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In the last decades, significant efforts have been made to investigate possible cytotoxic effects of metallic nanoparticles (NPs). Methodologies enabling precise information regarding uptake and intracellular distribution of NPs at the single cell level remain to be established. Mass cytometry (MC) has been developed for high-dimensional single cell analyses and is a promising tool to quantify NP−cell interactions. Here, we aim to establish a new MC-based quantification procedure to receive absolute numbers of NPs per single cell by using a calibration that considers the specific transmission efficiency (TE) of suspended NPs. The current MC-quantification strategy accept TE values of complementary metal solutions. In this study, we demonstrate the different transmission behavior of 50 nm silver NPs (AgNP) and silver nitrate solution. We have used identical AgNPs for calibration as for in vitro-differentiated macrophages (THP-1 cell line) in a time- and dose-dependent manner. Our quantification relies on silver intensities measuring AgNPs in the same detection mode as the cells. Results were comparable with the TE quantification strategy using AgNPs but differed when using ionic silver. Furthermore, intact and digested cell aliquots were measured to investigate the impact of MC sample processing on the amount of AgNPs/cell. Taken together, we have provided a MC-specific calibration procedure to precisely calculate absolute numbers of NPs per single cell. Combined with its unique feature of multiplexing up to 50 parameters, MC provides much more information on the single cell level than single cell-inductively coupled plasma mass spectrometry (SC-ICP-MS) and, therefore, offers new opportunities in nanotoxicology.
The impact of nanoparticles, NPs, at the single cell level has become a major field of toxicological research and different analytical methodologies are being investigated to obtain biological and toxicological information to better understand the mechanisms of cell–NP interactions. Here, inductively coupled plasma mass spectrometry in the single cell measurement mode (SC-ICP-MS) is proposed to study the uptake of silver NPs, AgNPs, with a diameter of 50 nm by human THP-1 monocytes in a proof-ofprinciple experiment. The main operating parameters of SC-ICP-MS have been optimized and applied for subsequent quantitative analysis of AgNPs to determine the number of particles in individual cells using AgNP suspensions for calibration. THP-1 cells were incubated with AgNP suspensions with concentrations of 0.1 and 1 µg/mL for 4 and 24 hours. The results reveal that the AgNP uptake by THP-1 monocytes is minimal at the lower dose of 0.1 µg/mL (roughly 1 AgNP per cell was determined), whereas a large cell-to-cell variance dependent on the exposure time is observed for a 10 times higher concentration (roughly 7 AgNPs per cell). The method was further applied to monitor the AgNP uptake by THP-1 cells differentiated macrophages incubated at the same AgNP concentration levels and exposure times demonstrating a much higher AgNP uptake (roughly from 9 to 45 AgNPs per cell) that was dependent on exposure concentration and remained constant over time. The results have been compared and validated by sample digestion followed by ICP-MS analysis as well as with other alternative promising techniques providing single cell analysis.
A simple metal staining procedure for identification and visualization of single cells by LA-ICP-MS
(2017)
High lateral resolution of metal detection in single cells by use of laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) demands powerful staining methods. In this work different staining procedures for the single cell analysis with LA-ICP-MS were optimized. An iridium intercalator was utilized to stain the cell nuclei whereas the whole cell was stained by the use of maleimido-mono-amide-DOTA (mDOTA) complexing lanthanide(III) ions. The content of the artificially introduced metals per cell was quantified using a matrix matched calibration approach based on cellulose membranes onto which standards were spotted by a microarray spotter. Absolute metal stain amounts in the range of 2.34 to 9.81 femtomole per cell were determined. The metal staining procedures allow direct identification and visualization of single cells and their cell compartments by element microscopy without the use of bright field images of the sample.
Mit einer langfristigen Forschungsstrategie begleiten die für die Sicherheit von Mensch und Umwelt zuständigen Bundesoberbehörden (Umweltbundesamt, Bundesinstitut für Risikobe-wertung, Bundesanstalt für Arbeitsschutz und Arbeitsmedizin, Bundesanstalt für Materialfor-schung und -prüfung und Physikalisch-Technische Bundesanstalt) die rasch voranschreiten-de Entwicklung neuer Materialien unter den Gesichtspunkten des Arbeits-, Verbraucher- und Umweltschutzes. Die Strategie steht daher in enger Verbindung zu den öffentlichen Förder-programmen für Nanomaterialien und andere innovative Werkstoffe, z. B. des BMBF („Vom Material zur Innovation“) und der EU („Horizon 2020“).
Die Forschungsstrategie baut auf den bisherigen Ergebnissen der 2008 begonnenen und 2013 erstmals bilanzierten gemeinsamen Forschungsstrategie der Bundesoberbehörden „Nanotechnologie - Gesundheits- und Umweltrisiken von Nanomaterialien"1 auf und erweitert den Blickwinkel auch auf andere Materialinnovationen, bei denen vergleichbare Risiken für Mensch und Umwelt bestehen oder abgeklärt werden müssen. Darüber hinaus greift sie die Idee „anwendungssichere chemische Produkte“2 aus der Initiative „Neue Qualität der Arbeit“ (INQA) des Bundesministeriums für Arbeit und Soziales (BMAS) und das Konzept der nach-haltigen Chemie3 auf, das vom Bundesministerium für Umwelt, Naturschutz, Bau und Reak-torsicherheit (BMUB) unterstützt wird. Durch eine anwendungssichere und umweltverträgli-che Gestaltung innovativer Materialien und ihrer Folgeprodukte sollen nicht akzeptable Risi-ken für Mensch und Umwelt von Anfang an weitgehend ausgeschlossen werden. Dies kann erreicht werden durch
1.
die Verwendung sicherer Materialien ohne Gefahreneigenschaften für Mensch und Umwelt (direkte Anwendungssicherheit) oder
2.
eine Produktgestaltung, die über den gesamten Lebenszyklus emissionsarm und umweltverträglich ist (integrierte Anwendungssicherheit) oder
3.
eine Unterstützung des Anwenders (product stewardship) durch den Hersteller bei technischen, organisatorischen und persönlichen Schutzmaßnahmen zur sicheren Verwendung und Entsorgung des Produktes (unterstützte Anwendungssicherheit).
Die Fortschreibung der Forschungsstrategie soll als Bestandteil des Nanoaktionsplans 2020 der Bundesregierung Beiträge der Ressortforschung zu folgenden Schwerpunkten leisten:
•
Charakterisierung und Bewertung der Risiken von Materialinnovationen
•
Unterstützung von Forschungseinrichtungen und Unternehmen
•
Fortschreiben von Rechtsvorschriften und Praxisempfehlungen
1
http://www.baua.de/nn_47716/de/Themen-von-A-Z/Gefahrstoffe/Nanotechnologie/pdf/Forschungsstrategie.pdf
2 http://www.baua.de/de/Themen-von-A-Z/Gefahrstoffe/Nachhaltige-Chemie/Nachhaltige-Chemie.html
3
http://www.umweltbundesamt.de/themen/chemikalien/chemikalien-management/nachhaltige-chemie
2
•
Gesellschaftliche Akzeptanz
Die Forschungsstrategie soll mit Projekten und anderen forschungsnahen Aktivitäten umge-setzt werden. Dies umfasst die eigene Forschung der Häuser, die extramurale Ausschrei-bung und Vergabe von Forschungsdienstleistungen sowie die Beteiligung an vorwiegend öffentlich geförderten Drittmittelprojekten. Hinzu kommen Aktivitäten im Rahmen der Politik-beratung und der hoheitlichen Aufgaben. Mit inter- und transdisziplinären Ansätzen soll die Risiko- und Sicherheitsforschung enger mit der Innovationsforschung und Materialentwick-lung verknüpft werden. Die Forschungsstrategie ist aufgrund der raschen Entwicklungen auf diesem Gebiet für den Zeitraum bis 2020 angelegt. Die Forschungsziele adressieren die in diesem Zeitraum voraussichtlich umsetzbaren Forschungsansätze. Die Forschungsstrategie wird durch einen Arbeitskreis begleitet und spätestens mit Ablauf des Nanoaktionsplans 2020 evaluiert und angepasst.
The design of bright and functional dye–protein conjugates requires hydrophilic and stable fluorophores with high molar absorption coefficients and high fluorescence quantum yields,which must not be prone to dimerization, as well as conservation of protein function and suppression of protein association. Although many synthetic dyes meet these needs, the influence of dye charge on bioconjugate performance is commonly neglected. This encouraged us to assess the spectroscopic properties, antibody functionality, binding behavior, folding, and association of conjugates of the therapeutic antibodies trastuzumab and cetuximab with the red cyanine dyes S0586, S2381,and 6SIDCC (bearing two, three, and six sulfonate Groups respectively.
Our results demonstrate a negligible effect of dye labeling on antibody folding, yet a strong influence of Label charge and density on antibody isoelectric points and association.Especially 6SIDCC decreased strongly the isoelectric points of both antibodies and their heavy or light chains even at low labeling degrees, thus favoring protein association. Although an increasingly negative dye charge reduces Antigen affinity as shown in a competitive immunoassay, all conjugates still bound to cells overexpressing the target of the respective antibody. Obviously, dyes that cause minimum dimerization with a small number of charged groups are best for conjugate brightness, minimum protein association, and strong target binding. This underlines the need to consider dye Charge for the rational design of conjugates with Optimum performance.
Increased use of nanomaterials in everyday products leads to their environmental release and therefore, the information need on their fate and behaviour. Nanomaterials have to be suspended with high repeatability and comparability for studies on environmental effects. They also have to be well characterised with a focus on the state of agglomeration and particle size distribution. Dynamic light-scattering (DLS) is a common technique used for these measurements. If suspensions are prepared in different laboratories, then concern has risen about the comparability of the measured results, especially when different DLS instruments are used. Therefore, for quality assurance, a round-robin test was conducted to assess the comparability of different DLS instruments and a dispersion protocol in ten independent laboratories. Polystyrene and TiO2 were chosen as test (nano)materials. For the comparability of the DLS instruments, the average sizes of the PSL and a stabilised TiO2 suspension were measured. The measured average hydrodynamic diameter shows an overall good inter-laboratory comparability. For the PSL suspension, an average hydrodynamic diameter of 201 ± 13 nm and for the TiO2 suspension an average diameter of 224 ± 24 nm were detected. For the TiO2 suspension that was prepared at each laboratory following an established suspension preparation protocol, an average hydrodynamic diameter of 211 ± 11 nm was detected. The measured average particle size (mode) increased up to 284 nm with a high standard deviation of 119 nm if the preparation protocol could not established and different procedures or different equipment were employed. This study shows that no significant differences between the employed DLS instrument types were determined. It was also shown that comparable measurements and suspension preparation could be achieved if well-defined suspension preparation protocols and comparable equipment can be used.
Carbohydrate functionalization of silver nanoparticles modulates cytotoxicity and cellular uptake
(2014)
Background
Increasing use of silver nanoparticles (Ag-NPs) in various products is resulting in a greater likelihood of human exposure to these materials. Nevertheless, little is still known about the influence of carbohydrates on the toxicity and cellular uptake of nanoparticles.
Methods
Ag-NPs functionalized with three different monosaccharides and ethylene glycol were synthesized and characterised. Oxidative stress and toxicity was evaluated by protein carbonylation and MTT assay, respectively. Cellular uptake was evaluated by confocal microscopy and ICP-MS.
Results
Ag-NPs coated with galactose and mannose were considerably less toxic to neuronal-like cells and hepatocytes compared to particles functionalized by glucose, ethylene glycol or citrate. Toxicity correlated to oxidative stress but not to cellular uptake.
Conclusions
Carbohydrate coating on silver nanoparticles modulates both oxidative stress and cellular uptake, but mainly the first has an impact on toxicity. These findings provide new perspectives on modulating the bioactivity of Ag-NPs by using carbohydrates.
Silver nanoparticles (SNP) are among the most commercialized nanoparticles. Here, we show that peptide-coated SNP cause functional impairment of human macrophages. A dose-dependent inhibition of phagocytosis is observed after nanoparticle treatment, and pretreatment of cells with N-acetyl cysteine (NAC) can counteract the phagocytosis disturbances caused by SNP.
Using the surface-sensitive mode of time-of-flight secondary ion mass spectrometry, in combination with multivariate statistical methods, we studied the composition of cell membranes in human macrophages upon exposure to SNP with and without NAC preconditioning. This method revealed characteristic changes in the lipid pattern of the cellular membrane outer leaflet in those cells challenged by SNP. Statistical analyses resulted in 19 characteristic ions, which can be used to distinguish between NAC pretreated and untreated macrophages. The present study discusses the assignments of surface cell membrane phospholipids for the identified ions and the resulting changes in the phospholipid pattern of treated cells. We conclude that the adverse effects in human macrophages caused by SNP can be partially reversed through NAC administration. Some alterations, however, remained.
Silver nanoparticles (SNPs) are among the most commercialized nanoparticles because of their antibacterial effects. Besides being employed, e.g. as a coating material for sterile surfaces in household articles and appliances, the particles are also used in a broad range of medical applications. Their antibacterial properties make SNPs especially useful for wound disinfection or as a coating material for prostheses and surgical instruments. Because of their optical characteristics, the particles are of increasing interest in biodetection as well. Despite the widespread use of SNPs, there is little knowledge of their toxicity. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) and laser post-ionization secondary neutral mass spectrometry (Laser-SNMS) were used to investigate the effects of SNPs on human macrophages derived from THP-1 cells in vitro. For this purpose, macrophages were exposed to SNPs. The SNP concentration ranges were chosen with regard to functional impairments of the macrophages. To optimize the analysis of the macrophages, a special silicon wafer sandwich preparation technique was employed; ToF-SIMS was employed to characterize fragments originating from macrophage cell membranes. With the use of this optimized sample preparation method, the SNP-exposed macrophages were analyzed with ToF-SIMS and with Laser-SNMS. With Laser-SNMS, the three-dimensional distribution of SNPs in cells could be readily detected with very high efficiency, sensitivity, and submicron lateral resolution. We found an accumulation of SNPs directly beneath the cell membrane in a nanoparticular state as well as agglomerations of SNPs inside the cells.
Silica and silver nanoparticles are relevant materials for new applications in optics, medicine, and analytical chemistry. We have previously reported the synthesis of pH responsive, peptide-templated, chiral silver nanoparticles. The current report shows that peptide-stabilized nanoparticles can easily be coated with a silica shell by exploiting the ability of the peptide coating to hydrolyze silica precursors such as TEOS or TMOS. The resulting silica layer protects the nanoparticles from chemical etching, allows their inclusion in other materials, and renders them biocompatible. Using electron and atomic force microscopy, we show that the silica shell thickness and the particle aggregation can be controlled simply by the reaction time. Small-angle X ray scattering confirms the Ag/peptide@silica core–shell structure. UV–vis and circular dichroism spectroscopy prove the conservation of the silver nanoparticle chirality upon silicification. Biological tests show that the biocompatibility in simple bacterial systems is significantly improved once a silica layer is deposited on the silver particles.
Silver nanoparticles (SNP) are the subject of worldwide commercialization because of their antimicrobial effects. Yet only little data on their mode of action exist. Further, only few techniques allow for visualization and quantification of unlabeled nanoparticles inside cells. To study SNP of different sizes and coatings within human macrophages, we introduce a novel laser postionization secondary neutral mass spectrometry (Laser-SNMS) approach and prove this method superior to the widely applied confocal Raman and transmission electron microscopy. With time-of-flight secondary ion mass spectrometry (TOF-SIMS) we further demonstrate characteristic fingerprints in the lipid pattern of the cellular membrane indicative of oxidative stress and membrane fluidity changes. Increases of protein carbonyl and heme oxygenase-1 levels in treated cells confirm the presence of oxidative stress biochemically. Intriguingly, affected phagocytosis reveals as highly sensitive end point of SNP-mediated adversity in macrophages. The cellular responses monitored are hierarchically linked, but follow individual kinetics and are partially reversible.
Silver nanoparticles (SNP) are among the most commercialized nanoparticles worldwide. They can be found in many diverse products, mostly because of their antibacterial properties. Despite its widespread use only little data on possible adverse health effects exist. It is difficult to compare biological data from different studies due to the great variety in sizes, coatings or shapes of the particles. Here, we applied a novel synthesis approach to obtain SNP, which are covalently stabilized by a small peptide. This enables a tight control of both size and shape. We applied these SNP in two different sizes of 20 or 40 nm (Ag20Pep and Ag40Pep) and analyzed responses of THP-1-derived human macrophages. Similar gold nanoparticles with the same coating (Au20Pep) were used for comparison and found to be non-toxic. We assessed the cytotoxicity of particles and confirmed their cellular uptake via transmission electron microscopy and confocal Raman microscopy. Importantly a majority of the SNP could be detected as individual particles spread throughout the cells. Furthermore we studied several types of oxidative stress related responses such as induction of heme oxygenase I or formation of protein carbonyls. In summary, our data demonstrate that even low doses of SNP exerted adverse effects in human macrophages.
Biomimetic synthesis of chiral erbium-doped silver/peptide/silica core-shell nanoparticles (ESPN)
(2011)
Peptide-modified silver nanoparticles have been coated with an erbium-doped silica layer using a method inspired by silica biomineralization. Electron microscopy and small-angle X-ray scattering confirm the presence of an Ag/peptide core and silica shell. The erbium is present as small Er2O3 particles in and on the silica shell. Raman, IR, UV-Vis, and circular dichroism spectroscopies show that the peptide is still present after shell formation and the nanoparticles conserve a chiral plasmon resonance. Magnetic measurements find a paramagnetic behavior. In vitro tests using a macrophage cell line model show that the resulting multicomponent nanoparticles have a low toxicity for macrophages, even on partial dissolution of the silica shell.