PD Dr. Annette Kraegeloh

Superparamagnetic iron oxide nanoparticles (SPION) are widely used both clinically and experimentally for diverse in vivo applications, such as contrast enhancement in magnetic resonance imaging, hyperthermia and drug delivery. Biomedical applications require particles to have defined physical and chemical properties, and to be stable in biological media. Despite a suggested low cytotoxic action, adverse reactions of SPION in concentrations relevant for biomedical use have not yet been studied in sufficient detail. In the present work we employed Endorem®, dextran-stabilized SPION approved as an intravenous contrast agent, and compared its action to a set of other nanoparticles with potential for magnetic resonance imaging applications. SPION in concentrations relevant for in vivo applications were rapidly taken up by endothelial cells and exhibited no direct cytotoxicity. Electric cell impedance sensing measurements demonstrated that SPION, but not BaSO4/Gd nanoparticles, impaired endothelial integrity, as was confirmed by increased intercellular gap formation in endothelial monolayers. These structural changes induced the subcellular translocation and inhibition of the cytoprotective and anti-atherosclerotic enzyme endothelial NO-synthase and reduced NO production. Lipopolysaccharide-induced inflammatory NO production of macrophages was not affected by SPION. In conclusion, our data suggest that SPION might substantially alter endothelial integrity and function at therapeutically relevant doses, which are not cytotoxic.

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.

Background: Beside the promising application potential of nanotechnologies in engineering, the use of nanomaterials in medicine is growing. New therapies employing innovative nanocarrier systems to increase specificity and efficacy of drug delivery schemes are already in clinical trials. However the influence of the nanoparticles themselves is still unknown in medical applications, especially for complex interactions in neural systems. The aim of this study was to investigate in vitro effects of coated silver nanoparticles (cAgNP) on the excitability of single neuronal cells and to integrate those findings into an in silico model to predict possible effects on neuronal circuits. Methods: We first performed patch clamp measurements to investigate the effects of nanosized silver particles, surrounded by an organic coating, on excitability of single cells. We then determined which parameters were altered by exposure to those nanoparticles using the Hodgkin-Huxley model of the sodium current. As a third step, we integrated those findings into a well-defined neuronal circuit of thalamocortical interactions to predict possible changes in network signaling due to the applied cAgNP, in silico. Results: We observed rapid suppression of sodium currents after exposure to cAgNP in our in vitro recordings. In numerical simulations of sodium currents we identified the parameters likely affected by cAgNP. We then examined the effects of such changes on the activity of networks. In silico network modeling indicated effects of local cAgNP application on firing patterns in all neurons in the circuit.
Conclusion: Our sodium current simulation shows that suppression of sodium currents by cAgNP results primarily by a reduction in the amplitude of the current. The network simulation shows that locally cAgNP-induced changes result in changes in network activity in the entire network, indicating that local application of cAgNP may influence the activity throughout the network.

Nanoparticles may be taken up into cells via endocytotic processes whereby the foreign particles are encapsulated in vesicles formed by lipid bilayers. After uptake into these endocytic vesicles, intracellular targeting processes and vesicle fusion might cause transfer of the vesicle cargo into other vesicle types, e.g., early or late endosomes, lysosomes, or others. In addition, nanoparticles might be taken up as single particles or larger agglomerates and the agglomeration state of the particles might change during vesicle processing. In this study, liposomes are regarded as simple models for intracellular vesicles. We compared the energetic balance between two liposomes encapsulating each a single silica nanoparticle and a large liposome containing two silica nanoparticles. Analytical expressions were derived that show how the energy of the system depends on the particle size and the distance between the particles. We found that the electrostatic contributions to the total energy of the system are negligibly small. In contrast, the van der Waals term strongly favors arrangements where the liposome snugly fits around the nanoparticle(s). Thus the two separated small liposomes have a more favorable energy than a larger liposome encapsulating two nanoparticles.

This paper proposes an advanced image enhancement method that is specifically tailored towards 3-D confocal and STED microscopy imagery. Our approach unifies image denoising, deblurring and interpolation in one joint method to handle the typical weaknesses of these advanced microscopy techniques: out-of-focus blur, Poisson noise and low axial resolution. In detail, we propose the combination of (i) Richardson–Lucy deconvolution, (ii) image restoration and (iii) anisotropic inpainting in one single scheme. To this end, we develop a novel PDE-based model that realizes these three ideas. First we consider a basic variational image restoration functional that is turned into a joint interpolation scheme by extending the regularization domain. Next, we integrate the variational representation of Richardson–Lucy deconvolution into our model, and illustrate its relation to Poisson distributed noise. In the following step, we supplement the components of our model with sub-quadratic penalization strategies that increase the robustness of the overall method. Finally, we consider the associated minimality conditions, where we exchange the occurring scalar-valued diffusivity function by a so-called diffusion tensor. This leads to an anisotropic regularization that is aligned with structures in the evolving image. As a further contribution of this paper, we propose a more efficient and faster semi-implicit iteration scheme that also increases the stability. Our experiments on real data sets demonstrate that this joint model achieves a superior reconstruction quality of the recorded cell.

Inflammatory activation of alveolar macrophages by ambient particles can be facilitated via Toll-like receptors (TLR). The action of TLR agonists and antagonists has been reported to depend on the formation of nanoparticulate structures. Aim of the present study was to identify the signaling pathways induced by nanoparticulate structures in human macrophages, which might be critical for inflammatory cell activation. Methods Studies were performed in primary human alveolar macrophages or in differentiated THP-1 macrophages. Silica nanoparticles were prepared by Stöber synthesis and characterized by dynamic light scattering and scanning electron microscopy. Mycobacterial DNA was isolated from Mycobacterium bovis BCG, and nanoparticle formation was assessed by atomic force microscopy and dynamic light scattering. Actin polymerization was measured by phalloidin-TRITC staining, and cell activation was determined by reverse transcription quantitative PCR analysis, L929 cytotoxicity assay (cytokine induction), and pull-down assays (Rho GTPases). Results In contrast to immune stimulatory sequence ISS 1018, BCG DNA spontaneously formed nanoparticulate structures and induced actin polymerization as did synthetic silica nanoparticles. Co-incubation with silica nanoparticles amplified the responsiveness of macrophages toward the TLR9 ligand ISS 1018. The activation of Rac1 was induced by silica nanoparticles as well as BCG DNA and is suggested as the critical signaling event inducing both cytoskeleton changes as well as inflammatory cell activation. Conclusion Nanoparticles can induce signaling pathways, which amplify an inflammatory response in macrophages.

For a detailed analysis of the biological effects of silver nanoparticles, discrimination between effects related to the nano-scale size of the particles and effects of released silver ions is required. Silver ions are either present in the initial particle dispersion or released by the nanoparticles over time. The aim of this study is to monitor the free silver ion activity {Ag+} in the presence of silver nanoparticles using a silver ion selective electrode. Therefore, silver in the form of silver nanoparticles, 4.2 ± 1.4 nm and 2-30 nm in size, or silver nitrate was added to cell culture media in the absence or presence of A549 cells as a model for human type II alveolar epithelial cells. The free silver ion activity measured after the addition of silver nanoparticles was determined by the initial ionic silver content. The p {Ag+} values indicated that the cell culture media decrease the free silver ion activity due to binding of silver ions by constituents of the media. In the presence of A549 cells, the free silver ion activity was further reduced. The morphology of A549 cells, cultivated in DME medium containing 9.1% (v/v) FBS, was affected by adding AgNO3 at concentrations of ≥30 μM after 24 h. In comparison, silver nanoparticles up to a concentration of 200 μM Ag did not affect cellular morphology. Our experiments indicate that the effect of silver nanoparticles is mainly mediated by silver ions. An effect of silver on cellular morphology was observed at p {Ag+} ≤ 9.2.

Localization and intracellular migration of 32 and 83 nm SiO2 nanoparticles in relation to the nucleus was evaluated in vitro on undifferentiated human colon carcinoma (Caco-2) cells. The fluorescence dye Atto647N was incorporated into the particles, which enabled detection by high resolution, nondiffraction limited stimulated emission depletion (STED) microscopy. The distribution and agglomeration of nanoparticles was measured with STED microscopy after 5, 24, 48, and 72 h. Analyses revealed that only 32 nm silica particles penetrated into the nucleus of Caco-2 cells upon exposure for 24 h. Here, they formed agglomerates up to 300 nm after 72 h of incubation. Quantitative evaluation of the migration of 32 nm compared to 83 nm particles demonstrated that 32 nm particles obviously migrated faster into and through the cell in the beginning (5 h time point). The presence and agglomeration inside the cells and the penetration into the nucleus were considered to potentially activate cytotoxic responses. Therefore, the cytotoxic (WST-1 assay) and genotoxic (comet assay) effects of the silica nanoparticles were evaluated. Even though 32 nm silica particles are penetrating into the nucleus, neither cytotoxic nor genotoxic effects were detected for either particle size.