Polymorphism of human telomeric quadruplexes with drugs: a multi-technique biophysical study.

The human telomeric G-quadruplex structural motif of DNA has come to be known as a new and stimulating target for anticancer drug discovery. Small molecules that interact with G-quadruplex structures in a selective way have gained impressive interest in recent years as they may serve as potential therapeutic agents. Here, we show how circular dichroism, UV resonance Raman and small angle X-ray scattering spectroscopies can be effectively combined to provide insights into structural and molecular aspects of the interaction between human telomeric quadruplexes and ligands. This study focuses on the ability of berberine and palmatine to bind with human telomeric quadruplexes and provides analysis of the conformational landscape visited by the relevant complexes upon thermal unfolding. With increasing temperature, both free and bound G-quadruplexes undergo melting through a multi-state process, populating different intermediate states. Despite the structural similarity of the two ligands, valuable distinctive features characterising their interaction with the G-quadruplex emerged from our multi-technique approach.

In-situ aerosol nanoparticle characterization by small angle X-ray scattering at ultra-low volume fraction.

State-of-the-art aerosol nanoparticle techniques all have one feature in common: for analysis they remove the nanoparticles from their original environment. Therefore, physical and chemical properties of the particles might be changed or cannot be measured correctly. To overcome these shortcomings, we apply synchrotron based small angle X-ray scattering (SAXS) as an in-situ measurement technique. Contrasting other aerosol studies using SAXS, we focus on particle concentrations which allow direct comparison to common aerosol nanoparticle analyzers. To this end, we analyze aerosol nanoparticles at ambient pressure and concentrations of slightly above ~106 cm-3. A differential mobility particle sizer (DMPS) is operated in parallel. We find that SAXS enables measurement of the primary particles and the aggregates, whereas the DMPS detects only aggregates. We conclude that in-situ nanoparticle characterization with ultra-low volume fractions of ~10-10 is feasible with SAXS. Our technique opens up a doorway to the in-situ analysis of aerosol nanoparticles under atmospheric conditions.

Author Correction: Salt concentration and charging velocity determine ion charge storage mechanism in nanoporous supercapacitors.

The original version of this Article contained an error in the Acknowledgements, which incorrectly omitted from the end the following: 'The research leading to these results has received funding from the European Community's Horizon 2020 Framework Programme under grant agreement nº 730872.' This has been corrected in both the PDF and HTML versions of the Article.

Salt concentration and charging velocity determine ion charge storage mechanism in nanoporous supercapacitors.

A fundamental understanding of ion charge storage in nanoporous electrodes is essential to improve the performance of supercapacitors or devices for capacitive desalination. Here, we employ in situ X-ray transmission measurements on activated carbon supercapacitors to study ion concentration changes during electrochemical operation. Whereas counter-ion adsorption was found to dominate at small electrolyte salt concentrations and slow cycling speed, ion replacement prevails for high molar concentrations and/or fast cycling. Chronoamperometry measurements reveal two distinct time regimes of ion concentration changes. In the first regime the supercapacitor is charged, and counter- and co-ion concentration changes align with ion replacement and partially co-ion expulsion. In the second regime, the electrode charge remains constant, but the total ion concentration increases. We conclude that the initial fast charge neutralization in nanoporous supercapacitor electrodes leads to a non-equilibrium ion configuration. The subsequent, charge-neutral equilibration slowly increases the total ion concentration towards counter-ion adsorption.

Formation of highly ordered liquid crystalline coatings - an in situ GISAXS study.

Functional coatings based on self-assembled lyotropic liquid crystals have the potential for applications such as biosensing, drug delivery and nanotemplating. Here we demonstrate the design and in-depth characterization of glycerol monooleate based liquid crystalline coatings on silicon wafers using drop casting and spin coating techniques. In situ time-resolved grazing incidence small angle X-ray scattering (GISAXS) measurements were used to monitor the coating formation and its response to increasing relative humidity conditions between 5 and 100%. Additional atomic force microscopy (AFM) measurements were applied to visualize the coating nanostructure. Structural transformations through ordered intermediate phases to the sponge- and lamellar phase were observed during ethanol evaporation. Relative humidity dependent GISAXS results revealed gradual phase transitions from the lamellar via the gyroid type cubic phase to the diamond type bicontinuous cubic structure between 5 and 100% relative humidity. The detailed insights into the formation and transformation of the coating nanostructures in this system may provide essential knowledge for the comprehensive design of functional nanostructured surfaces in biomedical applications.

A water-soluble, bay-functionalized perylenediimide derivative - correlating aggregation and excited state dynamics.

The aggregation and the photophysics of a water soluble perylenediimide (PDI) derivative that features two bromine substituents in the bay positions has been probed. Non-fluorescent aggregates were found to be present at concentrations of 1.0 × 10-5 M. In situ small-angle X-ray scattering (SAXS) measurements and complementary molecular modeling showed the presence of PDI aggregates. In their singlet excited states, the PDI aggregates are characterized by distinct transient fingerprints and rapid deactivation, as revealed by pump-probe experiments on the femto-, pico-, nano-, and microsecond timescales. The product of this deactivation is a PDI triplet excited state. The efficiency of the triplet formation depends on the concentration, and hence on the degree of aggregation. Notably, for PDI concentrations in the range of the critical micelle concentration, the efficiency of intersystem crossing is close to zero. In short, we have demonstrated, for the first time, aggregation-induced formation of triplet excited states for PDI derivatives.

Direct monitoring of lipid transfer on exposure of citrem nanoparticles to an ethanol solution containing soybean phospholipids by combining synchrotron SAXS with microfluidics.

Lipid exchange among citrem nanoparticles and an ethanol micellar solution containing soy phosphatidylcholine was investigated in situ by coupling small angle X-ray scattering with a microfluidic device. The produced soy phosphatidylcholine/citrem nanoparticles have great potential in the development of hemocompatible nanocarriers for drug delivery.

A carbon nanopore model to quantify structure and kinetics of ion electrosorption with in situ small-angle X-ray scattering.

A new carbon model derived from in situ small-angle X-ray scattering (SAXS) enables a quantitative description of the voltage-dependent arrangement and transport of ions within the nanopores of carbon-based electric double-layer capacitors. In the first step, ex situ SAXS data for nanoporous carbon-based electrodes are used to generate a three-dimensional real-space model of the nanopore structure using the concept of Gaussian random fields. This pore model is used to derive important pore size characteristics, which are cross-validated against the corresponding values from gas sorption analysis. In the second step, simulated in situ SAXS patterns are generated after filling the model pore structure with an aqueous electrolyte and rearranging the ions via a Monte Carlo simulation for different applied electrical potentials. These simulated SAXS patterns are compared with in situ SAXS patterns recorded during voltage cycling. Experiments with different cyclic voltammetry scan rates revealed a systematic time lag between ion transport processes and the applied voltage signal. Global transport into and out of nanopores was found to be faster than the accommodation of the local equilibrium arrangement in favor of sites with a high degree of confinement.

Monodisperse Iron Oxide Nanoparticles by Thermal Decomposition: Elucidating Particle Formation by Second-Resolved in Situ Small-Angle X-ray Scattering.

The synthesis of iron oxide nanoparticles (NPs) by thermal decomposition of iron precursors using oleic acid as surfactant has evolved to a state-of-the-art method to produce monodisperse, spherical NPs. The principles behind such monodisperse syntheses are well-known: the key is a separation between burst nucleation and growth phase, whereas the size of the population is set by the precursor-to-surfactant ratio. Here we follow the thermal decomposition of iron pentacarbonyl in the presence of oleic acid via in situ X-ray scattering. This method allows reaction kinetics and precursor states to be followed with high time resolution and statistical significance. Our investigation demonstrates that the final particle size is directly related to a phase of inorganic cluster formation that takes place between precursor decomposition and particle nucleation. The size and concentration of clusters were shown to be dependent on precursor-to-surfactant ratio and heating rate, which in turn led to differences in the onset of nucleation and concentration of nuclei after the burst nucleation phase. This first direct observation of prenucleation formation of inorganic and micellar structures in iron oxide nanoparticle synthesis by thermal decomposition likely has implications for synthesis of other NPs by similar routes.

Effects of resveratrol on the structure and fluidity of lipid bilayers: a membrane biophysical study.

Resveratrol is a natural active compound which has been attracting increasing interest due to its several pharmacological effects in cancer prevention, cardiovascular protection and treatment of neurodegenerative disorders and diabetes. The current work investigates how resveratrol affects membrane order and structure, gathering information determined by X-ray scattering analysis, derivative spectrophotometry, fluorescence quenching and fluorescence anisotropy studies. The results indicate that resveratrol is able to be incorporated into DMPC liposome model systems, either fluidizing or stiffening the bilayer, which largely depends on the membrane fluidity state. These findings suggest that the effects of resveratrol resemble cholesterol action on biological membranes, thereby contributing to the regulation of cell membrane structure and fluidity, which may influence the activity of transmembrane proteins and hence control the cell signaling pathways. The regulation of a number of cellular functions, thus may contribute to the pharmacological and therapeutic activities of this compound, explaining its pleiotropic action.

Probing hemoglobin confinement inside submicron silica tubes using synchrotron SAXS and electrochemical response.

The configuration of hemoglobin in solution and confined inside silica nanotubes has been studied using synchrotron small angle X-ray scattering and electrochemical activity. Confinement inside submicron tubes of silica aid in preventing protein aggregation, which is vividly observed for unconfined protein in solution. The radius of gyration (R g) and size polydispersity (p) of confined hemoglobin was found to be lower than that in solution. This was also recently demonstrated in case of confined hemoglobin inside layered polymer capsules. The confined hemoglobin displayed a higher thermal stability with R g and p showing negligible changes in the temperature range 25-75 °C. The differences in configuration between the confined and unconfined protein were reflected in their electrochemical activity. Reversible electrochemical response (from cyclic voltammograms) obtained in case of the confined hemoglobin, in contrary to the observance of only a cathodic response for the unconfined protein, gave direct indication of the differences between the residences of the electroactive heme center in a different orientation compared to that in solution state. The confined Hb showed loss of reversibility only at higher temperatures. The electron transfer coefficient (α) and electron transfer rate constant (k s) were also different, providing additional evidence regarding structural differences between the unconfined and confined states of hemoglobin. Thus, absence of any adverse effects due to confinement of proteins inside the inorganic matrices such as silica nanotubes opens up new prospects for utilizing inorganic matrices as protein "encapsulators", as well as sensors at varying temperatures.

X-ray lithography and small-angle X-ray scattering: a combination of techniques merging biology and materials science.

The advent of micro/nanotechnology has blurred the border between biology and materials science. Miniaturization of chemical and biological assays, performed by use of micro/nanofluidics, requires both careful selection of the methods of fabrication and the development of materials designed for specific applications. This, in turn, increases the need for interdisciplinary combination of suitable microfabrication and characterisation techniques. In this review, the advantages of combining X-ray lithography, as fabrication technique, with small-angle X-ray scattering measurements will be discussed. X-ray lithography enables the limitations of small-angle X-ray scattering, specifically time resolution and sample environment, to be overcome. Small-angle X-ray scattering, on the other hand, enables investigation and, consequently, adjustment of the nanostructural morphology of microstructures and materials fabricated by X-ray lithography. Moreover, the effect of X-ray irradiation on novel materials can be determined by use of small-angle X-ray scattering. The combination of top-down and bottom-up methods to develop new functional materials and structures with potential in biology will be reported.

The role played by salts in the formation of SBA-15, an in situ small-angle X-ray scattering/diffraction study.

The influence of salts (NaCl, NaBr, and NaI) on the formation of mesoporous silica SBA-15 was studied in situ by small-angle X-ray scattering and diffraction. Pluronic P104 was used as structure director. The micellar properties and the dynamics of formation were clearly dependent on the presence of salt. It was also shown that the kinetics of mesophase formation, the initial value of the cell parameters, and the extent of long-range order were all influenced by salt additions. The observations are explained to primarily originate from the influence of the anions on the ethylene oxide part of the polymer, i.e., the corona region of the Pluronic micelles. Two effects are identified: a general ion effect causing dehydration of the ethylene oxide part and consequently inducing micellar growth, and a specific ion effect that counterbalances this. The study provides the basis for understanding the means by which addition of simple Na-salts influence the formation of mesoscopically ordered silicas synthesized using nonionic surfactants as structure directors, hence advancing the knowledge base toward a more rational design of mesoporous materials.

Mesostructured silica aerosol particles: comparison of gas-phase and powder deposit X-ray diffraction data.

We report on the characterization of mesostructured aerosol silica particles in the gas phase using in situ synchrotron small-angle X-ray scattering (SAXS) in order to unveil the influence of the basic production parameters. The investigated system was based on tetraethylorthosilicate (TEOS) as the inorganic precursor and on cetyltrimethyl-ammonium bromide (CTAB) as the surfactant. The heating temperature, surfactant to silicate ratio, and particle flow rate were thoroughly investigated, and for this purpose, an in-house-built aerosol reactor equipped with a special X-ray observation chamber was used. Complementary fine structural analysis was applied on dried deposits of the silica aerosols comprising direct Fourier transforms as well as simple two-phase model fits. This resulted in robust estimates for the silica wall thickness and surfactant core radius of the hexagonally ordered mesostructure. The particle shape and size distribution were examined by scanning electron microscopy (SEM). The quality of the inner nanostructure was revealed from an analysis of the peak width. The comparison of data from the gas phase and powder deposit shows that, in general, slower drying conditions (heating temperature about 80 °C) and a medium surfactant to Si ratio (about 0.14) lead to nanostructures of the best quality in terms of well-defined long-range organization.

Role of temperature-independent lipoplex-cell membrane interactions in the efficiency boost of multicomponent lipoplexes.

Multicomponent lipoplexes have recently emerged as especially promising transfection candidates, as they are from 10 to 100 times more efficient than binary complexes usually employed for gene delivery purposes. Previously, we investigated a number of chemical-physical properties of DNA-lipid complexes that were proposed to affect transfection efficiency (TE) of lipoplexes, such as nanoscale structure, size, surface potential, DNA-protection ability and DNA release from complexes upon interaction with cellular lipids. Although some minor differences between multicomponent and binary lipoplexes were found, they did not correlate clearly with efficiency. Instead, here we show that a marked difference between the cell internalization mechanism of binary and multicomponent lipoplexes does exist. Multicomponent lipoplexes significantly transfect cells at 4 °C, when endocytosis does not take place suggesting that they can enter cells via a temperature-independent mechanism. Confocal fluorescence microscopy experiments showed the existence of a correlation between endosomal escape and TE. Multicomponent lipoplexes exhibited a distinctive ability of endosomal escape and release DNA into the nucleus, whereas, poorly efficient binary lipoplexes exhibited minor, if any, endosomal rupture ability and remained confined in perinuclear late endosomes. Stopped-flow mixing measurements showed that the fusion rates of multicomponent cationic liposomes with anionic vesicles, used as model systems of cell membranes, were definitely shorter than those of binary liposomes. As either lipoplex uptake and endosomal escape involve fusion between lipoplex and cellular membranes, we suggest that a mechanism of lipoplex-cellular membrane interaction, driven by lipid mixing between cationic and anionic cellular lipids, does explain the TE boost of multicomponent lipoplexes.

Guanosine quadruplexes in solution: a small-angle x-ray scattering analysis of temperature effects on self-assembling of deoxyguanosine monophosphate.

We investigated quadruplex formation in aqueous solutions of 2'-deoxyriboguanosine 5'-monophosphate, d(pG), which takes place in the absence of the covalent axial backbone. A series of in-solution small angle X-ray scattering experiments on d(pG) have been performed as a function of temperature in the absence of excess salt, at a concentration just above the critical one at which self-assembling occurs. A global fit approach has been used to derive composition and size distribution of the scattering particles as a function of temperature. The obtained results give thermodynamical justification for the observed phase-behavior, indicating that octamer formation is essential for quadruplex elongation. Our investigation shows that d(pG) quadruplexes are very suitable to assess the potential of G-quadruplex formation and to study the self-assembling thermodynamics.

Mesoporous aluminophosphate thin films with cubic pore arrangement.

Mesoporous aluminophosphate thin films with 3D cubic (Im3m) pore arrangement were synthesized for the first time. Thin films were templated with block copolymer nonionic templates Pluronic F127 and F108 and deposited on a glass substrate by dip-coating. In situ SAXS investigations show the formation of a highly ordered mesostructure upon the dip-coating process, which remains stable up to at least 670 K. A cubic mesostructure was observed also by TEM. Template removal process was monitored by TG and FT-IR. A transition from an amorphous aluminophosphate gel to a well-defined aluminophosphate framework was observed by MAS NMR.

Novel in situ setup to study the formation of nanoparticles in the gas phase by small angle x-ray scattering.

An in-house built aerosol generator setup for in situ gas phase studies of aerosol and nanoparticles is described. The aerosol generator with an ultrasonic ceramic disk mist maker provides high enough particle concentrations for structural gas phase analysis by synchrotron small angle x-ray scattering (for water approximately 4 x 10(8) droplets/s with a droplet size of approximately 2.5 microm). The working principle was proved by scattering of gold nanoparticles. For evaporation induced self-assembly studies of nanostructured particles, an additional thermal treatment chamber was included in the setup. The first on-line gas phase data with our setup for mesostructured silica particles are presented for different thermal treatments. Scanning electron microscope imaging revealed the average particle size to be approximately 1 microm. Furthermore, to quantify their internal nanostructure, diffraction experiments of deposited silica aerosols were carried out and the corresponding electron density map indicates a silica wall thickness of about 1 nm.

Interaction of LL-37 with model membrane systems of different complexity: influence of the lipid matrix.

As the main difference between bacterial and mammalian cell membranes is their net charge, the focal point of consideration in many model membrane experiments with antimicrobial peptides is lipid headgroup charge. We studied the interaction of the human multifunctional peptide LL-37 with single phospholipid monolayers, bilayers, and bilayers composed of binary mixtures of the four phospholipid species predominantly used in model membrane experiments (phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylserine). We found that 1), the effects on single lipid monolayers are not comparable to those on the corresponding bilayers; 2), there are four different effects of LL-37 on bilayers of the four lipids; 3), the preference of LL-37 for the specific lipids is roughly inversely related to chain packing density; and 4), in the binary lipid mixtures, one lipid-and not necessarily the charged one--generally governs the mode of lipid/peptide interaction. Thus, our results show that lipid net charge is not the decisive factor determining the membrane-perturbing mechanism of LL-37, but only one of several parameters, among them packing density, the ability to form intermolecular H-bonds, and lipid molecular shape, which emphasizes how profoundly the choice of the model system can influence the outcome of a study of lipid/peptide interaction.

Discovery of new hexagonal supramolecular nanostructures formed by squalenoylation of an anticancer nucleoside analogue.

In this study, the dynamically folded conformation of squalene (SQ) is taken advantage of to link this natural compound to the anticancer nucleoside analogue gemcitabine (gem) in order to achieve the spontaneous formation of nanoassemblies (SQgem) in water. Cryogenic transmission electron microscopy examination reveals particles (104 nm) with a hexagonal or multifaceted shape that display an internal structure made of reticular planes, each particle being surrounded by an external shell. X-ray diffraction evidences the hexagonal molecular packing of SQgem, resulting from the stacking of direct or inverse cylinders. The respective volumes of the gem and SQ molecules as well as molecular modeling of SQgem suggest the stacking of inverse hexagonal phases, in which the central aqueous core, consisting of water and gem molecules, is surrounded by SQ moieties. These SQgem nanoassemblies also exhibit impressively greater anticancer activity than gem against a solid subcutaneously grafted tumor, following intravenous administration. To our knowledge, this is the first demonstration of hexagonal phase organization with a SQ derivative.