Browsing by Author "Kraegeloh, Annette"
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- ItemBright fluorescent silica-nanoparticle probes for high-resolution STED and confocal microscopy(Frankfurt am Main : Beilstein-Institut, 2017) Tavernaro, Isabella; Cavelius, Christian; Peuschel, Henrike; Kraegeloh, AnnetteIn recent years, fluorescent nanomaterials have gained high relevance in biological applications as probes for various fluorescencebased 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.
- ItemA correlative analysis of gold nanoparticles internalized by A549 cells(Hoboken, NJ : Wiley, 2014) Böse, Katharina; Koch, Marcus; Cavelius, Christian; Kiemer, Alexandra K.; Kraegeloh, AnnetteFluorescently labeled nanoparticles are widely used to investigate nanoparticle cell interactions by fluorescence microscopy. Owing to limited lateral and axial resolution, nanostructures (<100 nm) cannot be resolved by conventional light microscopy techniques. Especially after uptake into cells, a common fate of the fluorescence label and the particle core cannot be taken for granted. In this study, a correlative approach is presented to image fluorescently labeled gold nanoparticles inside whole cells by correlative light and electron microscopy (CLEM). This approach allows for detection of the fluorescently labeled particle shell as well as for the gold core in one sample. In this setup, A549 cells are exposed to 8 nm Atto 647N-labeled gold nanoparticles (3.3 × 109 particles mL−1, 0.02 μg Au mL−1) for 5 h and are subsequently imaged by confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM). Eight fluorescence signals located at different intracellular positions are further analyzed by TEM. Five of the eight fluorescence spots are correlated with isolated or agglomerated gold nanoparticles. Three fluorescence signals could not be related to the presence of gold, indicating a loss of the particle shell.
- ItemDigital research data: from analysis of existing standards to a scientific foundation for a modular metadata schema in nanosafety(London : BioMed Central, 2022) Elberskirch, Linda; Binder, Kunigunde; Riefler, Norbert; Sofranko, Adriana; Liebing, Julia; Minella, Christian Bonatto; Mädler, Lutz; Razum, Matthias; van Thriel, Christoph; Unfried, Klaus; Schins, Roel P. F.; Kraegeloh, AnnetteBackground: Assessing the safety of engineered nanomaterials (ENMs) is an interdisciplinary and complex process producing huge amounts of information and data. To make such data and metadata reusable for researchers, manufacturers, and regulatory authorities, there is an urgent need to record and provide this information in a structured, harmonized, and digitized way. Results: This study aimed to identify appropriate description standards and quality criteria for the special use in nanosafety. There are many existing standards and guidelines designed for collecting data and metadata, ranging from regulatory guidelines to specific databases. Most of them are incomplete or not specifically designed for ENM research. However, by merging the content of several existing standards and guidelines, a basic catalogue of descriptive information and quality criteria was generated. In an iterative process, our interdisciplinary team identified deficits and added missing information into a comprehensive schema. Subsequently, this overview was externally evaluated by a panel of experts during a workshop. This whole process resulted in a minimum information table (MIT), specifying necessary minimum information to be provided along with experimental results on effects of ENMs in the biological context in a flexible and modular manner. The MIT is divided into six modules: general information, material information, biological model information, exposure information, endpoint read out information and analysis and statistics. These modules are further partitioned into module subdivisions serving to include more detailed information. A comparison with existing ontologies, which also aim to electronically collect data and metadata on nanosafety studies, showed that the newly developed MIT exhibits a higher level of detail compared to those existing schemas, making it more usable to prevent gaps in the communication of information. Conclusion: Implementing the requirements of the MIT into e.g., electronic lab notebooks (ELNs) would make the collection of all necessary data and metadata a daily routine and thereby would improve the reproducibility and reusability of experiments. Furthermore, this approach is particularly beneficial regarding the rapidly expanding developments and applications of novel non-animal alternative testing methods.
- ItemDiscussion on Existing Standards and Quality Criteria in Nanosafety Research : Summary of the NanoS-QM Expert Workshop(Zenodo, 2021) Binder, Kunigunde; Bonatto Minella, Christian; Elberskirchen, Linda; Kraegeloh, Annette; Liebing, Julia; Petzold, Christiane; Razum, Matthias; Riefler, Norbert; Schins, Roel; Sofranko, Adriana; van Thriel, Christoph; Unfried, KlausThe 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.
- ItemDistribution of SiO2 nanoparticles in 3D liver microtissues(Macclesfield : Dove Medical Press, 2019) Fleddermann, Jana; Susewind, Julia; Peuschel, Henrike; Koch, Marcus; Tavernaro, Isabella; Kraegeloh, AnnetteIntroduction: Nanoparticles (NPs) are used in numerous products in technical fields and biomedicine; their potential adverse effects have to be considered in order to achieve safe applications. Besides their distribution in tissues, organs, and cellular localization, their impact and penetration during the process of tissue formation occurring in vivo during liver regeneration are critical steps for establishment of safe nanomaterials. Materials and methods: In this study, 3D cell culture of human hepatocarcinoma cells (HepG2) was used to generate cellular spheroids, serving as in vitro liver microtissues. In order to determine their differential distribution and penetration depth in HepG2 spheroids, SiO2 NPs were applied either during or after spheroid formation. The NP penetration was comprehensively studied using confocal laser scanning microscopy and scanning electron microscopy. Results: Spheroids were exposed to 100 µg mL-1 SiO2 NPs either at the beginning of spheroid formation, or during or after formation of spheroids. Microscopy analyses revealed that NP penetration into the spheroid is limited. During and after spheroid formation, SiO2 NPs penetrated about 20 µm into the spheroids, corresponding to about three cell layers. In contrast, because of the addition of SiO2 NPs simultaneously to cell seeding, NP agglomerates were located also in the spheroid center. Application of SiO2 NPs during the process of spheroid formation had no impact on final spheroid size. Conclusion: Understanding the distribution of NPs in tissues is essential for biomedical applications. The obtained results indicate that NPs show only limited penetration into already formed tissue, which is probably caused by the alteration of the tissue structure and cell packing density during the process of spheroid formation.
- ItemEstimating the modulatory effects of nanoparticles on neuronal circuits using computational upscaling(Milton Park : Taylor & Francis, 2013) Busse, Michael; Stevens, David; Kraegeloh, Annette; Cavelius, Christian; Vukelic, Mathias; Arzt, Eduard; Strauss, Daniel J.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.
- ItemFormation mechanism for stable hybrid clusters of proteins and nanoparticles(Washington D.C. : American Chemical Society, 2015) Moerz, Sebastian T.; Kraegeloh, Annette; Chanana, Munish; Kraus, TobiasCitrate-stabilized gold nanoparticles (AuNP) agglomerate in the presence of hemoglobin (Hb) at acidic pH. The extent of agglomeration strongly depends on the concentration ratio [Hb]/[AuNP]. Negligible agglomeration occurs at very low and very high [Hb]/[AuNP]. Full agglomeration and precipitation occur at [Hb]/[AuNP] corresponding to an Hb monolayer on the AuNP. Ratios above and below this value lead to the formation of an unexpected phase: stable, microscopic AuNP–Hb agglomerates. We investigated the kinetics of agglomeration with dynamic light scattering and the adsorption kinetics of Hb on planar gold with surface-acoustic wave-phase measurements. Comparing agglomeration and adsorption kinetics leads to an explanation of the complex behavior of this nanoparticle–protein mixture. Agglomeration is initiated either when Hb bridges AuNP or when the electrostatic repulsion between AuNP is neutralized by Hb. It is terminated when Hb has been depleted or when Hb forms multilayers on the agglomerates that stabilize microscopic clusters indefinitely.
- ItemHigh-dose intranasal application of titanium dioxide nanoparticles induces the systemic uptakes and allergic airway inflammation in asthmatic mice(London : BioMed Central, 2020) Harfoush, Shaza Abdulnasser; Hannig, Matthias; Le, Duc Dung; Heck, Sebastian; Leitner, Maximilian; Omlor, Albert Joachim; Tavernaro, Isabella; Kraegeloh, Annette; Kautenburger, Ralf; Kickelbick, Guido; Beilhack, Andreas; Bischoff, Markus; Nguyen, Juliane; Sester, Martina; Bals, Robert; Dinh, Quoc ThaiBackground Titanium dioxide nanoparticles (TiO2 NPs) have a wide range of applications in several industrial and biomedical domains. Based on the evidence, the workers exposed to inhaled nanosized TiO2 powder are more susceptible to the risks of developing respiratory diseases. Accordingly, this issue has increasingly attracted the researchers’ interest in understanding the consequences of TiO2 NPs exposure. Regarding this, the present study was conducted to analyze the local effects of TiO2 NPs on allergic airway inflammation and their uptake in a mouse model of ovalbumin (OVA)-induced allergic airway inflammation. Methods For the purpose of the study, female BALB/c mice with or without asthma were intranasally administered with TiO2 NPs. The mice were subjected to histological assessment, lung function testing, scanning electron microscopy (SEM), inductively coupled plasma mass spectrometry (ICP-MS), and NP uptake measurement. In addition, T helper (Th) 1/Th2 cytokines were evaluated in the lung homogenate using the enzyme-linked immunosorbent assay. Results According to the results, the mice receiving OVA alone or OVA plus TiO2 NPs showed eosinophilic infiltrates and mucus overproduction in the lung tissues, compared to the controls. Furthermore, a significant elevation was observed in the circulating Th2 cytokines, including interleukin (IL)-4, IL-5, and IL-13 after NP exposure. The TiO2 NPs were taken up by alveolar macrophages at different time points. As the results of the SEM and ICP-MS indicated, TiO2 NPs were present in most of the organs in both asthmatic and non-asthmatic mice. Conclusion Based on the findings of the current study, intranasally or inhalation exposure to high-dose nanosized TiO2 particles appears to exacerbate the allergic airway inflammation and lead to systemic uptake in extrapulmonary organs. These results indicate the very important need to investigate the upper limit of intranasally or inhalation exposure to nanosized TiO2 particles in occupational and environmental health policy.
- ItemHow Structured Metadata Acquisition Contributes to the Reproducibility of Nanosafety Studies: Evaluation by a Round-Robin Test(Basel : MDPI, 2022) Elberskirch, Linda; Sofranko, Adriana; Liebing, Julia; Riefler, Norbert; Binder, Kunigunde; Bonatto Minella, Christian; Razum, Matthias; Mädler, Lutz; Unfried, Klaus; Schins, Roel P.F.; Kraegeloh, Annette; van Thriel, ChristophIt has been widely recognized that nanosafety studies are limited in reproducibility, caused by missing or inadequate information and data gaps. Reliable and comprehensive studies should be performed supported by standards or guidelines, which need to be harmonized and usable for the multidisciplinary field of nanosafety research. The previously described minimal information table (MIT), based on existing standards or guidelines, represents one approach towards harmonization. Here, we demonstrate the applicability and advantages of the MIT by a round-robin test. Its modular structure enables describing individual studies comprehensively by a combination of various relevant aspects. Three laboratories conducted a WST-1 cell viability assay using A549 cells to analyze the effects of the reference nanomaterials NM101 and NM110 according to predefined (S)OPs. The MIT contains relevant and defined descriptive information and quality criteria and thus supported the implementation of the round-robin test from planning, investigation to analysis and data interpretation. As a result, we could identify sources of variability and justify deviating results attributed to differences in specific procedures. Consequently, the use of the MIT contributes to the acquisition of reliable and comprehensive datasets and therefore improves the significance and reusability of nanosafety studies
- ItemImpact of mucus modulation by N-acetylcysteine on nanoparticle toxicity(Amsterdam : Elsevier, 2023) Meziu, Enkeleda; Shehu, Kristela; Koch, Marcus; Schneider, Marc; Kraegeloh, AnnetteHuman respiratory mucus is a biological hydrogel that forms a protective barrier for the underlying epithelium. Modulation of the mucus layer has been employed as a strategy to enhance transmucosal drug carrier transport. However, a drawback of this strategy is a potential reduction of the mucus barrier properties, in particular in situations with an increased exposure to particles. In this study, we investigated the impact of mucus modulation on its protective role. In vitro mucus was produced by Calu-3 cells, cultivated at the air-liquid interface for 21 days and used for further testing as formed on top of the cells. Analysis of confocal 3D imaging data revealed that after 21 days Calu-3 cells secrete a mucus layer with a thickness of 24 ± 6 μm. Mucus appeared to restrict penetration of 500 nm carboxyl-modified polystyrene particles to the upper 5–10 μm of the layer. Furthermore, a mucus modulation protocol using aerosolized N-acetylcysteine (NAC) was developed. This treatment enhanced the penetration of particles through the mucus down to deeper layers by means of the mucolytic action of NAC. These findings were supported by cytotoxicity data, indicating that intact mucus protects the underlying epithelium from particle-induced effects on membrane integrity. The impact of NAC treatment on the protective properties of mucus was probed by using 50 and 100 nm amine-modified and 50 nm carboxyl-modified polystyrene nanoparticles, respectively. Cytotoxicity was only induced by the amine-modified particles in combination with NAC treatment, implying a reduced protective function of modulated mucus. Overall, our data emphasize the importance of integrating an assessment of the protective function of mucus into the development of therapy approaches involving mucus modulation.
- ItemImplementation of safe-by-design for nanomaterial development and safe innovation: Why we need a comprehensive approach(Basel : MDPI, 2018) Kraegeloh, Annette; Suarez-Merino, Blanca; Sluijter, Teun; Micheletti, ChristianManufactured nanomaterials (MNMs) are regarded as key components of innovations in various fields with high potential impact (e.g., energy generation and storage, electronics, photonics, diagnostics, theranostics, or drug delivery agents). Widespread use of MNMs raises concerns about their safety for humans and the environment, possibly limiting the impact of the nanotechnology-based innovation. The development of safe MNMs and nanoproducts has to result in a safe as well as functional material or product. Its safe use, and disposal at the end of its life cycle must be taken into account too. However, not all MNMs are similarly useful for all applications, some might bear a higher hazard potential than others, and use scenarios could lead to different exposure probabilities. To improve both safety and efficacy of nanotechnology, we think that a new proactive approach is necessary, based on pre-regulatory safety assessment and dialogue between stakeholders. On the basis of the work carried out in different European Union (EU) initiatives, developing and integrating MNMs Safe-by-Design and Trusted Environments (NANoREG, ProSafe, and NanoReg2), we present our point of view here. This concept, when fully developed, will allow for cost effective industrial innovation, and an exchange of key information between regulators and innovators. Regulators are thus informed about incoming innovations in good time, supporting a proactive regulatory action. The final goal is to contribute to the nanotechnology governance, having faster, cheaper, effective, and safer nano-products on the market.
- ItemThe intracellular localization of inorganic engineered versus biogenic materials: a comparison(Saarbrücken : Leibniz-Institut für Neue Materialien, 2011) Kucki, Melanie; Kraegeloh, AnnetteThe uptake of engineered nanoobjects into cells is assumed to significantly account for their potential toxicity. By internalisation, nanoparticles are at least temporarily trapped in the confined volume of a single cell and come into close contact with cellular components, like organelles, structural proteins, enzymes or signalling molecules. As cells are highly structured entities, exhibiting various types of chemically and biologically distinct compartments, first of all the uptake mechanism determines which types of molecules are encountered. In this review, an introduction into the compartmentalisation of cells as well as some uptake processes is given. The localisation of engineered materials within cells of human and animal origin is exemplified. On the other hand, many living organisms are known for their ability to intracellularly precipitate inorganic structures. Some of these biogenic materials are chemically and structurally similar to artificially generated nanostructures. Therefore, the localisation of some biogenic structures within cells is also illustrated. Finally, the relevance of the specific cellular localisation for toxicity is discussed.
- ItemM2 polarization enhances silica nanoparticle uptake by macrophages(Lausanne : Frontiers Media, 2015) Hoppstädter, Jessica; Seif, Michelle; Dembek, Anna; Cavelius, Christian; Huwer, Hanno; Kraegeloh, Annette; Kiemer, Alexandra KWhile silica nanoparticles have enabled numerous industrial and medical applications, their toxicological safety requires further evaluation. Macrophages are the major cell population responsible for nanoparticle clearance in vivo. The prevailing macrophage phenotype largely depends on the local immune status of the host. Whereas M1-polarized macrophages are considered as pro-inflammatory macrophages involved in host defense, M2 macrophages exhibit anti-inflammatory and wound-healing properties, but also promote tumor growth. We employed different models of M1 and M2 polarization: granulocyte-macrophage colony-stimulating factor/lipopolysaccharide (LPS)/interferon (IFN)-γ was used to generate primary human M1 cells and macrophage colony-stimulating factor (M-CSF)/interleukin (IL)-10 to differentiate M2 monocyte-derived macrophages (MDM). PMA-differentiated THP-1 cells were polarized towards an M1 type by LPS/IFN-γ and towards M2 by IL-10. Uptake of fluorescent silica nanoparticles (Ø26 and 41 nm) and microparticles (Ø1.75 μm) was quantified. At the concentration used (50 μg/ml), silica nanoparticles did not influence cell viability as assessed by MTT assay. Nanoparticle uptake was enhanced in M2-polarized primary human MDM compared with M1 cells, as shown by flow cytometric and microscopic approaches. In contrast, the uptake of microparticles did not differ between M1 and M2 phenotypes. M2 polarization was also associated with increased nanoparticle uptake in the macrophage-like THP-1 cell line. In accordance, in vivo polarized M2-like primary human tumor-associated macrophages obtained from lung tumors took up more nanoparticles than M1-like alveolar macrophages isolated from the surrounding lung tissue. In summary, our data indicate that the M2 polarization of macrophages promotes nanoparticle internalization. Therefore, the phenotypical differences between macrophage subsets should be taken into consideration in future investigations on nanosafety, but might also open up therapeutic perspectives allowing to specifically target M2 polarized macrophages.
- ItemNon-Canonical activation of the epidermal growth factor receptor by carbon nanoparticles(Basel : MDPI, 2018) Stöckmann, Daniel; Spannbrucker, Tim; Ale-Agha, Niloofar; Jakobs, Phillipp; Goy, Christine; Dyballa-Rukes, Nadine; Hornstein, Tamara; Kümper, Alexander; Kraegeloh, Annette; Haendeler, Judith; Unfried, KlausThe 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.
- ItemPenetration of CdSe/ZnS quantum dots into differentiated vs undifferentiated Caco-2 cells(London : BioMed Central, 2016) Peuschel, Henrike; Ruckelshausen, Thomas; Kiefer, Silke; Silina, Yuliya; Kraegeloh, AnnetteBackground: 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.
- ItemPreventing carbon nanoparticle-induced lung inflammation reduces antigen-specific sensitization and subsequent allergic reactions in a mouse model(London : BioMed Central, 2015) Kroker, Matthias; Sydlik, Ulrich; Autengruber, Andrea; Cavelius, Christian; Weighardt, Heike; Kraegeloh, Annette; Unfried, KlausBackground Exposure of the airways to carbonaceous nanoparticles can contribute to the development of immune diseases both via the aggravation of the allergic immune response in sensitized individuals and by adjuvant mechanisms during the sensitization against allergens. The cellular and molecular mechanisms involved in these adverse pathways are not completely understood. We recently described that the reduction of carbon nanoparticle-induced lung inflammation by the application of the compatible solute ectoine reduced the aggravation of the allergic response in an animal system. In the current study we investigated the influence of carbon nanoparticles on the sensitization of animals to ovalbumin via the airways. Ectoine was used as a preventive strategy against nanoparticle-induced neutrophilic lung inflammation. Methods Balb/c mice were repetitively exposed to the antigen ovalbumin after induction of airway inflammation by carbon nanoparticles, either in the presence or in the absence of ectoine. Allergic sensitization was monitored by measurement of immunoglobulin levels and immune responses in lung and lung draining lymph nodes after challenge. Furthermore the role of dendritic cells in the effect of carbon nanoparticles was studied in vivo in the lymph nodes but also in vitro using bone marrow derived dendritic cells. Results Animals exposed to antigen in the presence of carbon nanoparticles showed increased effects with respect to ovalbumin sensitization, to the allergic airway inflammation after challenge, and to the specific TH2 response in the lymph nodes. The presence of ectoine during the sensitization significantly reduced these parameters. The number of antigen-loaded dendritic cells in the draining lymph nodes was identified as a possible cause for the adjuvant effect of the nanoparticles. In vitro assays indicate that the direct interaction of the particles with dendritic cells is not able to trigger CCR7 expression, while this endpoint is achieved by lung lavage fluid from nanoparticle-exposed animals. Conclusions Using the intervention strategy of applying ectoine into the airways of animals we were able to demonstrate the relevance of neutrophilic lung inflammation for the adjuvant effect of carbon nanoparticles on allergic sensitization.n.
- ItemQuantification of internalized silica nanoparticles via STED microscopy(London : Hindawi, 2015) Peuschel, Henrike; Ruckelshausen, Thomas; Cavelius, Christian; Kraegeloh, AnnetteThe development of safe engineered nanoparticles (NPs) requires a detailed understanding of their interaction mechanisms on a cellular level. Therefore, quantification of NP internalization is crucial to predict the potential impact of intracellular NP doses, providing essential information for risk assessment as well as for drug delivery applications. In this study, the internalization of 25 nm and 85 nm silica nanoparticles (SNPs) in alveolar type II cells (A549) was quantified by application of super-resolution STED (stimulated emission depletion) microscopy. Cells were exposed to equal particle number concentrations (9.2 x 10^10 particles mL^-1) of each particle size and the sedimentation of particles during exposure was taken into account. Microscopy images revealed that particles of both sizes entered the cells after 5 h incubation in serum supplemented and serum-free medium. According to the in vitro sedimentation, diffusion, and dosimetry (ISDD) model 20–27 of the particles sedimented. In comparison, 102-103 NPs per cell were detected intracellularly serum-containing medium. Furthermore, in the presence of serum, no cytotoxicity was induced by the SNPs. In serum-free medium, large agglomerates of both particle sizes covered the cells whereas only high concentrations (≥ 3.8 × 10^12 particles mL^-1) of the smaller particles induced cytotoxicity.
- ItemThe role of the intestinal microvasculature in inflammatory bowel disease: studies with a modified Caco-2 model including endothelial cells resembling the intestinal barrier in vitro(Auckland : DOVE Medical Press, 2016) Kasper, Jennifer Y.; Hermanns, Maria Iris; Cavelius, Christian; Kraegeloh, Annette; Jung, Thomas; Danzebrink, Rolf; Unger, Ronald E.; Kirkpatrick, Charles JamesThe microvascular endothelium of the gut barrier plays a crucial role during inflammation in inflammatory bowel disease. We have modified a commonly used intestinal cell model based on the Caco-2 cells by adding microvascular endothelial cells (ISO-HAS-1). Transwell filters were used with intestinal barrier-forming Caco-2 cells on top and the ISO-HAS-1 on the bottom of the filter. The goal was to determine whether this coculture mimics the in vivo situation more closely, and whether the model is suitable to evaluate interactions of, for example, prospective nanosized drug vehicles or contrast agents with this coculture in a physiological and inflamed state as it would occur in inflammatory bowel disease. We monitored the inflammatory responsiveness of the cells (release of IL-8, soluble intercellular adhesion molecule 1, and soluble E-selectin) after exposure to inflammatory stimuli (lipopolysaccharide, TNF-α, INF-γ, IL1-β) and a nanoparticle (Ba/Gd: coprecipitated BaSO4 and Gd(OH)3), generally used as contrast agents. The barrier integrity of the coculture was evaluated via the determination of transepithelial electrical resistance and the apparent permeability coefficient (Papp) of NaFITC. The behavior of the coculture Caco-1/ISO-HAS-1 was compared to the respective monocultures Caco-2 and ISO-HAS-1. Based on transepithelial electrical resistance, the epithelial barrier integrity of the coculture remained stable during incubation with all stimuli, whereas the Papp decreased after exposure to the cytokine mixture (TNF-α, INF-γ, IL1-β, and Ba/Gd). Both the endothelial and epithelial monocultures showed a high inflammatory response in both the upper and lower transwell-compartments. However, in the coculture, inflammatory mediators were only detected on the epithelial side and not on the endothelial side. Thus in the coculture, based on the Papp, the epithelial barrier appears to prevent a potential inflammatory overreaction in the underlying endothelial cells. In summary, this coculture model exhibits in vivo-like features, which cannot be observed in conventional monocultures, making the former more suitable to study interactions with external stimuli.
- ItemSafe-by-Design part II: A strategy for balancing safety and functionality in the different stages of the innovation process(Amsterdam : Elsevier, 2021) Tavernaro, Isabella; Dekkers, Susan; Soeteman-Hernández, Lya G.; Herbeck-Engel, Petra; Noorlander, Cornelle; Kraegeloh, AnnetteManufactured nanomaterials have the potential to impact an exceedingly wide number of industries and markets ranging from energy storage, electronic and optical devices, light-weight construction to innovative medical approaches for diagnostics and therapy. In order to foster the development of safer nanomaterial-containing products, two main aspects are of major interest: their functional performance as well as their safety towards human health and the environment. In this paper a first proposal for a strategy is presented to link the functionality of nanomaterials with safety aspects. This strategy first combines information on the functionality and safety early during the innovation process and onwards, and then identifies Safe-by-Design (SbD) actions that allow for optimisation of both aspects throughout the innovation process. The strategy encompasses suggestions for the type of information needed to balance functionality and safety to support decision making in the innovation process. The applicability of the strategy is illustrated using a literature-based case study on carbon nanotube-based transparent conductive films. This is a first attempt to identify information that can be used for balancing functionality and safety in a structured way during innovation processes.
- ItemSilica nanoparticles for intracellular protein delivery: A novel synthesis approach using green fluorescent protein(London : BioMed Central, 2017) Schmidt, Sarah; Tavernaro, Isabella; Cavelius, Christian; Weber, Eva; Kümper, Alexander; Schmitz, Carmen; Fleddermann, Jana; Kraegeloh, AnnetteIn 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.