Developing an in situ LED irradiation system for small-angle X-ray scattering at B21, Diamond Light Source.

Beamline B21 at the Diamond Light Source synchrotron in the UK is a small-angle X-ray scattering (SAXS) beamline that specializes in high-throughput measurements via automated sample delivery systems. A system has been developed whereby a sample can be illuminated by a focused beam of light coincident with the X-ray beam. The system is compatible with the highly automated sample delivery system at the beamline and allows a beamline user to select a light source from a broad range of wavelengths across the UV and visible spectrum and to control the timing and duration of the light pulse with respect to the X-ray exposure of the SAXS measurement. The intensity of the light source has been characterized across the wavelength range enabling experiments where a quantitative measure of dose is important. Finally, the utility of the system is demonstrated via measurement of several light-responsive samples.

Self-Assembly and Antimicrobial Activity of Lipopeptides Containing Lysine-Rich Tripeptides.

The conformation and self-assembly of two pairs of model lipidated tripeptides in aqueous solution are probed using a combination of spectroscopic methods along with cryogenic-transmission electron microscopy (cryo-TEM) and small-angle X-ray scattering (SAXS). The palmitoylated lipopeptides comprise C16-YKK or C16-WKK (with two l-lysine residues) or their respective derivatives containing d-lysine (k), i.e., C16-Ykk and C16-Wkk. All four molecules self-assemble into spherical micelles which show structure factor effects in SAXS profiles due to intermicellar packing in aqueous solution. Consistent with micellar structures, the tripeptides in the coronas have a largely unordered conformation, as probed using spectroscopic methods. The molecules are found to have good cytocompatibility with fibroblasts at sufficiently low concentrations, although some loss of cell viability is noted at the highest concentrations examined (above the critical aggregation concentration of the lipopeptides, determined from fluorescence dye probe measurements). Preliminary tests also showed antimicrobial activity against both Gram-negative and Gram-positive bacteria.

Controlling the formation and alignment of low molecular weight gel 'noodles'.

We show how to control the formation and alignment of gel 'noodles'. Nanostructure alignment can be achieved reproducibly by extensional deformation as the filaments form. Using a spinning technique, very long and highly aligned filaments can be made. The Young's moduli of the gel noodles are similar to that of a bulk gel. By using two syringe pumps in a concentric flow setup, we show that a filament-in-filament morphology can be created.

Design of a multipurpose sample cell holder for the Diamond Light Source high-throughput SAXS beamline B21.

The design of a multipurpose sample cell holder for the high-throughput (HT) beamline B21 is presented. The device is compatible with the robot bioSAXS sample changer currently installed on BM29, ESRF, and P12 Petra IV synchrotrons. This work presents an approach that uses 3D-printing to make hardware alterations which can expand the versatility of HT beamlines at low cost.

Light-responsive self-assembly of a cationic azobenzene surfactant at high concentration.

The formation of high-concentration mesophases by a cationic azobenzene photosurfactant is described for the first time. Using a combination of polarised optical microscopy and small-angle X-ray scattering, optically anisotropic, self-assembled structures with long-range order are reported. The mesophases are disrupted or lost upon UV irradiation.

Beamline B21: high-throughput small-angle X-ray scattering at Diamond Light Source.

B21 is a small-angle X-ray scattering (SAXS) beamline with a bending magnet source in the 3 GeV storage ring at the Diamond Light Source Ltd synchrotron in the UK. The beamline utilizes a double multi-layer monochromator and a toroidal focusing optic to deliver 2 × 1012 photons per second to a 34 × 40 µm (FWHM) focal spot at the in-vacuum Eiger 4M (Dectris) detector. A high-performance liquid chromatography system and a liquid-handling robot make it possible to load solution samples into a temperature-controlled in-vacuum sample cell with a high level of automation. Alternatively, a range of viscous or solid materials may be loaded manually using a range of custom sample cells. A default scattering vector range from 0.0026 to 0.34 Å-1 and low instrument background make B21 convenient for measuring a wide range of biological macromolecules. The beamline has run a full user programme since 2013.

Isotopic Control over Self-Assembly in Supramolecular Gels.

It is common to switch between H2O and D2O when examining peptide-based systems, with the assumption being that there are no effects from this change. Here, we describe the effect of changing from H2O to D2O in a number of low-molecular-weight dipeptide-based gels. Gels are formed by decreasing the pH. In most cases, there is little difference in the structures formed at high pH, but this is not universally true. On lowering the pH, the kinetics of gelation are affected and, in some cases, the structures underpinning the gel network are different. Where there are differences in the self-assembled structures, the resulting gel properties are different. We, therefore, show that isotopic control over gel properties is possible.

Nanoparticulate Gels for Cutaneous Administration of Caffeic Acid.

Caffeic acid is a natural antioxidant, largely distributed in plant tissues and food sources, possessing anti-inflammatory, antimicrobial, and anticarcinogenic properties. The object of this investigation was the development of a formulation for caffeic acid cutaneous administration. To this aim, caffeic acid has been loaded in solid lipid nanoparticles by hot homogenization and ultrasonication, obtaining aqueous dispersions with high drug encapsulation efficiency and 200 nm mean dimension, as assessed by photon correlation spectroscopy. With the aim to improve the consistence of the aqueous nanodispersions, different types of polymers have been considered. Particularly, poloxamer 407 and hyaluronic acid gels containing caffeic acid have been produced and characterized by X-ray and rheological analyses. A Franz cell study enabled to select poloxamer 407, being able to better control caffeic acid diffusion. Thus, a nanoparticulate gel has been produced by addition of poloxamer 407 to nanoparticle dispersions. Notably, caffeic acid diffusion from nanoparticulate gel was eight-fold slower with respect to the aqueous solution. In addition, the spreadability of nanoparticulate gel was suitable for cutaneous administration. Finally, the antioxidant effect of caffeic acid loaded in nanoparticulate gel has been demonstrated by ex-vivo evaluation on human skin explants exposed to cigarette smoke, suggesting a protective role exerted by the nanoparticles.

Controlling the properties of the micellar and gel phase by varying the counterion in functionalised-dipeptide systems.

The micellar aggregates formed at high pH for dipeptide-based gelators can be varied by using different alkali metal salts to prepare the solutions. The nature of the micellar aggregates directly affects the properties of the resulting gels.

Using Small Angle X-Ray Scattering (SAXS) to Characterize the Solution Conformation and Flexibility of Matrix Metalloproteinases (MMPs).

Small angle X-ray scattering (SAXS) provides information about the conformation and flexibility of proteins in solution, and hence provides complementary structural information to that obtained from X-ray crystallography and nuclear magnetic resonance spectroscopy. In this chapter, we describe the methods for the preparation of matrix metalloproteinase (MMP) samples for SAXS analyses, and for the acquisition, processing and interpretation of the SAXS data.