Size Dependence of Steric Shielding and Multivalency Effects for Globular Binding Inhibitors

Competitive binding inhibitors based on multivalent nanoparticles have shown great potential for preventing virus infections. However, general design principles of highly efficient inhibitors are lacking as the quantitative impact of factors such as virus concentration, inhibitor size, steric shielding, or multivalency effects in the inhibition process is not known. Based on two complementary experimental inhibition assays we determined size- dependent steric shielding and multivalency effects. This allowed us to adapt the Cheng–Prusoff equation for its application to multivalent systems. Our results show that the particle and volume normalized IC50 value of an inhibitor at very low virus concentration predominantly depends on its multivalent association constant, which itself exponentially increases with the inhibitor/virus contact area and ligand density. Compared to multivalency effects, the contribution of steric shielding to the IC50 values is only minor, and its impact is only noticeable if the multivalent dissociation constant is far below the virus concentration, which means if all inhibitors are bound to the virus. The dependence of the predominant effect, either steric shielding or multivalency, on the virus concentration has significant implications on the in vitro testing of competitive binding inhibitors and determines optimal inhibitor diameters for the efficient inhibition of viruses

A Water-Processable and Bioactive Multivalent Graphene Nanoink for Highly Flexible Bioelectronic Films and Nanofibers

The capabilities of conductive nanomaterials to be produced in liquid form with well-defined chemical, physical, and biological properties are highly important for the construction of next-generation flexible bioelectronic devices. Although functional graphene nanomaterials can serve as attractive liquid nanoink platforms for the fabrication of bioelectronics, scalable synthesis of graphene nanoink with an integration of high colloidal stability, water processability, electrochemical activity, and especially bioactivity remains a major challenge. Here, a facile and scalable synthesis of supramolecular-functionalized multivalent graphene nanoink (mGN-ink) via [2+1] nitrene cycloaddition is reported. The mGN-ink unambiguously displays a well- defined and flat 2D morphology and shows good water processability and bioactivity. The uniquely chemical, physical, and biological properties of mGN-ink endow the constructed bioelectronic films and nanofibers with high flexibility and durability, suitable conductivity and electrochemical activity, and most importantly, good cellular compatibility and a highly efficient control of stem-cell spreading and orientation. Overall, for the first time, a water-processable and bioactive mGN-ink is developed for the design of flexible and electrochemically active bioelectronic composites and devices, which not only presents manifold possibilities for electronic- cellular applications but also establishes a new pathway for adapting macroscopic usages of graphene nanomaterials in bionic, biomedical, electronic, and even energy fields

The Cytotoxic Effects of Betulin-Conjugated Gold Nanoparticles as Stable Formulations in Normal and Melanoma Cells

Gold nanoparticles are currently investigated as theranostics tools in cancer therapy due to their proper biocompatibility and increased efficacy related to the ease to customize the surface properties and to conjugate other molecules. Betulin, [lup-20(29)-ene-3β, 28-diol], is a pentacyclic triterpene that has raised scientific interest due to its antiproliferative effect on several cancer types. Herein we described the synthesis of surface modified betulin-conjugated gold nanoparticles using a slightly modified Turkevich method. Transmission electron microscopy (TEM) imaging, dynamic light scattering (DLS), scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX) were used for the characterization of obtained gold nanoparticles. Cytotoxic activity and apoptosis assessment were carried out using the MTT and Annexin V/PI apoptosis assays. The results showed that betulin coated gold nanoparticles presented a dose-dependent cytotoxic effect and induced apoptosis in all tested cell lines

The bubble snails (Gastropoda, Heterobranchia) of Mozambique: an overlooked biodiversity hotspot

This first account, dedicated to the shallow water marine heterobranch gastropods of Mozambique is presented with a focus on the clades Acteonoidea and Cephalaspidea. Specimens were obtained as a result of sporadic sampling and two dedicated field campaigns between the years of 2012 and 2015, conducted along the northern and southern coasts of Mozambique. Specimens were collected by hand in the intertidal and subtidal reefs by snorkelling or SCUBA diving down to a depth of 33 m. Thirty-two species were found, of which 22 are new records to Mozambique and five are new for the Western Indian Ocean. This account raises the total number of shallow water Acteonoidea and Cephalaspidea known in Mozambique to 39 species, which represents approximately 50 % of the Indian Ocean diversity and 83 % of the diversity of these molluscs found in the Red Sea. A gap in sampling was identified in the central swamp/mangrove bio-region of Mozambique, and therefore, we suggest that future research efforts concentrate on or at least consider this region.publishedVersio

Multivalent glycoconjugates as vaccines and potential drug candidates

Pathogens adhere to the host cells during the first steps of infection through multivalent interactions which involve protein–glycan recognition. Multivalent interactions are also involved at different stages of immune response. Insights into these multivalent interactions generate a way to use suitable carbohydrate ligands that are attached to a basic scaffold consisting of e.g., dendrimer, polymer, nanoparticle, etc., with a suitable linker. Thus a multivalent architecture can be obtained with controllable spatial and topology parameters which can interfere with pathogen adhesion. Multivalent glycoconjugates bearing natural or unnatural carbohydrate antigen epitopes have also been used as carbohydrate based vaccines to stimulate an innate and adaptive immune response. Designing and synthesizing an efficient multivalent architecture with optimal ligand density and a suitable linker is a challenging task. This review presents a concise report on the endeavors to potentially use multi- and polyvalent glycoconjugates as vaccines as well as anti-infectious and anti-inflammatory drug candidates

Multivalenz-assoziierte Eigenschaften von polysulfatierten Nanomaterialien in biomedizinischen Anwendungen

1\. Introduction 1.1 Multivalency and binding inhibition 1.1.1 Definition and quantification of multivalency 1.1.2 Multivalency in competitive binding inhibition 1.2 Polysulfates and their biological interactions 1.2.1 Anti- inflammatory properties of polysulfates 1.2.3 Polyanions and their application as viral entry inhibitors 1.3 Inorganic nanomaterials for biomedical applications 1.3.1 Stability of colloidal dispersions – The DLVO theory 1.3.2 Biocompatibility of colloids 1.3.3 Synthesis of inorganic nanoparticles 1.3.4 Stabilizing ligands and functionalization of inorganic nanoparticles 1.3.5 Material-derived properties and applications of inorganic nanomaterials 1.3.6 Dimension-derived properties and applications of inorganic nanomaterials 2\. Motivation and Objective 3\. Publications and Manuscripts 3.1 Polyglycerolsulfate functionalized gold nanorods as optoacoustic signal nanoamplifiers for in vivo bioimaging of rheumatoid arthritis 3.2 Virus inhibition induced by polyvalent nanoparticles of different sizes 3.3 Size Dependence of Steric Shielding and Multivalency Effects for Globular Binding Inhibitors 4\. Summary and Conclusion 5\. Outlook 6\. Abstract and Kurzzusammenfassung 6.1 Abstract 6.2 Kurzzusammenfassung 7\. ReferencesWithin the scope of this thesis, gold nanomaterials of different sizes and shapes were synthesized and functionalized with polyglycerolsulfates. Their targeting properties for inflammatory diseases as well as their application as virus entry inhibitors were evaluated, with special focus on the multivalent enhancement of their binding affinity to biological surfaces. The first part of my thesis was concerned with the synthesis and evaluation of polyglycerolsulfate functionalized gold nanorods (AuNR-dPGS) as contrast agent for the imaging of rheumatoid arthritis via multispectral optoacoustic tomography (MSOT). A novel procedure for the anionic functionalization of gold nanorods was established by a thermally induced ligand exchange of PEG1000-thiol with thioctic acid-dPGS 10 kDa. As a result, the prepared nanorods were significantly less cytotoxic than other polyanionic nanorods in the literature. The active targeting properties of AuNR-dPGS for inflammatory diseases could be affirmed in vitro and in vivo, which successfully showed that the large contact area of colloids with the biological surface results in strong binding affinities. The second part of my thesis was focused on a detailed investigation on the mechanism of globular binding inhibition and the possible application of polysulfated gold nanoparticles (AuNP-PGS) as viral entry inhibitors. The investigations on the size dependent inhibition of vesicular stomatitis virus (VSV) with AuNP-PGS indicated that the inhibition potential of globular inhibtors is strongly size dependent and that the infection inhibition is based on the inhibition of the virus’ binding to the cells. The follow-up study elucidated the mechanism of competitive binding inhibition in detail. The effect of steric shielding and the multivalent enhancement of binding affinities due to larger contacts areas of inhibitors and binders on the competitive binding inhibition could be quantitatively described theoretical models. It is now possible to predict the optimal inhibitor sizes for mass efficient virus inhibition and rationally design competitive binding inhibitors.In der vorliegenden Arbeit wurden Goldkolloide unterschiedlicher Form und Größe synthetisiert und mit Polyglycerinsulfaten beschichtet. Der mögliche Einsatz der Nanomaterialien zur gezielten Diagnostik von Entzündungskrankheiten sowie als Virusinhibitoren wurden untersucht, wobei ein besonderes Augenmerk auf die Untersuchung der Multivalenz bedingten Verstärkung der Bindungsaffinitäten gelegt wurde. Der erste Teil meiner Arbeit beschäftigte sich mit der Synthese und Evaluation von Polyglycerinsulfat- funktionalisierten Goldnanostäbchen (AuNR-dPGS) als Kontrastmittel für die Abbildung von rheumatoider Arthritis mittels multispektraler optoakustischer Tomographie (MSOT). Auf Basis einer thermisch-induzierten Ligandenaustauschreaktion von PEG1000-Thiol mit Thioctsäure-dPGS 10 kDa wurde eine neuartige Methode zur anionischen Beschichtung von Goldnanostäbchen eingeführt, welche die Zytotoxizität der Stäbchen im Vergleich zu anderen Methoden in der Literatur signifikant verringerte. Die aktive Zielführung von AuNR-dPGS zu Entzündungskrankheiten konnte in in vitro sowie in vivo Experimenten bestätigt werden, wodurch erfolgreich gezeigte wurde, dass die großen Kontaktfläche von Kolloiden mit biologischen Oberflächen die Bindungsaffinitäten stark verstärken. Der zweite Teil meiner Arbeit war auf die mechanistische Aufklärung der kompetitiven Bindungsinhibition durch globuläre Inhibitoren und der möglichen Anwendung von polysulfatierten Goldnanopartikeln (AuNP-PGS) für die Inhibition von Virus-Zell Bindungen fokussiert. Die Untersuchungen zur größenabhängige Inhibition des Vesicular Stomatitis Virus (VSV) mittels AuNP-dPGS haben gezeigt, dass das Inhibitionspotential von globulären Inhibitoren stark größenabhängig ist und dass die Inhibition der Infektion hauptsächlich auf der Inhibition der Virus- Zell Bindung beruht. In dem darauffolgendem Projekt wurde der Mechanismus der kompetitiven Bindungsinhibition aufgeklärt. Die Auswirkungen der sterischen Abschirmung von Viren durch Inhibitoren sowie von Multivalenzeffekten auf die Bindungsinhibition konnten quantifiziert und mit theoretischen Modellen aufgeklärt werden. Optimale Durchmesser von globulären Inhibitoren können nun vorhergesagt werden, womit die Möglichkeit zur rationellen Entwicklung von Virusinhibitoren gegeben ist

Rekonstruktion der Phylogenie der Opisthobranchia (Mollusca, Gastropoda) mit Hilfe molekularer Methoden

Der Schwerpunkt der vorliegenden Arbeit liegt in der Rekonstruktion der Phylogenie der Opisthobranchia (Mollusca, Gastropoda) mit Hilfe von 18S, 28S und 16S rDNA-Sequenzen. Der Datensatz umfasst die Sequenzen von 78 Vertretern der Opisthobranchia, Pulmonata und Pyramidellidae, sowie von 13 "Prosobranchiern" (Außengruppe). Die mit Hilfe von Maximum Parsimonie-, Distanz-, $\textit {maximum likelihood-}$ und Bayes'schen Verfahren rekonstruierten Stammbäume zeigen mit Ausnahme der Nudipleura (Nudibranchia + Pleurobranchoidea) eine deutliche Variabilität in den postulierten Verwandtschaftsbeziehungen der opisthobranchiaten Gruppierungen. Die einzelnen Taxa können dagegen im Wesentlichen als Monophyla gestützt werden. Abschließend betrachtet müssen sowohl die Stellung der Opisthobranchia innerhalb der Heterobranchia, als auch die phylogenetischen Beziehungen der meisten ihrer Subtaxa als ungeklärt angesehen werden

The influence of surface charge on serum protein interaction and cellular uptake: studies with dendritic polyglycerols and dendritic polyglycerol-coated gold nanoparticles

Tony Bewersdorff,1 Jonathan Vonnemann,2 Asiye Kanik,1 Rainer Haag,2 Andrea Haase1 1Department of Chemical and Product Safety, German Federal Institute for Risk Assessment (BfR), Berlin, Germany; 2Institute of Chemistry and Biochemistry, Freie Universität Berlin, Berlin, Germany Abstract: Nanoparticles (NPs) have gained huge interest in the medical field, in particular for drug delivery purposes. However, binding of proteins often leads to fast NP uptake and rapid clearance, thereby hampering medical applications. Thus, it is essential to determine and control the bio–nano interface. This study investigated the serum protein interactions of dendritic polyglycerols (dPGs), which are promising drug delivery candidates by means of two dimensional gel electrophoresis (2DE) in combination with mass spectrometry. In order to investigate the influence of surface charge, sulfated (sulfated dendritic polyglycerol [dPGS]) and non-sulfated (dPGOH) surfaces were applied, which were synthesized on a gold core allowing for easier separation from unbound biomolecules through centrifugation. Furthermore, two different sizes for dPGS were included. Although size had only a minor influence, considerable differences were detected in protein affinity for dPGS versus dPGOH surfaces, with dPGOH binding much less proteins. Cellular uptake into human CD14+ monocytes was analyzed by flow cytometry, and dPGOH was taken up to a much lower extent compared to dPGS. By using a pull-down approach, possible cellular interaction partners of serum pre-incubated dPGS-Au20 NPs from the membrane fraction of THP-1 cells could be identified such as for instance the transferrin receptor or an integrin. Clathrin-mediated endocytosis was further investigated using chlorpromazine as an inhibitor, which resulted in a 50% decrease of the cellular uptake of dPGS. This study could confirm the influence of surface charge on protein interactions and cellular uptake of dPGS. Furthermore, the approach allowed for the identification of possible uptake receptors and insights into the uptake mechanism. Keywords: sulfated dendritic polyglycerols, protein corona, cellular uptake, uptake receptors, clathrin-mediated endocytosi