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

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

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

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

Synthesis of tumor-associated MUC1-glycopeptides and their multivalent presentation by functionalized gold colloids

The mucin MUC1 is a glycoprotein involved in fundamental biological processes, which can be found over-expressed and with a distinctly altered glycan pattern on epithelial tumor cells; thus it is a promising target structure in the quest for effective carbohydrate-based cancer vaccines and immunotherapeutics. Natural glycopeptide antigens indicate only a low immunogenicity and a T-cell independent immune response; however, this major drawback can be overcome by coupling of glycopeptide antigens multivalently to immunostimulating carrier platforms. In particular, gold nanoparticles are well suited as templates for the multivalent presentation of glycopeptide antigens, due to their remarkably high surface-to-volume ratio in combination with their high biostability. In this work the synthesis of novel MUC1-glycopeptide antigens and their coupling to gold nanoparticles of different sizes are presented. In addition, the development of a new dot-blot immunoassay to test the potential antigen-antibody binding is introduced

Influence of nanoparticle encapsulation and encoding on the surface chemistry of polymer carrier beads

Surface-functionalized polymer beads encoded with molecular luminophores and nanocrystalline emitters such as semiconductor nanocrystals, often referred to as quantum dots (QDs), or magnetic nanoparticles are broadly used in the life sciences as reporters and carrier beads. Many of these applications require a profound knowledge of the chemical nature and total number of their surface functional groups (FGs), that control bead charge, colloidal stability, hydrophobicity, and the interaction with the environment and biological systems. For bioanalytical applications, also the number of groups accessible for the subsequent functionalization with, e.g., biomolecules or targeting ligands is relevant. In this study, we explore the influence of QD encoding on the amount of carboxylic acid (COOH) surface FGs of 2 µm polystyrene microparticles (PSMPs). This is done for frequently employed oleic acid and oleylamine stabilized, luminescent core/shell CdSe QDs and two commonly used encoding procedures. This included QD addition during bead formation by a thermally induced polymerization reaction and a post synthetic swelling procedure. The accessible number of COOH groups on the surface of QD-encoded and pristine beads was quantified by two colorimetric assays, utilizing differently sized reporters and electrostatic and covalent interactions. The results were compared to the total number of FGs obtained by a conductometric titration and Fourier transform infrared spectroscopy (FTIR). In addition, a comparison of the impact of QD and dye encoding on the bead surface chemistry was performed. Our results demonstrate the influence of QD encoding and the QD-encoding strategy on the number of surface FG that is ascribed to an interaction of the QDs with the carboxylic acid groups on the bead surface. These findings are of considerable relevance for applications of nanoparticle-encoded beads and safe-by-design concepts for nanomaterials

Popularization of health in public television: the case of RTVE in 2016

La televisión es el principal medio al que los ciudadanos están expuestos para informarse sobre temas de ciencia y tecnología (FECYT, 2015). El servicio pú- blico radiotelevisivo es el ente, que, por ley, debe atender a las necesidades de información, cultura, educación y entretenimiento de la ciudadanía. Esta investigación tiene como objetivo conocer el tratamiento informativo y audiovisual de los programas especializados en salud de RTVE. La muestra cuenta con aquellos emitidos en 2016: La mañana (incluye Saber Vivir), Centro Médico, Esto es Vida y El Ojo Clínico. La metodología empleada es el estudio de caso: se elabora una triangulación a partir de una exploración descriptivo-analítica de los programas seleccionados, un cuestionario sobre la recepción informativa de los mismos y un análisis de contenido de una serie de episodios y, a su vez, de los tweets emitidos por sus respectivas cuentas durante el último mes. Las variables miden aspectos del tratamiento informativo y audiovisual de los programas, así como la funcionalidad, el tratamiento informativo de los tuits y el contenido extra que aportan. Los resultados presentan como rasgo común la conducción de la información a través de fuentes personales. Asimismo, destaca El Ojo Clínico y Esto es Vida por su tratamiento divulgativo, tanto en pantalla como en Twitter.Citizens inform themselves about science and technology mainly through television (FECYT, 2015) and the public broadcasting service is the entity that by law must offer the information, culture, education and entertainment that citizens need. This research aims to know the informative and audiovisual treatment of RTVE’s specialized health programs. The sample consists of the programmes broadcasted in 2016: La mañana (includes Saber Vivir), Centro Médico, Esto es Vida and El Ojo Clínico. A case study methodology will be implemented, a triangulation of methods is applied: a descriptive-analytical exploration of the selected programs, a content analysis of some episodes and of a sample of official tweets issued by the tv programmes accounts and a questionnaire on the informative reception by the audience. The variables analyse aspects of the informative and audiovisual treatment of the tv programmes, as well as tweet’s aims and reporting style. An overall result is the common usage of personal information as scientific sources. El Ojo Clínico and Esto es Vida need to be highlighted for their positive informative reporting, both on screen and on Twitter

Targeted T1 Magnetic Resonance Imaging Contrast Enhancement with Extraordinarily Small CoFe2O4 Nanoparticles

Extraordinarily small (2.4 nm) cobalt ferrite nanoparticles (ESCIoNs) were synthesized by a one-pot thermal decomposition approach to study their potential as magnetic resonance imaging (MRI) contrast agents. Fine size control was achieved using oleylamine alone, and annular dark-field scanning transmission electron microscopy revealed highly crystalline cubic spinel particles with atomic resolution. Ligand exchange with dimercaptosuccinic acid rendered the particles stable in physiological conditions with a hydrodynamic diameter of 12 nm. The particles displayed superparamagnetic properties and a low r2/r1 ratio suitable for a T1 contrast agent. The particles were functionalized with bile acid, which improved biocompatibility by significant reduction of reactive oxygen species generation and is a first step toward liver-targeted T1 MRI. Our study demonstrates the potential of ESCIoNs as T1 MRI contrast agents

Multivalent presentation of potential inhibitors of the selectin-ligand interaction by bioactive nanoparticles

Neue Fortschritte im Verständnis des Entzündungsprozesses und dessen Beteiligung in einigen ernsten Erkrankungen, wie z.B. der Krebs-Metastasierung und verschiedenen (Auto)Immunerkrankungen (Asthma, Allergien, Morbus Crohn, rheumatoide Arthritis), haben die Entwicklung neuer antiinflammatorischer Therapieansätze in den letzten zwei Jahrzehnten rasant vorangetrieben. Dabei handelt es sich beim Entzündungsprozess um eine Kaskade verschiedener Reaktionen, deren Schlüsselschritt die Adhäsion der Leukozyten am vaskulären Endothel und deren Extravasation aus dem Blutstrom in das entzündete Gewebe darstellt. Eine entscheidende Rolle spielen hierbei die Wechselwirkungen einer Gruppe von kohlenhydratbindenden Adhäsionsmolekülen, die als Selektine bezeichnet werden, weswegen diese als interessante Wirkstofftargets gelten. Untersuchungen bekannter natürlicher Selektinliganden, wie z.B. Heparin und der P–Selektin Glycoprotein Ligand–1 (PSGL-1), konnten zeigen, dass die geringe Affinität der Bindung zwischen Selektinen und ihren entsprechenden Liganden durch eine multivalente Präsentation der funktionellen Gruppen überwunden werden kann. Aus diesem Grund besitzen Nanopartikel wegen ihres enormen Oberflächen-zu-Volumen-Verhältnisses großes Potenzial als mögliche Template für die Entwicklung effektiverer Selektin-Inhibitoren. Ziel der vorliegenden Arbeit war es, die Oberfläche von kolloidalen Nanopartikeln (Edelmetall-, Halbleiter-, Eisenoxidnanopartikel) mit verschiedenen Strukturen zu funktionalisieren. Neben der Stabilität der Ligandenhülle hängen die Eigenschaften der Nanopartikel stark von ihrer Partikelgröße, Partikelform, der Dispersität und ihrem Agglomerationsgrad ab, weswegen die Grundlage für die angestrebten Ziele die Synthese möglichst monodisperser, sphärischer Kolloide in unterschiedlichen Größen war. Im ersten Teil dieser Arbeit wurden durch Verbesserungen literaturbekannter und Entwicklung neuer Synthesewege monodisperse Kolloide mit Partikelgrößen je nach Material im Bereich von 1,5-25 nm und einer Größendispersität < 10% hergestellt. Die Charakterisierung der erhaltenen Nanopartikel wurde dabei mithilfe von TEM-Aufnahmen, DLS-Messungen und Absorptions- bzw. Emissionsspektroskopie durchgeführt, während die Charakterisierung der organischen Ligandenhülle durch NMR- und IR-Spektroskopie erfolgte. Im zweiten Teil der vorliegenden Arbeit wurde die Funktionalisierung der aus der Synthese erhaltenen, monodispersen Nanopartikel mit potenziellen Selektin-Inhibitoren vorgestellt. Die Modifikation der Partikeloberfläche erfolgte dabei durch Ligandenaustauschreaktionen und Reaktionen an der Ligandenperipherie durch Amidbindungsknüpfung. Dabei gelang es die aus früheren Arbeiten des Arbeitskreises bekannten azyklischen Aminoalkohole und Kohlenhydratmimetika unter Funktionserhalt auf neue Kernmaterialien (Halbleiter- und Eisenoxidnanopartikel) zu übertragen. Auf diese Weise lassen sich die guten Bindungseigenschaften mit einer Erhöhung der Biokompatibilität und einer besseren Detektierbarkeit im biologischen System kombinieren. Ausgehend von den bekannten Vorbildern sLex und Heparinoide wurden im Rahmen dieser Arbeit weitere Strukturen als potenzielle Inhibitoren der Selektin-Ligand-Wechselwirkung auf der Nanopartikeloberfläche immobilisiert. Beginnend mit der Klasse der zyklischen Oligosaccharide wurde beta-Cyclodextrin wegen seiner hohen Ladungsdichte auf der Partikeloberfläche immobilisiert und im Anschluss daran sulfatiert. Außerdem boten sich als zu testende Ligandenklasse vereinfachte, abgeleitete Strukturen der Kakao-Polyphenole (Clovamide) an, da sie einerseits für eine Hemmung der P-Selektin Expression in der Literatur bekannt sind und andererseits zusätzlich Tyrosin-Reste enthalten, die in sulfatierter Form auch in natürlichen Selektinliganden vorkommen. Eine dritte Substanzgruppe waren MUC1-Glycopeptide-Antigene, die zur Entwicklung von Krebsvakzinen eingesetzt werden könnten. Durch Variation des Kernmaterials, der Partikelgröße, der Ankergruppe und der Linkerlänge wurde ein breites Spektrum an unterschiedlichen funktionalisierten Nanopartikeln erhalten. Diese wurden im Rahmen der Arbeit ebenfalls auf ihre Biokompatibilität hin untersucht. Dieser Einfluss wurde an einer einzelnen Charge HUVEC-Zellen getestet. Dabei zeigte sich, dass die zytotoxische und zellproliferationshemmende Wirkung der funktionalisierten Nanopartikel nicht auf das Kernmaterial und nur bedingt auf die Partikelgröße zurückzuführen war. Neben der Konzentration hatte die Stabilität der gebundenen Liganden den größten Einfluss auf die Toxizität. Hingegen zeigten Tests an mesenchymalen Stammzellen auch einen Einfluss der Partikelgröße. Zusätzlich wurden erste Untersuchungen zur Aufnahme der Partikel in die Zelle vorgestellt. Dabei konnte nachgewiesen werden, dass unabhängig von der Partikelgröße und der Ligandenhülle die Nanopartikel über Endozytose in die Zellen aufgenommen werden.Recently inflammation has become one of the most rapidly developing areas of medical research, due to new studies indicating that inflammation mediate most chronic diseases (e.g. cancer, asthma, allergies, Crohn´s disease, rheumatoid arthritis), which are accompanied by an ongoing extravasation of leukocytes from the blood vessels into the inflammed tissue. This involves a multistep adhesion process initialized by different selectins and their respective carbohydrate ligands. Because of this selectins are promising targets for an anti-inflammatory therapy to inhibit this key step of the inflammation casscade. Since selectins tend to bind only weakly to their complementary receptors, stronger binding affinities can be achieved by the use of multivalent interaction. Due to their remarkably high surface-to-volume ratio in combination with their high stability, nanoparticles are well suited as scaffolds for the multivalent immobilization of potential selectin-inhibitors. The aim of this doctoral thesis was the synthesis of different types of monodisperse nanoparticles and their surface modification with bioactive molecules. These potential ligands have to fulfill some requirements, like good dispersibility and stability in a physiological environment to protect the particle core against agglomeration. In addition, they also should indicate a relative high binding affinity to selectins. The properties of functionalized nanoparticles dependent on their size, their shape, their dispersity and their agglomeration degree. Therefore the first part of this thesis describes the syntheses of various types of spherical, nearly monodisperse nanoparticles. For this purpose literature known methods were improved and new synthetic strategies were developed, so that depending on the type, synthesized nanoparticles ranging in size from 1.5 to 25 nm with a dispersity < 10% were obtained. The characterization of the synthesized nanoparticles was carried out by various methods. TEM images, UV/Vis spectra and dynamic light scattering measurements were used to determine the average diameter, the shape, the agglomeration state and the size distribution of the synthesized nanoparticles, while the characterization of the organic ligand shell was performed by NMR and IR spectroscopy. In the second part of this thesis the functionalization of the synthesized nanoparticles with selectin binding ligands was presented. The modification of the particle surface was achieved by ligand exchange reactions or by chemical binding of the ligands through formation of a stable amide bond. It was demonstrated that the transfer of a ligand shell to other nanostructured core materials (quantum dots, iron oxide nanoparticles) is possible under preservation of its function. This was tested using acyclic amino alcohols and aminopyrans already known from previous work. Thereby the good binding properties have been combined with further desirable attributes, like a higher biocompatibility and better detectability in biological systems. Using the tetrasaccharide sLex and heparinoids as lead compounds for selectin-binding ligands, further structures were identified as potential inhibitors and immobilized on the nanoparticle surface. Due to its high charge density the cyclic oligosaccharide beta-cyclodextrine was selected and bond on the nanoparticle surface and sulfated subsequently.Following the concept of simplified binding epitopes, a series of structures derieved from cocoa polyphenols (clovamides), were also tested as potential selectin-binding ligands. Recent studies indicate that these clovamides are able to inhibit P-selectin expression and that they are derivatives of the amino acid L-tyrosine, which occur in sulfated form also in natural selectin ligands. A third group of substances were MUC1 glycopeptides antigens that may be used for the development of cancer vaccines. Another scope of this thesis was the investigation of the biocompatibility of the functionalized nanoparticles. For this purpose several parameters like the core material, the particle diameter and a variety of surface modifications were taken into account to evaluate the potential cytotoxic effects of the functionalized nanoparticles on HUVECs of one single batch. The test results demonstrates that the core material have no influence on the cytotoxicity and cell proliferation, whereas the particle size show only small effects. The main influence of nanoparticles on HUVECs was accounted for the nanoparticle concentration and the stability of the ligands. On the other hand, in vitro tests on mesenchymal stem cells also indicate a toxic effect of the particle size. Beside the cytotoxicity of nanoparticles, the cell uptake of the nanoparticles was investigated in this thesis. It was demonstrated, that the nanoparticles penetrate into the cells by endocytosis. This is independent from their size and ligand shell

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 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