Time-resolved Synchrotron Small Angle Research Articles

A novel droplet-based approach to study phase transformations in lyotropic liquid crystalline systems

HypothesisLyotropic liquid crystals (LLC) and their phase transformations in response to stimuli have gathered much interest for controlled and ‘on-demand’ drug applications. Bulk methods of preparation impose limitations on studying the transformations, especially induced by compositional changes, such as enzymatic changes to lipid structure. Here we hypothesise that controlled microfluidic production and coalescence of dissimilar aqueous and lipid droplets emulsified in a third mutually immiscible liquid will provide a new approach to the spatio-temporal study of structure formation in lyotropic liquid crystalline materials. ExperimentsSeparate lipid and aqueous droplets, dispersed in a fluorocarbon oil were generated using a microfluidic format. The chip, prepared as a hybrid polydimethylsiloxane (PDMS) and glass microfluidic device, was constructed to enable in-situ acquisition of time-resolved synchrotron small angle X-ray scattering (SAXS) and crossed polarised light microscopy of the coalesced droplets to determine the structures present during aging. FindingsJanus-like droplets formed upon coalesce, with distinct lipid and aqueous portions with a gradient between the two sides of the merged droplet. SAXS and polarised light microscopy revealed a progression of mesophases as the lipid portion was hydrated by the aqueous portion via the diffusion limited interface which separated the portions. Thus demonstrating, on a droplet scale, a new approach for studying the phase transformation kinetics and identification of non-equilibrium phase in droplet-based lyotropic liquid systems.

Early stages of nucleation and growth in melt crystallized polyethylene

Time-resolved synchrotron small angle X-ray scattering (SAXS) was used to investigate the early stages of crystallization in melt crystallized polyethylene. Classic Gibbs nucleation or density fluctuation theory can be used to describe the primary nucleation mechanism. At 110 °C, no signal of crystallization can be detected by SAXS for 30 min. When it is lower than 110 °C, the low q scattering intensity (0.008 < q < 0.03 Å−1) begins to upturn, and the primary nucleation process starts. The measured fractal dimension of the critical nuclei is in the vicinity of 3 which is close to the prediction of classic Gibbs nucleation theory. The growth rate of density fluctuations R(q) at different scattering vector q for different temperatures was obtained by analyzing the increase of scattering intensities. The results show that the growth rate of density fluctuation gets much bigger with the decrease of the isothermal crystallization temperature, but there is no signal of spinodal decomposition mechanism, in which there should be a linear relationship between R(q)/q2 and q2.

Transfer of lipid between triglyceride dispersions and lyotropic liquid crystal nanostructured particles using time-resolved SAXS

Lipid-based lyotropic liquid crystalline nanostructured phases, such as the reverse bicontinuous cubic phase (V2) and the reverse hexagonal phase (H2), can be dispersed into submicron sized particles with often preserved internal nanostructure, termed cubosomes and hexosomes respectively. The internal nanostructure of the particles imparts desirable characteristics such as the ability to provide sustained release of active ingredients but is susceptible to change upon the incorporation of other lipids used as an active ingredient delivery system. These are complex systems in which the direction of lipid transfer, role of the stabiliser and formation of different phase structures, such as the frustrated H2 phase are all likely to play a role but are not yet completely understood. Consequently, we have investigated the interparticle transfer of lipids between cubosomes/hexosomes and other dispersed lipids, namely triglycerides and vitamin E acetate emulsions. Time-resolved synchrotron small angle X-ray scattering was used to follow changes to nanostructure, attributed to the transfer of lipids into or out of the cubosomes. It was found that transfer of lipid occurred due to compositional ripening via a micelle-mediated mechanism, evident from the rate of lipid transfer increasing with Pluronic concentration. The rate of transfer of alkanes between liquid crystalline particles and emulsions has been shown previously to depend inversely on chain length. However in the triglyceride systems in the current study, the rate of lipid transfer (trilaurin > tricaprylin > supercooled trimyristin ≫ crystallised trimyristin and tristearin) did not decrease with increase in the chain length of triglyceride as might be anticipated. The equilibrium phase diagrams of triglyceride + phytantriol in excess water were determined. These diagrams showed that the transfer of phytantriol away from cubosomes to the emulsions, which increases the local triglyceride to phytantriol concentration in the cubosomes, would require formation of H2 phase more readily for tricaprylin than for trilaurin. Therefore, the lack of correlation of transfer rate with chain length of the triglyceride was attributed to the system seeking to avoid the unfavourable formation of the H2 phase, competing with the driving force from the entropy of mixing, and was therefore attributed as the reason for the greater rate of transfer of trilaurin compared to the less lipophilic tricaprylin.

Time-Resolved Small-Angle X-Ray Scattering Study of the Early Formation of Amyloid Protofibrils on a Apomyoglobin Mutant

Early prefibrillar intermediates are a key issue in amyloid researches, as they show the highest cytotoxicity with respect to mature fibrils, which are less toxic or even harmless. However, the early aggregation process is largely unknown, since nucleation events are rare, and elongation and reorganization processes are very fast. Time-resolved synchrotron Small Angle X-ray Scattering (SAXS), coupled to extended model fitting analysis, has been used to provide in a millisecond temporal range the structural and aggregational properties of the early and soluble protofibrils formed by a model protein after a pH jump. The amyloidogenic apomyoglobin mutant W7FW14F has been investigated as fibril former: this mutant, which is in a monomeric, partly folded state at acidic pH, undergoes at neutral pH a nucleation-dependent polymerization reaction, resulting in the formation of cytotoxic amyloid fibrils similar to those detected for proteins involved in amyloid diseases. SAXS evidenced that oligomerization of W7FW14F in solution happens in less than 100 ms after the pH jump from 4.0 to 7.0., while the resulting pattern of protein prefibrillation reveals the simultaneous presence of worm-like species and of cylindrically-shaped aggregates, whose structural features mainly change after 20 ms from the solution pH jump. Model fitting analysis gives the composition of the different oligomers and their relative concentration as a function of time, suggesting that protofibril formation occurs through the presence of aggregation and rearrangement competing processes and through the contribution of multiple coexisting elongated and worm-like protein species. The possibility to use SAXS in monitoring the effects of cosolvents and/or pharmaceutical agents in modifying or preventing the early amyloid aggregation patterns is then demonstrated.

Transfer of lipid and phase reorganisation in self-assembled liquid crystal nanostructured particles based on phytantriol

The internal structure of dispersed liquid crystal nanostructured particles of the V(2) and H(2) phases, termed cubosomes and hexosomes respectively, is integral to their application in the pharmaceutical, agricultural and food industries. However the nanostructure is susceptible to change upon incorporation of other lipids and hence it is important to understand the potential for interparticle lipid transfer for such particles when they encounter a particle of dissimilar lipid content. Using time resolved synchrotron small angle X-ray scattering, we have investigated the transfer of material between cubosomes composed of phytantriol with three different particle types of dissimilar composition, (i) hexosomes and (ii) emulsified microemulsion composed of phytantriol and vitamin E acetate, and (iii) cubosomes prepared from glycerol monooleate. It was found that material was transferred between the different dispersed nanostructured particles, with the transfer being caused by compositional ripening. Somewhat counter-intuitively the transfer was bidirectional with phytantriol being more rapidly transferred than the minor component vitamin E acetate. The greater lipophilicity of vitamin E acetate supports previous studies suggesting greater mobility for the less lipophilic components, regardless of the more efficient transfer route to achieve uniform composition. When particles comprising lipids with similar lipophilicities were mixed, the transfer was limited and did not achieve completion; a phase change between cubic nanostructures required to achieve complete mixing provides an apparent barrier to further compositional ripening. The conclusions from this study provide additional support to lipid transfer mechanisms, and highlight some subtleties in using dissimilar lipid mixtures in e.g. food applications.

Inhomogeneous fibril stretching in antler starts after macroscopic yielding: Indication for a nanoscale toughening mechanism

Antler is a unique mineralized tissue, with extraordinary toughness as well as an ability to annually regenerate itself in its entirety. The high fracture resistance enables it to fulfill its biological function as a weapon and defensive guard during combats between deer stags in the rutting season. However, very little is quantitatively understood about the structural origin of the antler's high toughness. We used a unique combination of time-resolved synchrotron small angle X-ray diffraction together with tensile testing of antler cortical tissue under physiologically wet conditions. We measured the deformation at the nanoscale from changes in the meridional diffraction pattern during macroscopic stretch-to-failure tests. Our results show that on average fibrils are strained only half as much as the whole tissue and the fibril strain increases linearly with tissue strain, both during elastic and inelastic deformation. Most remarkably, following macroscopic yielding we observe a straining of some fibrils equal to the macroscopic tissue strain while others are hardly stretched at all, indicating an inhomogeneous fibrillar strain pattern at the nanoscale. This behavior is unlike what occurs in plexiform bovine bone and may explain the extreme toughness of antler compared to normal bone.

Time-resolved synchrotron small angle X-ray scattering studies of poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate- co-3-hydroxyvalerate) polymers

Time-resolved synchrotron small-angle X-ray scattering measurements have been performed during the heating and melting of poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate- co-3-hydroxyvalerate). A rapid, continuous rise in d-spacing was observed during melting, consistent with the disappearance of thinner, unstable crystals from stacks of lamellae possessing a broad distribution of thicknesses. The thickening of the remaining crystals has been characterized during the annealing of a copolymer close to its melting temperature. Differential scanning calorimetry measurements have indicated the presence of multiple melting endotherms in the case of the copolymer but not in the homopolymer. These multiple endotherms have been related to the effects of the hydroxyvalerate units in limiting the thickness of the lamellae at any given temperature.

Isothermal thickening and thinning processes in low molecular weight poly(ethylene oxide) fractions crystallized from the melt: 2. Crystals involving more than one fold

The existence of a non-integral folding chain (NIF) crystal in a poly (ethylene oxide) fraction with molecular weight of 7100 can be proved by differential scanning calorimetry and time-resolved synchrotron small angle X-ray scattering experiments. At this molecular weight, the fraction can crystallize to a final form comprised of integral folding chain (IF) crystals with extended ( n = 0), once-folded ( n = 1), twice-folded ( n = 2), and even triply-folded ( n = 3) chain crystals. The fold length of NIF crystals increases with crystallization temperature. When this fold length is between those of IF ( n = 2) and IF ( n = 1) crystals, the NIF crystal can be thickened into the IF ( n = 1) crystal, or thinned into the IF ( n = 2) crystal. In the same way, if the fold length of NIF crystal lies between those of IF ( n = 1) and IF ( n = 0) crystals, similar isothermal thickening and thinning processes can also be observed. Which process occurs depends upon the thermodynamic driving force between the NIF and IF crystals and upon kinetic effects. Detailed kinetic maps of the fold length changes with time will be discussed.