On the way to precision formulation additives: 2D-screening to select solubilizers with tailored host and release capabilities.

A 2-dimensional high-throughput screening method is presented to select peptide sequences from large peptide libraries for precision formulation additives, having a high capacity to specifically host a drug of interest and provide tailored drug release properties. The identified sequences are conjugated with poly(ethylene glycol) (PEG) to obtain peptide-PEG conjugates that proved to be valuable as solubilizers for small organic molecule drugs to overcome limitations of poor water-solubility and low bio-availability. The 2D-screening method selects peptide sequences on both (i) high loading capacities and (ii) preferred drug-release capabilities as demonstrated on an experimental Tau-protein aggregation inhibitor/Tau- deaggregator with potentials for an anti-Alzheimer disease drug (BB17). To enable 2D-screening, a one-bead one-compound (OBOC) peptide library was immobilized on a glass slide, allocating individual beads to permanent positions. While the first screening step involved incubation of the supported OBOC library with BB17 to identify beads with high drug binding capacities by fluorescence scanner readouts, the second step reveals release properties of the high capacity binders by incubation with blood plasma protein model solutions. Efficiently peptides with high BB17 capacities and either keeper or medium or fast releaser properties can be identified by direct sequence readouts from the glass slide supported resin beads via matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry. Four peptides are synthesized as peptide-PEG solubilizers representing strong, medium, weak releasers and non-binders. Loading capacities reached up to 1:3.4 (mol drug per mol carrier) and release kinetics (fast/medium/slow) are in agreement with the selection process as investigated by fluorescence anisotropy and fluorescence correlation spectroscopy. The ability of BB17/conjugate complexes to inhibit the aggregation of Tau4RDΔK (four repeat Tau ((M)Q244-E372 with deletion of K280), 129 residues) in N2a cells is studied by a Tau-pelleting assay showing the modulation of cellular Tau aggregation. Promising effects such as the reduction of 55% of total Tau load are observed for the strong releaser additive. Studies of in vitro Thioflavin S Tau-aggregation assays show half-maximal inhibitory activities (IC50 values) of BB17/conjugates in the low micro-molar range.

Precision compatibilizers for composites: in-between self-aggregation, surfaces recognition and interface stabilization.

Peptide-polymer conjugates are applied as interface stabilizers that are precisely tuned to recognize the surfaces of inorganic constituents in composites. A set of peptide sequences is usually selected through phage-display and a strategy is presented for the identification of the most effective sequences through the evaluation of secondary interactions, including not only surface binding but also solubility and self-aggregation tendency.

Easy access to triazolinedione-endcapped peptides for chemical ligation.

An efficient and easy route towards triazolinedione (TAD) endcapped peptides is described, in which a TAD-precursor was coupled to N-terminal amines on a solid support. Modified peptides readily reacted with diene end-functionalized poly(ε-caprolactone) of different molecular weights. The ligation proved to be orthogonal to a variety of functional groups present in natural amino acids.

Individual bottle brush molecules in dense 2D layers restoring high degree of extension after collapse-decollapse cycle: directly measured scaling exponent.

We prepared dense films of adsorbed brush-like macromolecules on mica substrate by transfer of compressed Langmuir monolayers from water subphase. The main macromolecular contours in the dense films were clearly resolved by SFM. The films were subjected to successive treatments by ethanol and water vapours. In accordance with previous results for isolated macromolecules, the films underwent collapse and subsequent decollapse morphological transformations in the changing vapour environment. Statistical analysis of the macromolecular dimensions in the films allowed us to measure the values of the scaling exponent nu determining the correlation between mean lateral and linear dimensions of the macromolecules. The analysis showed that the macromolecular conformations in the film as transferred were similar to the previously described conformations of the same macromolecules deposited directly on mica as isolated chains at much lower surface densities. The determined nu was close to the 0.75 value corresponding to the 2D SAW statistics. We assumed that the molecules retained the high degree of extension during the compression step due to suppressed reorganisation of the side chains. Differently from previous observations for isolated macromolecules, the restored conformations in the dense films after collapse-decollapse cycle were more extended with the nu of about 0.73 value. A theoretical explanation of the high degree of re-extension is proposed.

Investigation of the Neutron Quantum States in the Earth's Gravitational Field.

We studied the neutron quantum states in the potential well formed by the Earth's gravitational field and a horizontal mirror. The estimated characteristic sizes of the neutron wave functions in two lowest quantum states correspond to their expectations with an accuracy of ≈25 %. The spatial density distribution in a standing neutron wave above a mirror was measured for a set of a few lowest quantum states. A position-sensitive neutron detector with an extra high spatial resolution of 1 µm to 2 µm was developed and tested for this particular task. Although this experiment was not designed or optimized to search for an additional short-range force, nevertheless it allowed us to slightly improve the published boundary in the nanometer range of characteristic distances. We studied systematical uncertainties in the chosen "flow-through" method as well as the feasibility to improve further the accuracy in this experiment.

Conformational dynamics of single molecules visualized in real time by scanning force microscopy: macromolecular mobility on a substrate surface in different vapours.

We describe a technique to visualize and effect in real time motion and conformational transitions of single macromolecules. Two steps are involved. First, scanning force microscopy (SFM) was applied to detect in situ conformational transitions of single polymer molecules adsorbed on a substrate surface. Secondly, these changes were induced by controlled variations of environmental conditions in a microscope environmental chamber. In particular, we have revealed that exposure of a substrate with adsorbed macromolecules to vapours of different nature was able to increase molecular mobility and to stimulate conformational transitions of the polymer chains on the surface. Realization of SFM observation in a variable vapour environment was not as difficult as in liquid media. Variations of the vapour composition affected the oscillation dynamics of the cantilever with the scanning probe only to a small extent, and did not impede continuation of the scanning procedure. In fact, the characteristic times of the observed conformational changes were large enough (minutes to dozens of minutes) for sampling images repeatedly. Although recording of an SFM image was slow and required several minutes, we were able to visualize step-by-step the successive stages of the slow conformational transformation of the macromolecules adhering to the substrate, i.e. to investigate a molecular response to the environment changes in real time. Here, we studied the reversible collapse-decollapse transitions of cylindrical poly(methacrylate)-graft-poly(n-butyl acrylate) brush-like macromolecules exposed to different vapours. Single macromolecules on mica tended to assume a compacted globular conformation when exposed to the vapour of compounds, which due to their amphiphilic nature adsorb on mica and lower the surface energy of the substrate (e.g. alcohols). By contrast, the macromolecules adopted extended two-dimensional worm-like conformations in the vapours of compounds having high values of surface tension (such as water). In our opinion, the reason for the observed tendency was a competition in spreading on the substrate surface between the macromolecules and the co-adsorbed vapour molecules. If the brush-like macromolecules succeeded in the spreading, they acquired an extended conformation. Otherwise they collapsed to globuli in order to reduce the surface area per macromolecule. Thus, the enhanced mobility of synthetic macromolecules on a substrate observed in a vapour environment in combination with the possibility to manipulate the macromolecular conformation via changes in a vapour phase and the ability to visualize the transitions of the macromolecules individually, provides challenging prospects for SFM studies on the dynamics of single molecules under applied external stimuli.

The GRID Technique: Current Status and New Trends.

In the GRID technique one measures Doppler-broadened line profiles of γ transitions using the high resolution crystal spectrometers GAMS, which are installed at the high flux reactor of the ILL Grenoble. One of the essential applications of this technique is the measurement of nuclear state lifetimes. In the present contribution the precision and the principal limits of the technique are discussed.

Study of Interatomic Potentials in ZnS-Crystal-GRID Experiments Versus Ab Initio Calculations.

Crystal-GRID measurements have been performed with ZnS single crystals. For the first time, an asymmetric Crystal-GRID line shape could be observed. The preliminary data evaluation indicates that the reported lifetime of the 3221 keV level in (33)S is too short. A value of about 60 fs has been found. Due to this "long" lifetime the line shape is much less structured than calculated with the reported lifetime.

Study of Interatomic Potentials Using the Crystal-GRID Method on Oriented Single Crystals of Ni, Fe, and Cr.

The Crystal-GRID method is used to study interatomic collisions at low energy in metals and such to probe the repulsive interatomic potential. Line shapes of gamma rays, emitted by the recoiling (59)Ni isotope after thermal neutron capture in Ni single crystals, were measured and compared to results obtained by molecular dynamics simulations of the slowing down. The same procedure is also used for recoiling (57)Fe and (54)Cr atoms in Fe and Cr single crystals, respectively. Different potentials (including several from the embedded atom method) are investigated using the observed fine structure of the line shape which depends on the crystal orientations. From the detailed study of the lineshapes measured in two different orientations, a new potential is then derived for each element. Nuclear state lifetimes for the excited isotopes are also deduced with a higher precision than obtained with standard nuclear techniques.

Lifetime Measurements in (178)Hf.

Lifetimes of levels from K(π) = 2(+), K(π) = 4(+) and several K(π) = 0(+) bands have been measured in the (178)Hf nucleus using the GRID technique. Lifetimes of the 2(+) and 3(+) levels were measured within the K(π) = 2(+) γ band. A lower limit was established for the lifetime of the 4(+) level of the K(π) = 4(+) band. The resulting upper limits for the absolute B(E2) values exclude collective transitions from the K(π) = 4(+) to the ground state band but not to the K(π)= 2(+) band. Level lifetimes were also measured for several states within three separate K(π)= 0(+) bands. Evidence is presented for a previously unobserved case of two excited K(π)= 0(+) bands being connected via collective E2 transitions.

GAMS5.

The construction of the double-crystal γ-spectrometer GAMS5 was finished recently and the instrument is now operational. Measurements with double flat crystals were carried out and we will report here on the progress concerning the characteristics of the spectrometer.

Application of GRID to Foreign Atom Localization in Single Crystals.

The application of GRID (Gamma Ray Induced Doppler broadening) spectroscopy to the localization of foreign atoms in single crystals is demonstrated on erbium in YAP. By the investigation of the Doppler broadened secondary γ line for two crystalline directions, the Er was determined to be localized on the Y site. Conditions for the nuclear parameters of the impurity atoms used for the application of GRID spectroscopy are discussed.

The relative biological effectiveness in V79 Chinese hamster cells of the neutron capture reactions in boron and nitrogen.

V79 Chinese hamster cells were irradiated in the presence of different amounts of boric acid with thermal neutrons at the Medical Research Reactor at Brookhaven National Laboratory. From the linear dose-survival curves observed, a D0 value of 66 rad for the 10B(n, alpha) 7Li neutron capture reaction was obtained. No dependence of this value on the concentration of boric acid was found. Comparing this value to the D0 value of 150 rad obtained with 250 kVp X rays between 10 and 0.01% survival, an extrapolated RBE value of 2.3 was calculated. By irradiation of the same line of cells with cold neutrons at the Institut Laue - Langevin , a D0 value for the 14N(n,p)14C reaction of 77 rad was obtained, with a corresponding RBE value of 1.9. Comparison is made with previously published RBE values for the 10B(n, alpha) 7Li reaction.

Boron-loaded macromolecules in experimental physiology: tracing by neutron capture radiography.

The search for suitable methods of attaching the boron isotope 10B to tumour cells in the human body for the treatment of malignant disease has prompted a study of ways of optimising the localisation of 10B in mammalian tissues. Compounds rich in boron, linked to various carriers, have been studied in experimental animals and in systems of cultivated cells. The current experimental prerequisites and results are outlined. The method for 'neutron capture radiography' has significant potential for general application in experimental physiology. The techniques are presented from this viewpoint and compared with other methods for visualisation of macromolecular markers by the use of physical or chemical principles.