Structural Insights into the Mechanism of Heat-Set Gel Formation of Polyisocyanopeptide Polymers.

One of the key factors influencing the mechanical properties of natural and synthetic extracellular matrices (ECM) is how large-scale 3D gel-like structures emerge from the molecular self-assembly of individual polymers. Here, structural characterization using small-angle neutron scattering (SANS) of ECM-mimicking polyisocyanopeptide (PIC) hydrogels are reported as a function of background ions across the Hofmeister series. More specifically, the process of polymer assembly is examined by probing the structural features of the heat-set gels and correlating them with their rheological and micro-mechanical properties. The molecular parameters obtained from SANS clearly show changes in polymer conformation which map onto the temperature-induced changes in rheological and micro-mechanical behavior. The formation of larger structures are linked to the formation of cross-links (or bundles), whilst the onset of their detection in the SANS is putatively linked to their concentration in the gel. These insights provide support for the 'hot-spot' gelation mechanism of PIC heat-set gels. Finally, it is found that formation of cross-links and heat-set gelling properties can be strongly influenced by ions in accordance with Hofmeister series. In practice, these results have significance since ions are inherently present in high concentration during cell culture studies; this may therefore influence the structure of synthetic ECM networks.

The Curious Case of the OZ439 Mesylate Salt: An Amphiphilic Antimalarial Drug with Diverse Solution and Solid State Structures.

Efforts to develop orally administered drugs tend to place an exceptional focus on aqueous solubility as this is an essential criterion for their absorption in the gastrointestinal tract. In this work we examine the solid state behavior and solubility of OZ439, a promising single-dose cure for malaria being developed as the highly water-soluble mesylate salt. The aqueous phase behavior of the OZ439 mesylate salt was determined using a combination of small angle neutron and X-ray scattering (SANS and SAXS, respectively). It was found that this salt has low solubility at low concentrations with the drug largely precipitated in free base aggregates. However, with increasing concentration these crystalline aggregates were observed to dissociate into cationic micelles and lamellar phases, effectively increasing the dissolved drug concentration. It was also found that the dissolved OZ439 spontaneously precipitated in the presence of biologically relevant anions, which we attribute to the high lattice energies of most of the salt forms of the drug. These findings show that aqueous solubility is not always what it seems in the context of amphiphilic drug molecules and highlights that its use as the principal metric in selecting drug candidates for development can be perilous.

Influence of molecular weight on PNIPAM brush modified colloidal silica particles.

The effect of molecular weight and temperature on the phase transition and internal structure of poly(N-isopropylacrylamide) brush modified colloidal silica particles was investigated using dynamic light scattering (DLS) and small angle neutron scattering (SANS) between 15 and 45 °C. Dry particle analysis utilising transmission electron microscopy (TEM), Fourier-transform infrared spectroscopy (FTIR) and thermogravimetric analysis (TGA) all confirmed the thickness of the polymer brush shell increased as a function of polymerisation time. Hydrodynamic diameter and electrophoretic mobility results revealed that the brush modified particles transitioned from swollen shells to a collapsed conformation between 15 and 35 °C. The dispersions were electrosterically stabilised over the entire temperature range investigated, with minimal thermal hysteresis recorded. Modelling of the hydrodynamic diameter enabled the calculation of a lower critical solution temperature (LCST) which increased as a function of brush thickness. The internal structure determined via SANS showed a swollen brush at low temperatures (18 and 25 °C) which decayed radially away from the substrate, while a collapsed block-like conformation with 60% polymer volume fraction was present at 40 °C. Radial phase separation was evident at intermediate temperatures (30 and 32.5 °C) with the lower molecular weight sample having a greater volume fraction of polymer in the dense inner region at these temperatures.

Neutron scattering shows a droplet of oleic acid at the center of the BAMLET complex.

The anti-cancer complex, Bovine Alpha-lactalbumin Made LEthal to Tumors (BAMLET), has intriguing broad-spectrum anti-cancer activity. Although aspects of BAMLET's anti-cancer mechanism are still not known, it is understood that it involves the oleic acid or oleate component of BAMLET being preferentially released into cancer cell membranes leading to increased membrane permeability and lysis. The structure of the protein component of BAMLET has previously been elucidated by small angle X-ray scattering (SAXS) to be partially unfolded and dramatically enlarged. However, the structure of the oleic acid component of BAMLET and its disposition with respect to the protein component was not revealed as oleic acid has the same X-ray scattering length density (SLD) as water. Employing the difference in the neutron SLDs of hydrogen and deuterium, we carried out solvent contrast variation small angle neutron scattering (SANS) experiments of hydrogenated BAMLET in deuterated water buffers, to reveal the size, shape, and disposition of the oleic acid component of BAMLET. Our resulting analysis and models generated from SANS and SAXS data indicate that oleic acid forms a spherical droplet of oil incompletely encapsulated by the partially unfolded protein component. This model provides insight into the anti-cancer mechanism of this cache of lipid. The model also reveals a protein component "tail" not associated with the oleic acid component that is able to interact with the tail of other BAMLET molecules, providing a plausible explanation of how BAMLET readily forms aggregates. Proteins 2017; 85:1371-1378. © 2017 Wiley Periodicals, Inc.

Nonlinear Behavior of Gelatin Networks Reveals a Hierarchical Structure.

We investigate the strain hardening behavior of various gelatin networks-namely physical gelatin gel, chemically cross-linked gelatin gel, and a hybrid gel made of a combination of the former two-under large shear deformations using the pre-stress, strain ramp, and large amplitude oscillations shear protocols. Further, the internal structures of physical gelatin gels and chemically cross-linked gelatin gels were characterized by small angle neutron scattering (SANS) to enable their internal structures to be correlated with their nonlinear rheology. The Kratky plots of SANS data demonstrate the presence of small cross-linked aggregates within the chemically cross-linked network whereas, in the physical gelatin gels, a relatively homogeneous structure is observed. Through model fitting to the scattering data, we were able to obtain structural parameters, such as the correlation length (ξ), the cross-sectional polymer chain radius (R(c)) and the fractal dimension (d(f)) of the gel networks. The fractal dimension d(f) obtained from the SANS data of the physical and chemically cross-linked gels is 1.31 and 1.53, respectively. These values are in excellent agreement with the ones obtained from a generalized nonlinear elastic theory that has been used to fit the stress-strain curves. The chemical cross-linking that generates coils and aggregates hinders the free stretching of the triple helix bundles in the physical gels.

Hierarchical architecture of bacterial cellulose and composite plant cell wall polysaccharide hydrogels using small angle neutron scattering.

Small angle neutron scattering (SANS) has been applied to characterise the structure of pure bacterial cellulose hydrogels, and composites thereof, with two plant cell wall polysaccharides (arabinoxylan and xyloglucan). Conventional published models, which assume that bacterial cellulose ribbons are solid one-phase systems, fail to adequately describe the SANS data of pure bacterial cellulose. Fitting of the neutron scattering profiles instead suggests that the sub-structure of cellulose microfibrils contained within the ribbons results in the creation of regions with distinct values of neutron scattering length density, when the hydrogels are subjected to H2O/D2O exchange. This may be represented within a core-shell formalism that considers the cellulose ribbons to comprise a core containing impermeable crystallites surrounded by a network of paracrystalline cellulose and tightly bound water, and a shell containing only paracrystalline cellulose and water. Accordingly, a fitting function comprising the sum of a power-law term to account for the large scale structure of intertwined ribbons, plus a core-shell cylinder with polydisperse radius, has been applied; it is demonstrated to simultaneously describe all SANS contrast variation data of pure and composite bacterial cellulose hydrogels. In addition, the resultant fitting parameters indicate distinct interaction mechanisms of arabinoxylan and xyloglucan with cellulose, revealing the potential of this approach to investigate the role of different plant cell wall polysaccharides on the biosynthesis process of cellulose.

pH-responsive micelles based on caprylic acid.

Free fatty acids play a vital role as fuel for cells and in lipid metabolism. During lipid digestion in the gastrointestinal tract, triglycerides are hydrolyzed, resulting in free fatty acid and monoglyceride amphiphilic products. These components, together with bile salts, are responsible for the transport of lipids and poorly water-soluble nutrients and xenobiotics from the intestine into the circulatory system of the body. In this study, we show that the self-assembly of digestion products from medium-chain triglycerides (tricaprylin) in combination with bile salt and phospholipid is highly pH-responsive. Individual building blocks of caprylic acid within the mixed colloidal structures are mapped using a combination of small-angle X-ray and neutron scattering combined with both solvent contrast variation and selective deuteration. Modeling of the scattering data shows transitions in the size and shape of the micelles in combination with a transfer of the caprylic acid from the core of the micelles to the shell or into the bulk water upon increasing pH. The results help to understand the process of lipid digestion with a focus on colloidal structure formation and transformation for the delivery of triglyceride lipids and other hydrophobic functional molecules.

Phase transition and gelation in cellulose nanocrystal-based aqueous suspensions studied by SANS.

HYPOTHESIS: Aqueous suspensions of cellulose nanocrystals (CNC) form a re-entrant liquid crystal (LC) phase with increasing salinity. Phase separation occurs in this LC state leading to a biphasic gel with a flow programmable structure that can be used to form anisotropic soft materials. We term this state a Liquid Crystal Hydroglass (LCH). Defining the mechanisms by which the LCH forms requires detailed structural analysis at the mesoscopic length scale. EXPERIMENTS: By utilising Small Angle Neutron Scattering (SANS), we investigated the microstructure transitions in CNC suspensions, with a particular focus on the unique LC re-entrancy and gelation into the biphasic LCH. FINDINGS: Scattering from LCH gels comprises contributions from a dispersed liquid state and static heterogeneity, characterised using a Lorentzian-Gaussian model of inhomogeneity. This conceptually supports a gelation mechanism (spinodal decomposition) in CNC suspensions towards a biphasic structure of the LCH. It also demonstrates that, with increasing salinity, the non-monotonic variation in effective volume fraction of CNC rods fundamentally causes the LC re-entrancy. This work provides the first experimental characterisation of the LC-re-entrancy and formation of an anisotropic LCH gel. The proposed mechanism can be extended to understanding the general behaviour of anisotropic colloids.

Glucan affinity of starch synthase IIa determines binding of starch synthase I and starch-branching enzyme IIb to starch granules.

The sugary-2 mutation in maize (Zea mays L.) is a result of the loss of catalytic activity of the endosperm-specific SS (starch synthase) IIa isoform causing major alterations to amylopectin architecture. The present study reports a biochemical and molecular analysis of an allelic variant of the sugary-2 mutation expressing a catalytically inactive form of SSIIa and sheds new light on its central role in protein-protein interactions and determination of the starch granule proteome. The mutant SSIIa revealed two amino acid substitutions, one being a highly conserved residue (Gly522→Arg) responsible for the loss of catalytic activity and the inability of the mutant SSIIa to bind to starch. Analysis of protein-protein interactions in sugary-2 amyloplasts revealed the same trimeric assembly of soluble SSI, SSIIa and SBE (starch-branching enzyme) IIb found in wild-type amyloplasts, but with greatly reduced activities of SSI and SBEIIb. Chemical cross-linking studies demonstrated that SSIIa is at the core of the complex, interacting with SSI and SBEIIb, which do not interact directly with each other. The sugary-2 mutant starch granules were devoid of amylopectin-synthesizing enzymes, despite the fact that the respective affinities of SSI and SBEIIb from sugary-2 for amylopectin were the same as observed in wild-type. The data support a model whereby granule-bound proteins involved in amylopectin synthesis are partitioned into the starch granule as a result of their association within protein complexes, and that SSIIa plays a crucial role in trafficking SSI and SBEIIb into the granule matrix.

Snake venom-defined fibrin architecture dictates fibroblast survival and differentiation.

Fibrin is the provisional matrix formed after injury, setting the trajectory for the subsequent stages of wound healing. It is commonly used as a wound sealant and a natural hydrogel for three-dimensional (3D) biophysical studies. However, the traditional thrombin-driven fibrin systems are poorly controlled. Therefore, the precise roles of fibrin's biophysical properties on fibroblast functions, which underlie healing outcomes, are unknown. Here, we establish a snake venom-controlled fibrin system with precisely and independently tuned architectural and mechanical properties. Employing this defined system, we show that fibrin architecture influences fibroblast survival, spreading phenotype, and differentiation. A fine fibrin architecture is a key prerequisite for fibroblast differentiation, while a coarse architecture induces cell loss and disengages fibroblast's sensitivity towards TGF-ß1. Our results demonstrate that snake venom-controlled fibrin can precisely control fibroblast differentiation. Applying these biophysical principles to fibrin sealants has translational significance in regenerative medicine and tissue engineering.

Relations between molecular, crystalline, and lamellar structures of amylopectin.

Chain (branch) length distributions (CLD) from size-exclusion chromatography of a series of waxy starches were parametrized using both an empirical and a biosynthesis-based method and correlated with their crystalline-amorphous lamellar properties obtained from X-ray scattering. Correlations were best seen with the biosynthesis-based parametrization. This showed for the first time that the following links between the CLD and lamellar parameters, the average interlamellar repeat distance and the distribution of these distances, were decreased by an increase in the proportion of very short branches and were increased by an increase in the proportion of intermediate and longer chains; further, the shoulder and linear sections of the CLD were found to affect the lamellar repeat distance and distribution. These effects are rationalized in terms of branch-length effects on the production of crystallites and the presence of portions of longer branches in the amorphous regions.

Amorphous packing of amylose and elongated branches linked to the enzymatic resistance of high-amylose wheat starch granules.

To elucidate starch structural features underlying resistant starch formation, wheat starch granules with three (A-, B- and C- type) crystalline polymorphisms and a range of amylose contents were digested in vitro. The changes in multi-level structure of digestion residues were compared. In the residues of A- and C-type starches, the molecular fine structure (distributions of chain length and whole molecular size), as analyzed by size exclusion chromatography (SEC), remained similar during digestion. In contrast, B-type high amylose wheat starch (HAWS) showed distinct changes in multi-level structures of digestion-resistant fractions: (1) the peak of longer amylopectin branches shifted to a lower degree of polymerization (40 DP); (2) production of α-limit dextrin (~2 nm hydrodynamic radius) in the residues; (3) a small increase of double helix content during digestion, in contrast to 6 % reduction for the A-type starch; (4) a decrease (6 °C lower) in the melting temperature of amylose-lipid complexes. The comparison suggests that elongated branches in B-type starch contribute to the formation of resistant fraction (including α-limit dextrin) against α-amylase. The amorphous packing of starch polymers with elongated branches together with the absence of surface pores and channels is proposed to be the basis for the enzymatic resistance of granular HAWS.

Effects of thermal denaturation on the solid-state structure and molecular mobility of glycinin.

The effects of moisture and thermal denaturation on the solid-state structure and molecular mobility of soy glycinin powder were investigated using multiple techniques that probe over a range of length and time scales. In native glycinin, increased moisture resulted in a decrease in both the glass transition temperature and the denaturation temperature. The sensitivity of the glass transition temperature to moisture is shown to follow the Gordon-Taylor equation, while the sensitivity of the denaturation temperature to moisture is modeled using Flory's melting point depression theory. While denaturation resulted in a loss of long-range order, the principal conformational structures as detected by infrared are maintained. The temperature range over which the glass to rubber transition occurred was extended on the high temperature side, leading to an increase in the midpoint glass transition temperature and suggesting that the amorphous regions of the newly disordered protein are less mobile. (13)C NMR results supported this hypothesis.

Structure of high internal phase aqueous-in-oil emulsions and related inverse micelle solutions. 3. Variation of surfactant.

The small angle neutron scattering from high internal phase water-in-hexadecane and saturated ammonium nitrate-in-hexadecane emulsions is compared with that from related hexadecane-based inverse micellar solutions. Three molecular weights of the monodisperse polyisobutylene acid amide (PIBSA) surfactant 750, 1200, and 1700 were studied over a range of surfactant concentrations. As an additional comparison, emulsions based on sorbitan monooleate and isostearate surfactants were investigated. The scattering from molecular weight 1200 water-based PIBSA emulsions can be fitted at all concentrations to a model with a surfactant coated aqueous droplet-oil interface together with the majority of the surfactant in the oil phase of the emulsion in the form of inverse micelles. Variation of the molecular weight shows a variety of phases of increasing curvature: lamellar, sponge, and, most commonly, the emulsion structure described above. In addition, the molecular weight affects the oil component in the emulsions, which can contain either cylindrical micelles or spherical micelles of varying water but constant hexadecane content. Increased phase curvature is favored by both increased PIBSA molecular weight and ammonium nitrate dissolved in the water. These observations are consistent with "Wedge theory". The structures observed in the emulsions are close to those observed in related inverse micellar solutions made from hexadecane, the surfactant, and water. Lower concentrations of surfactant in the micellar solutions decrease micelle curvature, except where the inverse micelles are spherical and small; here, there is little effect of dilution. Substitution of sorbitan surfactants for PIBSAs produces slightly less organized but similar structures, with smaller spherical micelles containing proportionally more water. The aqueous-oil droplet interface has a relatively invariant monolayer of adsorbed surfactant. For all emulsions, we can infer from the mass balance that micelle concentrations are depressed in the inverse micellar solutions because up to half the added surfactant is present as individually dissolved molecules.

Structure of high internal phase aqueous-in-oil emulsions and related inverse micelle solutions. 4. Surfactant mixtures.

The effects of combinations of surfactants on the structure and stability of high internal phase water-in-hexadecane and saturated ammonium nitrate-in-hexadecane oil-based emulsions and oil-based inverse micellar solutions are reported. The combinations were 750, 1,200, and 1,700 molecular weight monodisperse and 450 and 1,000 molecular weight polydisperse polyisobutylene acid amides, and sorbitan monooleate. The samples made from mixtures have qualitatively similar nanostructures to emulsions made from single surfactants. Again, for the emulsions, micrometer-scale aqueous droplets are dispersed in a continuous oil phase, which contains inverse spherical micelles composed of surfactant, hexadecane, and water. In quantitative terms, lower average surfactant molecular weight, lower ammonium nitrate content, and lower surfactant content increased the swelling of micelles, their water content, and the tendency of the emulsion to be unstable and form a sponge phase. This instability also allows micelle plasticity such that their geometry and content in mixed surfactant systems are not simply predictable by interpolation from single surfactant systems. An example was found of a mixed micelle 3 times larger than either single component micelle. The observed behavior suggests that mixing surfactant molecules of very different molecular weights destabilizes the emulsions, while mixing surfactants close in molecular weight has the opposite effect. The synergistic effects of surfactant molecular weight polydispersity and binary mixing are most marked for 1:1 molecular mixtures of surfactant.

Adsorption isotherm studies on the interaction between polyphenols and apple cell walls: Effects of variety, heating and drying.

The adsorption capacity of principal phenolic compounds onto cell walls from three apple varieties was investigated. Isothermal adsorption modelled with Langmuir, Freundlich and Redlich-Peterson equations were carried out over a range of concentrations from 0.5 to 30 mM before and after cell walls were subjected to boiling, oven-drying or freeze-drying. The isotherm data were best fitted by the Langmuir model in all cases. Polyphenols selectively adsorbed onto cell walls with maximum binding capacities ranging from 140 to 580 µg/mg cell walls depending on surface charge. Increased pectin in apple cell walls caused a 129%-311% decrease in the adsorption of negatively charged polyphenols, presumably due to electrostatic repulsive forces. Boiling had limited effect on cell wall polysaccharides and polyphenol-cell wall interactions. However, more than twofold reduction in binding capacities of polyphenols was induced after drying by altering the structural (i.e. binding sites) and compositional (i.e. pectin degradation) characteristics of cell walls.

Effect of amyloglucosidase hydrolysis on the multi-scale supramolecular structure of corn starch.

The effects of amyloglucosidase digestion on the multi-scale supramolecular structural changes of native corn starch were examined by ultra-small angle neutron scattering (USANS), small angle X-ray scattering (SAXS), particle sizing, and scanning electron microscopy (SEM). Well-defined and spherical pores were formed upon amyloglucosidase digestion as revealed by SEM. The pore polydispersity was determined using USANS by assuming spherical pore morphology with log-normal distribution. Both USANS and SEM measurements demonstrated that the pores become larger and more polydisperse as the digestion time increased. Moreover, SAXS revealed that the lamellar peak area decreased gradually for both thermally and enzymatically treated starches, indicating partial loss of lamellar organisation. Overall, the results demonstrate structural changes occur on multiple length scales upon enzymatic digestion from granular to lamellar with small-angle scattering demonstrated to provide detailed characterization of the resultant microporous structures.

Multi-scale assembly of hydrogels formed by highly branched arabinoxylans from Plantago ovata seed mucilage studied by USANS/SANS and rheology.

The structures of two hydrogels formed by purified brush-like polysaccharides from Plantago ovata seed mucilage have been characterised from the nanometre to micrometre scale by using a combination of SANS and USANS techniques. These two hydrogels have distinctly different melting and rheological properties, but the structure of their gel networks bears striking similarity as revealed by USANS/SANS experiments. Surprisingly, we find that the dramatic changes in the rheological properties induced by temperature or change in the solvent quality are accompanied by a small alteration of the network structure as inferred from scattering curves recorded above melting or in a chaotropic solvent (0.7 M KOD). These results suggest that, in contrast to most gel-forming polysaccharides for which gelation depends on a structural transition, the rheological properties of Plantago ovata mucilage gels are dependent on variations in intermolecular hydrogen bonding. By enzymatically cleaving off terminal arabinose residues from the side chains, we have demonstrated that composition of side-chains has a strong effect on intermolecular interactions, which, in turn, has a profound effect on rheological and structural properties of these unique polysaccharides.

PEGylation and surface functionalization of liposomes containing drug nanocrystals for cell-targeted delivery.

Liposomal formulations have important therapeutic applications in anti-cancer treatments but current formulations suffer from serious side effects, high dosage requirements and prolonged treatment. In this study, PEGylated azide-functionalized liposomes containing drug nanocrystals were investigated with the aim of increasing the drug payload and achieving functionalization for targeted delivery. Liposomes were characterized using cryogenic transmission electron microscopy (cryo-TEM), dynamic light scattering (DLS), small and ultra-small angle neutron scattering (SANS/USANS) and small and wide angle X-ray scattering (SAXS/WAXS). Cryo-TEM experiments revealed the dimensions of the nanocrystal-loaded liposomes and the change of shape from spherical to elongated after the formation of nanocrystals. Results from SANS/USANS experiments confirmed the asymmetric particle shape. SAXS/WAXS experiments confirmed that the crystalline drug only occurred in freeze-thawed samples and correlated with a new unidentified polymorphic form of ciprofloxacin. Using a small molecule dye, dibenzocyclooctyne (DBCO)-cy5, specific conjugation between DBCO groups and surface azide groups on the liposomes was confirmed; this indicates the promise of this system for tumour-targeted delivery.

Deuterated phytantriol - A versatile compound for probing material distribution in liquid crystalline lipid phases using neutron scattering.

Phytantriol is an interfacially-active lipid that is chemically robust, non-digestible and forms particles with internal bicontinuous cubic phase structures (cubosomes) when dispersed with non-ionic surfactants at ambient and physiological temperatures. The liquid crystalline internal structure of phytantriol-based cubosomes can be changed to alter the interfacial contact area/topology with the aqueous dispersant to trigger bioactive payload release or to alter the local membrane curvature around bound or embedded proteins. To enable the study of payload distribution, structure and transformation kinetics within phytantriol particles by neutron scattering techniques it is desirable to have access to a deuterated version of this molecule but to date a synthetic route has not been available. The first successful synthesis of phytantriol-d39 is presented here alongside a preliminary physical characterisation of related particle structures when phytantriol-d39 is dispersed using two non-ionic surfactants, Tween® 80 and Pluronic® F127. Synchrotron small angle X-ray scattering (SAXS) was used to confirm that phytantriol-d39-based nanoparticles in D2O form similar liquid crystalline structures to those of their natural isotopic abundance (phytantriol/H2O) counterparts as a function of temperature. Finally, small angle neutron scattering (SANS) with solvent contrast to match out the phytantriol-d39 structuring was used to show that the spatial correlations between the Tween® and Pluronic® non-ionic surfactant molecules are different within dispersed phytantriol-d39 particles with different liquid crystalline structures in D2O. The surfactant molecules in phytantriol-d39/Tween® 80 particles with Im3m cubic structures were found to follow a self-avoiding walk, whereas in phytantriol-d39/Pluronic® F127 particles with Pn3m cubic structures they were found to follow a more rod-like packing arrangement.