Stephan Block

Complex formation with nucleic acids and aptamers alters the antigenic properties of platelet factor 4

The tight electrostatic binding of the chemokine platelet factor 4 (PF4) to polyanions induces heparin-induced thrombocytopenia, a prothrombotic adverse drug reaction caused by immunoglobulin G directed against PF4/polyanion complexes. This study demonstrates that nucleic acids, including aptamers, also bind to PF4 and enhance PF4 binding to platelets. Systematic assessment of RNA and DNA constructs, as well as 4 aptamers of different lengths and secondary structures, revealed that increasing length and double-stranded segments of nucleic acids augment complex formation with PF4, while single nucleotides or single-stranded polyA or polyC constructs do not. Aptamers were shown by circular dichroism spectroscopy to induce structural changes in PF4 that resemble those induced by heparin. Moreover, heparin-induced anti-human-PF4/heparin antibodies cross-reacted with human PF4/nucleic acid and PF4/aptamer complexes, as shown by an enzyme immunoassay and a functional platelet activation assay. Finally, administration of PF4/44mer-DNA protein C aptamer complexes in mice induced anti-PF4/aptamer antibodies, which cross-reacted with murine PF4/heparin complexes. These data indicate that the formation of anti-PF4/heparin antibodies in postoperative patients may be augmented by PF4/nucleic acid complexes. Moreover, administration of therapeutic aptamers has the potential to induce anti-PF4/polyanion antibodies and a prothrombotic diathesis.

Determination of Exosome Concentration in Solution Using Surface Plasmon Resonance Spectroscopy

Exosomes are cell-secreted nanometer-sized extracellular vesicles that have been reported to play an important role in intercellular communication. They are also considered potential diagnostic markers for various health disorders, and intense investigations are presently directed toward their use as carriers in drug-delivery and gene-therapy applications. This has generated a growing need for sensitive methods capable of accurately and specifically determining the concentration of exosomes in complex biological fluids. Here, we explore the use of label-free surface-based sensing with surface plasmon resonance (SPR) read-out to determine the concentration of exosomes in solution. Human mast cell secreted exosomes carrying the tetraspanin membrane protein CD63 were analyzed by measuring their diffusion-limited binding rate to an SPR sensor surface functionalized with anti-CD63 antibodies. The concentration of suspended exosomes was determined by first converting the SPR response into the surface-bound mass. The increase in mass uptake over time was then related to the exosome concentration in solution using a formalism describing diffusion-limited binding under controlled flow conditions. The proposed quantification method is based on a calibration and control measurements performed with proteins and synthetic lipid vesicles and takes into account (i) the influence of the broad size distribution of the exosomes on the surface coverage, (ii) the fact that their size is comparable to the ∼150 nm probing depth of SPR, and (iii) possible deformation of exosomes upon adsorption. Under those considerations, the accuracy of the concentration determination was estimated to be better than ±50% and significantly improve if the exosome deformation is negligible.

Effect of 3D-scaffold formation on differentiation and survival in human neural progenitor cells

Background3D-scaffolds have been shown to direct cell growth and differentiation in many different cell types, with the formation and functionalisation of the 3D-microenvironment being important in determining the fate of the embedded cells. Here we used a hydrogel-based scaffold to investigate the influences of matrix concentration and functionalisation with laminin on the formation of the scaffolds, and the effect of these scaffolds on human neural progenitor cells cultured within them.MethodsIn this study we used different concentrations of the hydrogel-based matrix PuraMatrix. In some experiments we functionalised the matrix with laminin I. The impact of concentration and treatment with laminin on the formation of the scaffold was examined with atomic force microscopy. Cells from a human fetal neural progenitor cell line were cultured in the different matrices, as well as in a 2D culture system, and were subsequently analysed with antibody stainings against neuronal markers. In parallel, the survival rate of the cells was determined by a live/dead assay.ResultsAtomic force microscopy measurements demonstrated that the matrices are formed by networks of isolated PuraMatrix fibres and aggregates of fibres. An increase of the hydrogel concentration led to a decrease in the mesh size of the scaffolds and functionalisation with laminin promoted aggregation of the fibres (bundle formation), which further reduces the density of isolated fibres. We showed that laminin-functionalisation is essential for human neural progenitor cells to build up 3D-growth patterns, and that proliferation of the cells is also affected by the concentration of matrix. In addition we found that 3D-cultures enhanced neuronal differentiation and the survival rate of the cells compared to 2D-cultures.ConclusionsTaken together, we have demonstrated a direct influence of the 3D-scaffold formation on the survival and neuronal differentiation of human neural progenitor cells. These findings emphasize the importance of optimizing 3D-scaffolds protocols prior to in vivo engraftment of stem and progenitor cells in the context of regenerative medicine.

Conformation of Poly(styrene sulfonate) Layers Physisorbed from High Salt Solution Studied by Force Measurements on Two Different Length Scales

The conformation of poly(styrene sulfonate) (PSS) layers physisorbed from 1 M NaCl is determined by force measurements and imaging on two length scales. With colloidal probe technique steric forces as predicted for neutral grafted brushes are observed. On decrease and increase of the NaCl concentration, the grafting density remains constant, yet the brush thickness swells and shrinks reversibly with the salt concentration with an exponent of -0.3. At low salt conditions, the brush length amounts to 30% of the contour length, a behavior known for polyelectrolyte brushes and attributed to the entropy of the counterions trapped in the brush. Between a PSS layer and a pure colloidal silica sphere, the same steric forces are observed, and additionally at large separations (beyond the range of the steric repulsion) an electrostatic force is found. A negatively charged AFM tip penetrates the brush--a repulsive electrostatic force between the tip and surface is found, and single chains can be imaged. Thus, with the nanometer-sized AFM tip, the flatly adsorbed fraction of the PSS chains is seen, whereas the micrometer-sized colloidal probe interacts with the fraction of the chains penetrating into solution.

Physical properties of homogeneous TiO2 films prepared by high power impulse magnetron sputtering as a function of crystallographic phase and nanostructure

Optical, photo-electrochemical, crystallographic and morphological properties of TiO2 thin films prepared by high power impulse magnetron sputtering at low substrate temperatures (<65 °C) without post-deposition thermal annealing are studied. The film composition—anatase, rutile or amorphous TiO2—is adjusted by the pressure (p ~ 0.75–15 Pa) in the deposition chamber. The different crystallographic phases were determined with grazing incidence x-ray diffractometry. The surface morphology and size of TiO2 grains/clusters were imaged with atomic force microscopy. Basic plasma parameters were determined by means of the time-resolved Langmuir probe technique. The power density influx on the substrate was estimated from calorimetric probe measurement. The data from calorimetric probe measurements and time-resolved Langmuir probe served as input parameters for the calculation of influx contributions of particular species. The band-gap energy Eg depends on the film composition and crystallographic phase. Optical parameters (refractive index n + ik, transmittance T, reflectance R and absorbance A) are measured as functions of photon energy in the UV–Vis range by spectroscopic ellipsometry. For the rutile and anatase films agreement with the respective bulk phase is found. Incident photon-current conversion efficiency determined by photo-electrochemical measurements reached the highest values (0.312) for the anatase film.

Binding of anti-platelet factor 4/heparin antibodies depends on the thermodynamics of conformational changes in platelet factor 4.

The chemokine platelet factor 4 (PF4) undergoes conformational changes when complexing with polyanions. This can induce the antibody-mediated adverse drug effect of heparin-induced thrombocytopenia (HIT). Understanding why the endogenous protein PF4 becomes immunogenic when complexing with heparin is important for the development of other negatively charged drugs and may also hint toward more general mechanisms underlying the induction of autoantibodies to other proteins. By circular dichroism spectroscopy, atomic force microscopy, and isothermal titration calorimetry we characterized the interaction of PF4 with unfractionated heparin (UFH), its 16-, 8-, and 6-mer subfractions, low-molecular-weight heparin (LMWH), and the pentasaccharide fondaparinux. To bind anti-PF4/heparin antibodies, PF4/heparin complexes require (1) an increase in PF4 antiparallel β-sheets exceeding ∼30% (achieved by UFH, LMWH, 16-, 8-, 6-mer), (2) formation of multimolecular complexes (UFH, 16-, 8-mer), and (3) energy (needed for a conformational change), which is released by binding of ≥11-mer heparins to PF4, but not by smaller heparins. These findings may help to synthesize safer heparins. Beyond PF4 and HIT, the methods applied in the current study may be relevant to unravel mechanisms making other endogenous proteins more vulnerable to undergo conformational changes with little energy requirement (eg, point mutations and post-translational modifications) and thereby predisposing them to become immunogenic.

The adhesion and spreading of thrombocyte vesicles on electrode surfaces

The interaction of thrombocyte vesicles with the surface of metal electrodes, i.e., mercury, gold and gold electrodes modified with self assembled monolayers (SAM), was studied with the help of chronoamperometry, atomic force microscopy, and quartz crystal microbalance measurements. The experimental results show that the interaction of the thrombocyte vesicles with the surface of the electrodes depends on the hydrophobicity of the latter: whereas on very hydrophobic surfaces (mercury and gold functionalized with SAM) the thrombocyte vesicles disintegrate and form a monolayer of lipids, on the less hydrophobic gold surface a bilayer is formed. The chronoamperometric measurements indicate the possibility of future applications to probe membrane properties of thrombocytes.

Temperature-induced transition from odd-even to even-odd effect in polyelectrolyte multilayers due to interpolyelectrolyte interactions.

Within a liquid cell the linear growth of polyelectrolyte multilayers from poly(styrenesulfonate) (PSS) and poly(allylamine hydrochloride) (PAH) is observed with multiple angle null ellipsometry. The salt content is varied between 1 and 4 mol/L NaCl and the temperature between 20 and 55 °C. In the linear growth regime, the outermost layer is investigated. At low temperature, a top PSS layer is twice as thick as a top PAH layer (odd-even effect), consistent with the respective monomer volumes and the same water content for both kinds of top polyelectrolyte layers as confirmed by refractive index measurements. On heating, the thickness of a polycation/polyanion bilayer increases. For temperatures exceeding a crossover temperature, a top PAH layer is thicker than a top PSS layer (even-odd effect). Simultaneously, the index of refraction of the respective top layers indicates a compact PSS and a swollen PAH layer. It is suggested that, at elevated temperature and high salt conditions, secondary forces gain importance in comparison to electrostatic forces: therefore, a transition from an odd-even to an even-odd effect occurs, as well as the decreased film stability on drying as described before (Cornelsen, M., et al. Macromolecules 2010, 43, 4300). The ellipsometric data indicate that PAH/PSS layer pairs exceeding 8.6 nm thickness in solution are unstable in air.

Dual-Wavelength Surface Plasmon Resonance for Determining the Size and Concentration of Sub-Populations of Extracellular Vesicles

Accurate concentration determination of subpopulations of extracellular vesicles (EVs), such as exosomes, is of importance both in the context of understanding their fundamental biological role and of potentially using them as disease biomarkers. In principle, this can be achieved by measuring the rate of diffusion-limited mass uptake to a sensor surface modified with a receptor designed to only bind the subpopulation of interest. However, a significant error is introduced if the targeted EV subpopulation has a size, and thus hydrodynamic diffusion coefficient, that differs from the mean size and diffusion coefficient of the whole EV population and/or if the EVs become deformed upon binding to the surface. We here demonstrate a new approach to determine the mean size (or effective film thickness) of bound nanoparticles, in general, and EV subpopulation carrying a marker of interest, in particular. The method is based on operating surface plasmon resonance simultaneously at two wavelengths with different sensing depths and using the ratio of the corresponding responses to extract the particle size on the surface. By estimating in this way the degree of deformation of adsorbed EVs, we markedly improved their bulk concentration determination and showed that EVs carrying the exosomal marker CD63 correspond to not more than around 10% of the EV sample.

Direct Visualization and Identification of Biofunctionalized Nanoparticles using a Magnetic Atomic Force Microscope

Because of its outstanding ability to image and manipulate single molecules, atomic force microscopy (AFM) established itself as a fundamental technique in nanobiotechnology. (1) We present a new modality that distinguishes single nanoparticles by the surrounding magnetic field gradient. Diamagnetic gold and superparamagnetic iron oxide nanoparticles become discernible under ambient conditions. Images of proteins, magnetolabeled with nanoparticles, demonstrate the first steps toward a magnetic analogue to fluorescence microscopy, which combines nanoscale lateral resolution of AFM with unambiguous detection of magnetic markers.