Kristina Riehemann

Nanomedicine – challenge and perspectives

The application of nanotechnology concepts to medicine joins two large cross-disciplinary fields with an unprecedented societal and economical potential arising from the natural combination of specific achievements in the respective fields. The common basis evolves from the molecular-scale properties relevant to the two fields. Local probes and molecular imaging techniques allow surface and interface properties to be characterized on a nanometer scale at predefined locations, while chemical approaches offer the opportunity to elaborate and address surfaces, for example, for targeted drug delivery, enhanced biocompatibility, and neuroprosthetic purposes. However, concerns arise in this cross-disciplinary area about toxicological aspects and ethical implications. This Review gives an overview of selected recent developments and applications of nanomedicine.

Regulation of NF-κB Activation by MAP Kinase Cascades

Transcription factor NF-kappa B plays a crucial role in the regulation of numerous genes involved in the inflammatory response and control of cell death. Activation of NF-kappa B is mediated through the phosphorylation of its inhibitory subunit I kappa B, followed by the subsequent degradation of I kappa B at the proteasome. A second level of control involves phosphorylation events of NF-kappa B in the cell nucleus. The kinases that regulate these processes are rather undefined. NF-kappa B activation is induced by a great variety of predominantly pathogenic and noxious stimuli. A similar spectrum of conditions triggers the activation of two mitogen-activated protein (MAP) kinase cascades, designated as the JNK and p38 kinase pathways. Several points of evidence suggest that MAP kinases can participate in the regulation of NF-kappa B transcriptional activity. Here, we will review very recent data demonstrating that both the JNK and the p38 pathways are involved in the activation of NF-kappa B in the cytoplasm as well as in modulation of its transactivating potential in the nucleus.

DIFFERENTIAL EXPRESSION OF ANNEXINS I, II AND IV IN HUMAN TISSUES : AN IMMUNOHISTOCHEMICAL STUDY

Abstract Annexins constitute a family of Ca2+- and phospholipid-binding proteins. Although their functions are still not clearly defined, several members of the annexin family have been implicated in membrane-related events along exocytotic and endocytotic pathways. To elucidate a possible correlation of those functional proposals with the tissue distribution of annexins, we analysed immunohistochemically the expression of annexins I, II and IV in a broad variety of human tissues. Annexins I and II were chosen for this study since their functionally relevant N-terminal domains are structurally closely related, whilst annexin IV is structurally less related to the former two proteins. The study revealed distinct expression patterns of annexins I, II and IV throughout the body. Annexin I was found in leucocytes of peripheral blood, tissue macrophages and T-lymphocytes and in certain epithelial cells (respiratory and urinary system, superficial cells of non-keratinised squamous epithelium), annexin II in endothelial cells, myoepithelial cells and certain epithelial cells (mainly respiratory and urinary system), whereas annexin IV was almost exclusively found in epithelial cells. Epithelia of the upper respiratory system, Bowman’s capsule, urothelial cells, mesothelial cells, peripheral nerves, the choroid plexus, ependymal cells and pia mater and arachnoid of meninges generally strongly expressed all three annexins investigated. The characteristic expression in different tissues and the intracellular distribution indicates that the three annexins investigated are involved in aspects of differentiation and/or physiological functions specific to these tissues.

Internalization Pathways of Anisotropic Disc‐Shaped Zeolite L Nanocrystals with Different Surface Properties in HeLa Cancer Cells

Information about the mechanisms underlying the interactions of nanoparticles with living cells is crucial for their medical application and also provides indications of the putative toxicity of such materials. Here the uptake and intracellular delivery of disc-shaped zeolite L nanocrystals as porous aminosilicates with well-defined crystal structure, uncoated as well as with COOH-, NH2 -, polyethyleneglycol (PEG)- and polyallylamine hydrochloride (PAH) surface coatings are reported. HeLa cells are used as a model system to demonstrate the relation between these particles and cancer cells. Interactions are studied in terms of their fates under diverse in vitro cell culture conditions. Differently charged coatings demonstrated dissimilar behavior in terms of agglomeration in media, serum protein adsorption, nanoparticle cytotoxicity and cell internalization. It is also found that functionalized disc-shaped zeolite L particles enter the cancer cells via different, partly not yet characterized, pathways. These in vitro results provide additional insight about low-aspect ratio anisotropic nanoparticle interactions with cancer cells and demonstrate the possibility to manipulate the interactions of nanoparticles and cells by surface coating for the use of nanoparticles in medical applications.

Feuchtes Autoklavieren Der einfachere Weg zur Antigendemaskierung

ZusammenfassungMit der Technik des feuchten Autoklavierens wird eine einfache, verläßliche und zeitsparende Methode zur Antigen-Demaskierung an Formalin-fixiertem und Paraffin-eingebettetem Gewebe vorgestellt. Anhand einer Reihe von Antikörpern (Östrogen- und Progesteronrezeptoren, Zytoskelettproteine, verschiedene p53-Antikörper, mdm-2, bcl-2, MIB-1 u. a.) verwendeter Antikörper werden die Vorteile dieser Methode beschrieben. Das feuchte Autoklavieren ermöglicht bei einigen sonst nur am Gefrierschnitt einsetzbaren Antikörpern auch deren Anwendung am Paraffinschnitt. Für den Routinepathologen ist die leichte Handhabung sowie die hohe Reproduzierbarkeit von Vorteil.SummaryWet autoclaving is a simple, reliable and time-effective method for antigen retrieval in routinely processed archival material. Both routine diagnostic (e. g., oestrogen and progesterone receptors, cytoskeletal proteins) and research antibodies (e. g. various p53 antibodies, mdm-2, bcl-2, MIB-1) are reported to demonstrate its application. Wet autoclaving may allow successful application of antibodies in paraffin-embedded tissues designed for use on frozen sections. The technique has the poten-tial to reliably handle up to 200 sections at a time, without evidence of any significant damage to the sections or nuclear morphology.

Reaction of human macrophages on protein corona covered TiO2 nanoparticles

UNLABELLED The cytokine secretion of primary cells of human macrophages during the interaction of TiO2 nanoparticles (with an average primary size of 100-120 nm) is investigated down to concentration levels suggested to be relevant for in vivo conditions. We find that high TiO2 concentrations induce the cytokines Interleukin IL-1β, IL-6, and IL-10 secretion, while at low concentrations only IL-6 secretion is observed. To obtain further evidence on in vivo conditions we investigated the development and structure of the protein corona of the nanoparticles. We demonstrated that the surface of TiO2 particles attract preferably secondary modified proteins which then induce cytokine secretion of macrophages. Our results indicate that concentration of corona covered TiO2 particles below 1 μg/ml induce IL-6 secretion which is reported to be responsible for the development of autoimmune diseases as well as for the secretion of acute phase proteins. FROM THE CLINICAL EDITOR This study investigates the effects of protein corona covered titanium dioxide nanoparticles on human macrophages, concluding that concentration of such particles below 1 μg/ml induces IL-6 secretion, which may be responsible for the development of autoimmune diseases as well as for the secretion of acute phase proteins. This finding has important implications on future applications of such nanoparticles.

Standardisierte AgNOR-Färbemethode für formalinfixiertes und paraffineingebettetes Material

ZusammenfassungIn dieser Studie wird eine Methode zum Nachweis von sog. argyrophilen Nukleolus Organisator Regionen assoziierten Proteinen (AgNORs) vorgestellt, die es erstmalig erlaubt, diese als Teilstrukturen des Nukleolus an formalinfixiertem und paraffineingebetteten Gewebe zu erkennen. Mit einer Vorbehandlung durch feuchte Autoklavierung werden am archivierten Material Färbeergebnisse erzielt, wie sie an alkoholfixiertem Frischmaterial erreicht wurden. Sämtliche Färbeartefakte entfallen mit dieser neuen Methode. Weitere Vorteile bieten die einfache Handhabung sowie die Tatsache, daß auch bei unterschiedlicher Fixations- und Einbettungsdauer standardisierte und somit vergleichbare Ergebnisse erzielt werden. Das vorgestellte Verfahren bietet somit die Möglichkeit, die Wertigkeit der AgNOR-Quantifizierung auch an archiviertem Paraffinmaterial in retrospektiven Studien reproduzierbar zu evaluieren.SummaryVisualization of proteins associated with nucleolar organizer regions proteins (AgNORs) on formalin-fixed and paraffin-embedded archival tissues is substantially improved after application of wet autoclave pretreatment. Silver staining results are comparable to those obtained on tissues processed in alcoholbased fixatives, illustrating AgNORs as substructures of the nucleoli without any staining artefacts. A highly reproducible staining quality was achieved irrespective of tissue origin or duration of formalin fixation. As a result of this novel and simple method, the grounds have been prepared for standardized AgNOR quantification on archival material.

Controlled Cell Growth and Cell Migration in Periodic Mesoporous Organosilica/Alginate Nanocomposite Hydrogels.

Nanocomposite (NC) hydrogels with different periodic mesoporous organosilica (PMO) concentrations and a NC hydrogel bilayer with various PMO concentrations inside the layers of the hydrogel matrix are prepared. The effect of the PMO concentration on cell growth and migration of cells is reported. The cells migrate in the bilayer NC hydrogel towards higher PMO concentrations and from cell culture plates to NC hydrogel scaffolds.

Filling knowledge gaps that distinguish the safety profiles of nano versus bulk materials.

The nanotechnology revolution will lead to large scale production of engineered nanomaterials that will impact every aspect of our lives. The unique physicochemical properties and functions of engineered nanomaterials could also pose new biological hazards to humans and the environment, including exposure through inhalation, ingestion, skin uptake, or the therapeutic use of these materials in the workplace and consumer products. Thus, an understanding of the science that underpins the biological hazard of nanomaterials is important for the sustainable development of this technology. Research into the hazards of engineered nanomaterials has spawned the development of a new multidisciplinary science—nanotoxicology—which aims to understand the toxicological

Comparison of cellular effects of starch-coated SPIONs and poly(lactic-co-glycolic acid) matrix nanoparticles on human monocytes

Within the last years, progress has been made in the knowledge of the properties of medically used nanoparticles and their toxic effects, but still, little is known about their influence on cellular processes of immune cells. The aim of our comparative study was to present the influence of two different nanoparticle types on subcellular processes of primary monocytes and the leukemic monocyte cell line MM6. We used core-shell starch-coated superparamagnetic iron oxide nanoparticles (SPIONs) and matrix poly(lactic-co-glycolic acid) (PLGA) nanoparticles for our experiments. In addition to typical biocompatibility testing like the detection of necrosis or secretion of interleukins (ILs), we investigated the impact of these nanoparticles on the actin cytoskeleton and the two voltage-gated potassium channels Kv1.3 and Kv7.1. Induction of necrosis was not seen for PLGA nanoparticles and SPIONs in primary monocytes and MM6 cells. Likewise, no alteration in secretion of IL-1β and IL-10 was detected under the same experimental conditions. In contrast, IL-6 secretion was exclusively downregulated in primary monocytes after contact with both nanoparticles. Two-electrode voltage clamp experiments revealed that both nanoparticles reduce currents of the aforementioned potassium channels. The two nanoparticles differed significantly in their impact on the actin cytoskeleton, demonstrated via atomic force microscopy elasticity measurement and phalloidin staining. While SPIONs led to the disruption of the respective cytoskeleton, PLGA did not show any influence in both experimental setups. The difference in the effects on ion channels and the actin cytoskeleton suggests that nanoparticles affect these subcellular components via different pathways. Our data indicate that the alteration of the cytoskeleton and the effect on ion channels are new parameters that describe the influence of nanoparticles on cells. The results are highly relevant for medical application and further evaluation of nanomaterial biosafety.