Synthese, Stabilität und biologische Wirkungen

Das Ziel dieser Arbeit bestand darin, Silber-Nanopartikel hinsichtlich ihres Verhaltens in einem biologischen Umfeld zu untersuchen. Dazu wurden zunächst mit verschiedenen nasschemischen Methoden Nanopartikel synthetisiert. Diese Partikel wiesen unterschiedliche Größen und Funktionalisierungen auf. Es wurden 70 nm große Partikel durch Reduktion mit Glucose dargestellt, die mit dem Polymer PVP funktionalisiert waren. Durch Reduktion mit Natriumcitrat konnten 45 nm große Partikel erhalten werden, die elektrostatisch stabilisiert waren. Durch die Zugabe von Gerbsäure konnte die Partikelgröße deutlich auf 20 nm verringert werden. Diese Nanopartikel konnten mit PVP oder dem Phosphin-Liganden TPPTS funktionalisiert werden und zeigten, ebenso wie die mit Glucose reduzierten Partikel, eine hohe kolloidale Stabilität. Die Nanopartikel wurden jeweils eingehend mit zahlreichen kolloidchemischen und weiteren Methoden charakterisiert. Dabei zeigten sich die Stärken, aber auch die Grenzen einzelner Methoden wie der Dynamischen Lichtstreuung und der analytischen Scheibenzentrifugation. Es konnte gezeigt werden, dass Silber-Nanopartikel in verschiedenen Medien Ionen freisetzen, sich also auflösen. Dabei wurde gelöster Sauerstoff als die wahrscheinliche Ursache für die Oxidation des metallischen Silbers identifiziert: In sauerstofffreiem Wasser lösten sich die Partikel nahezu nicht auf. Der Zusatz diverser Stoffe förderte (H2O2) oder inhibierte (Cystein, NaCl) die Auflösung der Nanopartikel. Auch anhand makroskopischen Silbers konnte gezeigt werden, dass die chemische Umgebung die Freisetzung von Silberionen aus dem metallischen Silber beeinflusst: Eine Funktionalisierung mit Cystein verminderte die Ionenfreisetzung signifikant. Die mit Glucose reduzierten, PVP-funktionalisierten Silber-Nanopartikel und die mit Citrat und Gerbsäure reduzierten und mit PVP oder TPPTS umfunktionalisierten Nanopartikel wiesen auch in biologischen Zellkulturmedien eine hohe kolloidale Stabilität auf. Sie waren zum Teil über drei Tage in dem stark salzhaltigen, mit FCS versetzten Medium stabil, ohne nennenswerte Agglomeration zu zeigen. Dies ist insbesondere relevant, wenn die Partikel für zellbiologische Untersuchungen verwendet werden sollen. Bei diesen Experimenten wurde eine merkliche Anhebung des hydrodynamischen Durchmessers der dispergierten Partikel festgestellt. Da vermutet wurde, dass dies auf die Bildung einer Protein-Corona um die Partikel zurückzuführen ist, wurde die Adsorption von BSA untersucht. Dabei konnte ebenfalls eine Zunahme des Partikeldurchmessers in Abhängigkeit von der BSA-Konzentration beobachtet werden. BSA bildete eine konzentrationsabhängige Multilage um die Nanopartikel herum aus. Die zellbiologische Untersuchung der Wirkung der synthetisierten Silber-Nanopartikel zeigte, dass sie toxisch auf humane mesenchymale Stammzellen (hMSC) wirken. Diese toxische Wirkung ließ sich mit der Auflösung der Nanopartikel korrelieren. Je älter die Nanopartikel waren, je länger sie also in wässriger Dispersion gelagert wurden und Ionen freisetzen konnten, desto toxischer waren sie. Die Lagerung unter inerten Bedingungen verringerte die Toxizität der Nanopartikel im Vergleich zur Lagerung in lufthaltigem Wasser. Es wurde mittels der FIB-Technik nachgewiesen, dass Silber-Nanopartikel sowohl von hMSC, als auch von Monozyten aufgenommen werden. Die Nanopartikel aktivieren die Zellen, was durch die Messung der veränderten Freisetzung von Interleukin-6 und Interleukin-8 gezeigt wurde. Da sich die Nanopartikel während der Lagerung in wässriger Dispersion dadurch verändern, dass sie sich langsam auflösen und zusätzlich kolloidal altern, wurde versucht, die Partikel mittels Tiefkühlung und Lyophilisation zu konservieren. Dies ist durch den Zusatz des Kryoprotektors Trehalose sehr gut möglich, ohne dass die Partikel nach dem Auftauen bzw. Redispergieren ihre Monodispersität verlieren

A multi-center study of their physicochemical characteristics, cell culture and in vivo experiments

PVP-capped silver nanoparticles with a diameter of the metallic core of 70 nm, a hydrodynamic diameter of 120 nm and a zeta potential of −20 mV were prepared and investigated with regard to their biological activity. This review summarizes the physicochemical properties (dissolution, protein adsorption, dispersability) of these nanoparticles and the cellular consequences of the exposure of a broad range of biological test systems to this defined type of silver nanoparticles. Silver nanoparticles dissolve in water in the presence of oxygen. In addition, in biological media (i.e., in the presence of proteins) the surface of silver nanoparticles is rapidly coated by a protein corona that influences their physicochemical and biological properties including cellular uptake. Silver nanoparticles are taken up by cell-type specific endocytosis pathways as demonstrated for hMSC, primary T-cells, primary monocytes, and astrocytes. A visualization of particles inside cells is possible by X-ray microscopy, fluorescence microscopy, and combined FIB/SEM analysis. By staining organelles, their localization inside the cell can be additionally determined. While primary brain astrocytes are shown to be fairly tolerant toward silver nanoparticles, silver nanoparticles induce the formation of DNA double-strand-breaks (DSB) and lead to chromosomal aberrations and sister-chromatid exchanges in Chinese hamster fibroblast cell lines (CHO9, K1, V79B). An exposure of rats to silver nanoparticles in vivo induced a moderate pulmonary toxicity, however, only at rather high concentrations. The same was found in precision-cut lung slices of rats in which silver nanoparticles remained mainly at the tissue surface. In a human 3D triple-cell culture model consisting of three cell types (alveolar epithelial cells, macrophages, and dendritic cells), adverse effects were also only found at high silver concentrations. The silver ions that are released from silver nanoparticles may be harmful to skin with disrupted barrier (e.g., wounds) and induce oxidative stress in skin cells (HaCaT). In conclusion, the data obtained on the effects of this well-defined type of silver nanoparticles on various biological systems clearly demonstrate that cell-type specific properties as well as experimental conditions determine the biocompatibility of and the cellular responses to an exposure with silver nanoparticles

Effect of silver nanoparticles on human mesenchymal stem cell differentiation

Background: Silver nanoparticles (Ag-NP) are one of the fastest growing products in nano-medicine due to their enhanced antibacterial activity at the nanoscale level. In biomedicine, hundreds of products have been coated with Ag-NP. For example, various medical devices include silver, such as surgical instruments, bone implants and wound dressings. After the degradation of these materials, or depending on the coating technique, silver in nanoparticle or ion form can be released and may come into close contact with tissues and cells. Despite incorporation of Ag-NP as an antibacterial agent in different products, the toxicological and biological effects of silver in the human body after long-term and low-concentration exposure are not well understood. In the current study, we investigated the effects of both ionic and nanoparticulate silver on the differentiation of human mesenchymal stem cells (hMSCs) into adipogenic, osteogenic and chondrogenic lineages and on the secretion of the respective differentiation markers adiponectin, osteocalcin and aggrecan.Results: As shown through laser scanning microscopy, Ag-NP with a size of 80 nm (hydrodynamic diameter) were taken up into hMSCs as nanoparticulate material. After 24 h of incubation, these Ag-NP were mainly found in the endo-lysosomal cell compartment as agglomerated material. Cytotoxicity was observed for differentiated or undifferentiated hMSCs treated with high silver concentrations (≥20 µg·mL−1 Ag-NP; ≥1.5 µg·mL−1 Ag+ ions) but not with low-concentration treatments (≤10 µg·mL−1 Ag-NP; ≤1.0 µg·mL−1 Ag+ ions). Subtoxic concentrations of Ag-NP and Ag+ ions impaired the adipogenic and osteogenic differentiation of hMSCs in a concentration-dependent manner, whereas chondrogenic differentiation was unaffected after 21 d of incubation. In contrast to aggrecan, the inhibitory effect of adipogenic and osteogenic differentiation was confirmed by a decrease in the secretion of specific biomarkers, including adiponectin (adipocytes) and osteocalcin (osteoblasts).Conclusion: Aside from the well-studied antibacterial effect of silver, little is known about the influence of nano-silver on cell differentiation processes. Our results demonstrate that ionic or nanoparticulate silver attenuates the adipogenic and osteogenic differentiation of hMSCs even at non-toxic concentrations. Therefore, more studies are needed to investigate the effects of silver species on cells at low concentrations during long-term treatment

Cytotoxic and proinflammatory effects of PVP-coated silver nanoparticles after intratracheal instillation in rats

Silver nanoparticles (AgNP) are among the most promising nanomaterials, and their usage in medical applications and consumer products is growing rapidly. To evaluate possible adverse health effects, especially to the lungs, the current study focused on the cytotoxic and proinflammatory effects of AgNP after the intratracheal instillation in rats. Monodisperse, PVP-coated AgNP (70 nm) showing little agglomeration in aqueous suspension were instilled intratracheally. After 24 hours, the lungs were lavaged, and lactate dehydrogenase (LDH), total protein, and cytokine levels as well as total and differential cell counts were measured in the bronchoalveolar lavage fluid (BALF). Instillation of 50 µg PVP-AgNP did not result in elevated LDH, total protein, or cytokine levels in BALF compared to the control, whereas instillation of 250 µg PVP-AgNP caused a significant increase in LDH (1.9-fold) and total protein (1.3-fold) levels as well as in neutrophil numbers (60-fold) of BALF. Furthermore, while there was no change in BALF cytokine levels after the instillation of 50 µg PVP-AgNP, instillation of 250 µg PVP-AgNP resulted in significantly increased levels of seven out of eleven measured cytokines. These finding suggest that exposure to inhaled AgNP can induce moderate pulmonary toxicity, but only at rather high concentrations

In vitro and in vivo interactions of selected nanoparticles with rodent serum proteins and their consequences in biokinetics

When particles incorporated within a mammalian organism come into contact with body fluids they will bind to soluble proteins or those within cellular membranes forming what is called a protein corona. This binding process is very complex and highly dynamic due to the plethora of proteins with different affinities and fractions in different body fluids and the large variation of compounds and structures of the particle surface. Interestingly, in the case of nanoparticles (NP) this protein corona is well suited to provide a guiding vehicle of translocation within body fluids and across membranes. This NP translocation may subsequently lead to accumulation in various organs and tissues and their respective cell types that are not expected to accumulate such tiny foreign bodies. Because of this unprecedented NP accumulation, potentially adverse biological responses in tissues and cells cannot be neglected a priori but require thorough investigations. Therefore, we studied the interactions and protein binding kinetics of blood serum proteins with a number of engineered NP as a function of their physicochemical properties. Here we show by in vitro incubation tests that the binding capacity of different engineered NP (polystyrene, elemental carbon) for selected serum proteins depends strongly on the NP size and the properties of engineered surface modifications. In the following attempt, we studied systematically the effect of the size (5, 15, 80 nm) of gold spheres (AuNP), surface-modified with the same ionic ligand; as well as 5 nm AuNP with five different surface modifications on the binding to serum proteins by using proteomics analyses. We found that the binding of numerous serum proteins depended strongly on the physicochemical properties of the AuNP. These in vitro results helped us substantially in the interpretation of our numerous in vivo biokinetics studies performed in rodents using the same NP. These had shown that not only the physicochemical properties determined the AuNP translocation from the organ of intake towards blood circulation and subsequent accumulation in secondary organs and tissues but also the the transport across organ membranes depended on the route of AuNP application. Our in vitro protein binding studies support the notion that the observed differences in in vivo biokinetics are mediated by the NP protein corona and its dynamical change during AuNP translocation in fluids and across membranes within the organism

Interaction of dermatologically relevant nanoparticles with skin cells and skin

The investigation of nanoparticle interactions with tissues is complex. High levels of standardization, ideally testing of different material types in the same biological model, and combinations of sensitive imaging and detection methods are required. Here, we present our studies on nanoparticle interactions with skin, skin cells, and biological media. Silica, titanium dioxide and silver particles were chosen as representative examples for different types of skin exposure to nanomaterials, e.g., unintended environmental exposure (silica) versus intended exposure through application of sunscreen (titanium dioxide) or antiseptics (silver). Because each particle type exhibits specific physicochemical properties, we were able to apply different combinations of methods to examine skin penetration and cellular uptake, including optical microscopy, electron microscopy, X-ray microscopy on cells and tissue sections, flow cytometry of isolated skin cells as well as Raman microscopy on whole tissue blocks. In order to assess the biological relevance of such findings, cell viability and free radical production were monitored on cells and in whole tissue samples. The combination of technologies and the joint discussion of results enabled us to look at nanoparticle–skin interactions and the biological relevance of our findings from different angles

PVP-coated, negatively charged silver nanoparticles: A multi-center study of their physicochemical characteristics, cell culture and in vivo experiments

PVP-capped silver nanoparticles with a diameter of the metallic core of 70 nm, a hydrodynamic diameter of 120 nm and a zeta potential of −20 mV were prepared and investigated with regard to their biological activity. This review summarizes the physicochemical properties (dissolution, protein adsorption, dispersability) of these nanoparticles and the cellular consequences of the exposure of a broad range of biological test systems to this defined type of silver nanoparticles. Silver nanoparticles dissolve in water in the presence of oxygen. In addition, in biological media (i.e., in the presence of proteins) the surface of silver nanoparticles is rapidly coated by a protein corona that influences their physicochemical and biological properties including cellular uptake. Silver nanoparticles are taken up by cell-type specific endocytosis pathways as demonstrated for hMSC, primary T-cells, primary monocytes, and astrocytes. A visualization of particles inside cells is possible by X-ray microscopy, fluorescence microscopy, and combined FIB/SEM analysis. By staining organelles, their localization inside the cell can be additionally determined. While primary brain astrocytes are shown to be fairly tolerant toward silver nanoparticles, silver nanoparticles induce the formation of DNA double-strand-breaks (DSB) and lead to chromosomal aberrations and sister-chromatid exchanges in Chinese hamster fibroblast cell lines (CHO9, K1, V79B). An exposure of rats to silver nanoparticles in vivo induced a moderate pulmonary toxicity, however, only at rather high concentrations. The same was found in precision-cut lung slices of rats in which silver nanoparticles remained mainly at the tissue surface. In a human 3D triple-cell culture model consisting of three cell types (alveolar epithelial cells, macrophages, and dendritic cells), adverse effects were also only found at high silver concentrations. The silver ions that are released from silver nanoparticles may be harmful to skin with disrupted barrier (e.g., wounds) and induce oxidative stress in skin cells (HaCaT). In conclusion, the data obtained on the effects of this well-defined type of silver nanoparticles on various biological systems clearly demonstrate that cell-type specific properties as well as experimental conditions determine the biocompatibility of and the cellular responses to an exposure with silver nanoparticles