Formation mechanism for stable hybrid clusters of proteins and nanoparticles

Citrate-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

Distribution of SiO2 nanoparticles in 3D liver microtissues

Introduction: 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

Exposure in chemical nanotechnology

Exposures to ultrafine dust due to naturally emitted and incidentally generated nanoscale particles are already known. The exposure to specifically engineered nanomaterials is a topic of actual interest. Keeping in mind the special and heterogeneous spectrum of exposure, exposed individuals underwent detailed examinations in our institute and at their workplace. We also analysed the chemical nanotechnology work sites

A correlative analysis of gold nanoparticles internalized by A549 cells

Fluorescently 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

Tätigkeitsparallele Diagnostik bei beruflicher Exposition gegenüber Nanopartikeln und neuen Materialien. — II

Eine Exposition gegenüber ultrafeinen Stäuben — zurückzuführen auf natürlich freigesetzte und unbeabsichtigt erzeugte nanoskalige Partikel — ist seit langem bekannt. Neu dagegen ist die Exposition gegenüber gezielt industriell synthetisierten Nanomaterialien. Vor dem Hintergrund eines solch speziellen und sehr heterogenen Expositionsspektrums erfolgte nach ausführlicher Individual-Diagnostik eine tätigkeitsparallele Untersuchung bei Beschäftigten in der chemischen Nanotechnologie

Bright fluorescent silica-nanoparticle probes for high-resolution STED and confocal microscopy

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

Preventing carbon nanoparticle-induced lung inflammation reduces antigen-specific sensitization and subsequent allergic reactions in a mouse model

Background 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

Safe-by-Design part II: A strategy for balancing safety and functionality in the different stages of the innovation process

Manufactured 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

M2 polarization enhances silica nanoparticle uptake by macrophages

While 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

Silica nanoparticles for intracellular protein delivery: A novel synthesis approach using green fluorescent protein

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