate that the differentiation status of Caco-2 cells is an important factor in internalization and localization studies using Caco-2 cells. Furthermore, a combination of microscopy analysis and sensitive detection techniques like ICP-MS are necessary for studying the interaction of cadmium containing QDs with cells.
