Structural and therapeutic properties of salicylic acid-solubilized Pluronic solutions and hydrogels.

Salicylic acid (SA) finds extensive applications in the treatment of rheumatic and skin diseases because of its analgesic, anti-inflammatory and exfoliating properties. As it is lipophilic in nature, there is a need for appropriate delivery systems to harness these properties for different applications. Herein, we examined the suitability of Pluronic P123/F127 micellar systems as delivery media by investigating the structural, flow and antimicrobial properties of P123/F127-SA solutions and hydrogels using DLS, SANS, rheological and zone inhibition measurement techniques. SA modulates the aggregation characteristics of these surfactant systems and brings about spherical-to-worm-like micelle-to-vesicular structural transitions in the hydrophobic Pluronic P123 system, a spherical-to-worm-like micellar transition in the mixed P123/F127 system and an onset of inter-micellar attraction in the hydrophilic Pluronic F127 system. SA-solubilized systems of both hydrophobic and hydrophilic Pluronics inhibit the growth of Gram-positive and Gram-negative bacteria with comparable MIC values. This suggests that the interaction of SA molecules with the bacterial cell membrane remains unobstructed upon encapsulation in Pluronic micelles. F127 hydrogel-based SA formulations with rheological properties suitable for topical applications and up to 15% SA loading were prepared. These will be useful SA ointments as F127 is an FDA-approved excipient for topical drug delivery applications. The results indicate that Pluronics remain effective as delivery agents for SA and exhibit interesting structural polymorphism upon its solubilization.

Crystal-to-Crystal Transition of Ultrasoft Colloids under Shear.

Ultrasoft colloids typically do not spontaneously crystallize, but rather vitrify, at high concentrations. Combining in situ rheo-small-angle-neutron-scattering experiments and numerical simulations we show that shear facilitates crystallization of colloidal star polymers in the vicinity of their glass transition. With increasing shear rate well beyond rheological yielding, a transition is found from an initial bcc-dominated structure to an fcc-dominated one. This crystal-to-crystal transition is not accompanied by intermediate melting but occurs via a sudden reorganization of the crystal structure. Our results provide a new avenue to tailor colloidal crystallization and the crystal-to-crystal transition at the molecular level by coupling softness and shear.

Mixing ratio dependent complex coacervation versus bicontinuous gelation of pectin with in situ formed zein nanoparticles.

We report on the competitive phenomenon of complex coacervation versus bicontinuous gelation between pectin (P, a polyanionic carbohydrate, [P] = 0.01-2% (w/v)) and zein nanoparticles (Z, a hydrophobic protein and a weak polyampholyte, [Z] = 0.1 and 0.5% (w/v), in an ethanolic solution of effective concentration 4 and 27% (v/v)), which was studied below (pH ≈ 4), and above (pH ≈ 7.4) the pI (≈ 6.2) of zein at room temperature, 25 °C. The uniqueness of this study arises from the interaction protocol used, where the pectin used was in the extended polyelectrolyte (persistence length ≈ 10 nm) conformation while zein was used as a charged globular nanoparticle (size ≈ 80-120 nm) that was formed in situ. Their mixing ratio, r = [P] : [Z] (w/w), was varied from 0.02 to 4.0 (for [Z] = 0.5% (w/v)), and from 0.1 to 7.5 (for [Z] = 0.1% (w/v)) in the ionic strength range 10-4 to 10-2 M NaCl. Zeta potential data revealed that at pH ≈ 4, the complementary binding condition, r = 1 : 1 (equivalent to 1 : 5 molecule/nanoparticle) demarcated the coacervate from the gel region. The measured rigidity (G0, low frequency storage modulus) of these materials revealed the following: for r < 1, (low pectin content samples, coacervate region) the material had lower values of Gcoac0, whereas for r > 1, an excess of pectin facilitated gelation with Ggel0 ≫ Gcoac0. Above pI, surface patch binding caused associative interactions and complex coacervation though both biopolymers had similar net charge. The network density was used as a descriptor to distinguish between the coacervate and gel samples. Their microstructures were probed by small angle neutron scattering (SANS), and viscoelastic properties by rheology. Simple modeling shows that formation of the interpolymer complex was favored in higher protein containing samples. Mixing ratio dependent selective coacervation (a kinetic process) and bicontinuous gelation (a thermodynamic process) are rarely seen to coexist in biopolymer interactions.

The effect of temperature, composition and alcohols on the microstructures of catanionic mixtures of sodium dodecylsulfate and cetyltrimethylammonium bromide in water.

The influence of mixing protocol, composition, temperature, ageing and added alcohols on the characteristics of the microstructures of sodium dodecylsulfate (SDS) + cetyltrimethylammonium bromide (CTAB) mixtures has been investigated in this paper. In this catanionic mixture (1 weight% total surfactant content) temperature induced microstructural transition occurs, which is (i) a micelle-to-vesicle transition (MVT) if αSDS (mole fraction of SDS) = 0.7, 0.8 or 0.9 and (ii) a vesicle-to-micelle transition (VMT) if αSDS = 0.1, 0.2 or 0.3. In the mixture of αSDS = 0.7, specific conductivity and dynamic light scattering measurements also support the occurrence of MVT. Transition electron microscopy and small angle neutron scattering measurements were also made to assess the characteristics of the microstructures. Alcohols added to the mixture of αSDS = 0.7 reduced the size of the vesicle, while only monohydric alcohols suppressed the temperature induced transition indicating that the number and location of -OH groups of the alcohols have a dramatic modulating influence on the structural transition occurring in catanionic mixtures. The influence of the alcohols is explained in terms of changes produced in the dielectric constant and hydrophobicity of the medium.

DNA ionogel: Structure and self-assembly.

DNA dissolved in ionic liquid (IL) solution (1-ethyl-3-methylimidazolium chloride, [C2mim][Cl]) showed a transition to the gel phase ([DNA] ≥ 1% (w/v)). The gelation time was 400 s for the 1% [IL] sample which reduced to 260 s for 5% [IL] concentration. Gelation times, obtained from the viscosity and ergodicity breaking from the dynamic structure factor data, were remarkably identical to each other. Correspondingly, the gelation temperature which was ∼60 °C increased to 67 °C with [IL] content. The small angle neutron scattering (SANS) structure factor profile revealed the presence of the following three distinct length scales: (a) mesh size, ξ ≈ 3 ± 0.5 nm for ionogels, and ≈0.73 ± 0.06 nm, for sol; (b) cross-sectional radius of DNA strand, Rc ≈ 1.6 ± 0.1 nm; and (c) the characteristic inter-cluster distance ≈33 ± 5 nm. Physical conformation of the DNA-IL complexes remained close to the Gaussian coil definition. It was observed that without IL, in the sol phase, the system was completely ergodic and did not gel, while on addition of IL a sudden transition to the non-ergodic (arrested) gel phase occurred. This was due to the formation of an amorphous network of DNA-IL complexes preceding gelation. In summary, it is shown that the DNA ionogels can be prepared with a tunable gel strength (27-70 Pa) and gelation temperature (60-67 °C). Further, the relaxation dynamics was found to be hierarchical in IL content of the gel, revealing considerable self-organization.

Small-angle neutron scattering study of temperature vs. salt dependence of clouding in charged micellar system.

Clouding is studied by small-angle neutron scattering (SANS) on a charged micellar system of sodium dodecyl sulphate (SDS) and tetrabutylammonium bromide (TBAB) with varying temperature and salt NaCl. We show that the clouding occurs as a result of increase in the attractive potential between the micelles mediated by the dehydrated TBA(+) counterions on increasing temperature and in the presence of salt. Both micelles and clusters coexist at cloud point temperature (CP) and beyond CP. The addition of salt can be used to obtain CP at room temperature (30° C). The relative effect of different salts on clouding has been found in the order CaCl(2) > MgSO(4) > Na(2)SO(4) > NaF > NaCl > KCl > CsCl > NaBr > NaNO(3). This order is explained on the basis of two important roles played by salt ions: i) counterion condensation that increases the size of the micelles and ii) dehydration of TBA(+) counterions by salt ions for bridging the micelles.

Structural properties of thermoresponsive poly(N-isopropylacrylamide)-poly(ethyleneglycol) microgels.

We present investigations of the structural properties of thermoresponsive poly(N-isopropylacrylamide) (PNiPAM) microgels dispersed in an aqueous solvent. In this particular work poly(ethyleneglycol) (PEG) units flanked with acrylate groups are employed as cross-linkers, providing an architecture designed to resist protein fouling. Dynamic light scattering (DLS), static light scattering (SLS), and small angle neutron scattering (SANS) are employed to study the microgels as a function of temperature over the range 10 °C ≤ T ≤ 40 °C. DLS and SLS measurements are simultaneously performed and, respectively, allow determination of the particle hydrodynamic radius, R(h), and radius of gyration, R(g), at each temperature. The thermal variation of these magnitudes reveals the microgel deswelling at the PNiPAM lower critical solution temperature (LCST). However, the hydrodynamic radius displays a second transition to larger radii at temperatures T ≤ 20 °C. This feature is atypical in standard PNiPAM microgels and suggests a structural reconfiguration within the polymer network at those temperatures. To better understand this behavior we perform neutron scattering measurements at different temperatures. In striking contrast to the scattering profile of soft sphere microgels, the SANS profiles for T ≤ LCST of our PNiPAM-PEG suspensions indicate that the particles exhibit structural properties characteristic of star polymer configurations. The star polymer radius of gyration and correlation length gradually decrease with increasing temperature despite maintenance of the star polymer configuration. At temperatures above the LCST, the scattered SANS intensity is typical of soft sphere systems.

Neutron optical beam splitter from holographically structured nanoparticle-polymer composites.

We report a breakthrough in the search for versatile diffractive elements for cold neutrons. Nanoparticles are spatially arranged by holographical means in a photopolymer. These grating structures show remarkably efficient diffraction of cold neutrons up to about 50% for effective thicknesses of only 200   µm. They open up a profound perspective for next generation neutron-optical devices with the capability to tune or modulate the neutron diffraction efficiency.

Osmotic shrinkage in star/linear polymer mixtures.

Multiarm star polymers were used as model grafted colloidal particles with long hairs, to study their size variation due to osmotic forces arising from added linear homopolymers of smaller size. This is the origin of the depletion phenomenon that has been exploited in the past as a means to melt soft colloidal glasses by adding linear chains and analyzed using dynamic light scattering experiments and an effective interactions analysis yielding the depletion potential. Shrinkage is a generic phenomenon for hairy particles, which affects macroscopic properties and state transitions at high concentrations. In this work we present a small-angle neutron scattering study of star/linear polymer mixtures with different size ratios (varying the linear polymer molar mass) and confirm the depletion picture, i.e., osmotic star shrinkage. Moreover, we find that as the linear/star polymer size ratio increases for the same effective linear volume fraction (c/c* with c* the overlapping concentration), the star shrinkage is reduced whereas the onset of shrinkage appears to take place at higher linear polymer volume fractions. A theoretical description of the force balance on a star polymer in solution, accounting for the classic Flory contributions, i.e. elastic and excluded volume, as well as the osmotic force due to the linear chains, accurately predicts the experimental findings of reduced star size as a function of linear polymer concentration. This is done in a parameter-free fashion, in which the size of the cavity created by the star, and from which the chains are excluded, is related to the radius of the former from first principles.

Structure and phase diagram of an adhesive colloidal dispersion under high pressure: a small angle neutron scattering, diffusing wave spectroscopy, and light scattering study.

We have applied small angle neutron scattering (SANS), diffusing wave spectroscopy (DWS), and dynamic light scattering (DLS) to investigate the phase diagram of a sterically stabilized colloidal system consisting of octadecyl grafted silica particles dispersed in toluene. This system is known to exhibit gas-liquid phase separation and percolation, depending on temperature T, pressure P, and concentration phi. We have determined by DLS the pressure dependence of the coexistence temperature and the spinodal temperature to be dP/dT=77 bar/K. The gel line or percolation limit was measured by DWS under high pressure using the condition that the system became nonergodic when crossing it and we determined the coexistence line at higher volume fractions from the DWS limit of turbid samples. From SANS measurements we determined the stickiness parameter tau(B)(P,T,phi) of the Baxter model, characterizing a polydisperse adhesive hard sphere, using a global fit routine on all curves in the homogenous regime at various temperatures, pressures, and concentrations. The phase coexistence and percolation line as predicted from tau(B)(P,T,phi) correspond with the determinations by DWS and were used to construct an experimental phase diagram for a polydisperse sticky hard sphere model system. A comparison with theory shows good agreement especially concerning the predictions for the percolation threshold. From the analysis of the forward scattering we find a critical scaling law for the susceptibility corresponding to mean field behavior. This finding is also supported by the critical scaling properties of the collective diffusion.

Small-angle neutron scattering study of structure and kinetics of temperature-induced protein gelation.

The phase diagram, structural evolution, and kinetics of temperature-induced protein gelation of protein Bovine Serum Albumin (BSA) have been studied as a function of solution pH and protein concentration. The protein gelation temperature represents the onset of turbidity in the protein solution, which increases significantly with increasing pH beyond the isoelectric pH of the protein molecule. On the other hand, the gelation temperature decreases with an increase in protein concentration only in the low-protein-concentration regime and shows a small increasing trend at higher protein concentrations. The structural evolution and kinetics of protein gelation have been studied using small-angle neutron scattering. The structure of the protein molecule remains stable up to temperatures very close to the gelation temperature. On increasing the temperature above the gelation temperature, the protein solution exhibits a fractal structure, an indication of gel formation due to aggregation. The fractal dimension of the gel increases with increasing temperature, suggesting an increase in branching between the aggregates, which leads to stronger gels. The increase in both solution pH and protein concentration is found to delay the growth in the fractal structure and its saturation. The kinetics of gelation has been studied using the temperature-jump process of heating. It is found that the structure of the protein gels remains invariant after the heating time ( approximately 1 min), indicating a rapid formation of gel structure within this time. The protein gels prepared through gradual and temperature-jump heating routes do not always show the same structure. In particular, at higher temperatures (e.g., 85 degrees C ), while gradual heating shows a fractal structure, there is collapse of such fractal structure during temperature-jump heating.

Small-angle neutron scattering study of protein unfolding and refolding.

Small-angle neutron scattering has been used to study protein unfolding and refolding in protein bovine serum albumin (BSA) due to perturbation in its native structure as induced by three different protein denaturating agents: urea, surfactant, and pressure. The BSA protein unfolds for urea concentrations greater than 4 M and is observed to be independent of the protein concentration. The addition of surfactant unfolds the protein by the formation of micellelike aggregates of surfactants along the unfolded polypeptide chains of the protein and depends on the ratio of surfactant to protein concentration. We make use of the dilution method to show the refolding of unfolded proteins in the presence of urea and surfactant. BSA does not show any protein unfolding up to the pressure of 450 MPa. The presence of urea and surfactant (for concentrations prior to inducing their own unfolding) has been used to examine pressure-induced unfolding of the protein at lower pressures. The protein unfolds at 200 MPa pressure in the presence of urea; however, no unfolding is observed with surfactant. The protein unfolding is shown to be reversible in all the above denaturating methods.

Grain-boundary-induced spin disorder in nanocrystalline gadolinium.

Based on experimental magnetic-field-dependent neutron scattering data, we have calculated the autocorrelation function of the spin misalignment of nanocrystalline (160)gadolinium. The analysis suggests the existence of two characteristic length scales in the spin system: the smaller one is about 5 nm and is attributed to the defect cores of the grain boundaries, whereas the larger length scale is of the order of the average crystallite size D = 21 nm and presumably describes the response of the magnetization to the magnetic anisotropy field of the individual crystallites.

Fluorescent complex coacervates of agar and in situ formed zein nanoparticles: Role of electrostatic forces.

Herein, the complex coacervation between in situ formed spherical fluorescent zein nanoparticles and polyanion agar as function of mixing ratio (R=[Agar]/[Zein]) was investigated. This interaction yielded two distinguishable regions (at pH 5.4): Region I (R < 0.2), where fully charge neutralized soluble complexes with protein denaturation was noticed, and Region II (R > 0.2), where overcharged complexes were formed, with R = 0.2 defining the optimum binding. Small angle neutron scattering studies demonstrated that in the low-q region, nanoparticles formed the crosslink junctions and in the persistence regime of high-q region, the data captured the cross-sectional radius ( = 3.5 nm) for agar-zein complexes. The coacervates became more viscoelastic in salt-free samples because both the low frequency storage modulus and crosslink density were found to decrease with mixing ratio. Systematic decrease in storage modulus with ionic strength (0-0.01 M) implied screened Coulomb interaction was responsible for the observed coacervation. Further, we seek to find universality in complex coacervation of zein nanoparticle with biopolymers, and polysaccharides in particular.

Structural evolution during protein denaturation as induced by different methods.

Small-angle neutron scattering (SANS) and dynamic light scattering (DLS) have been used to study conformational changes in protein bovine serum albumin (BSA) due to perturbation in its native structure as induced by varying temperature and pressure, and in presence of protein denaturating agents urea and surfactant. BSA has prolate ellipsoidal shape at ambient temperature and we observe no effect of temperature on its structure up to a temperature of about 60 degrees C . At temperatures beyond 60 degrees C , protein denaturation leads to aggregation. The protein solution exhibits a fractal structure at temperatures above 64 degrees C , and its fractal dimension increases with temperature. This is an indication of aggregation followed by gelation that evolves with increasing temperature. It is known for some of the proteins (e.g., Staphylococcal Nuclease) that pressure of 200 MPa can unfold the protein, whereas BSA does not show any protein unfolding even up to the pressure of 450 MPa . In presence of urea, the BSA protein unfolds for urea concentrations greater than 4M and acquires a random coil configuration. We make use of the dilution method to show the reversibility of protein unfolding with urea. The addition of surfactant denaturates the protein by the formation of micellelike aggregates of surfactants along the unfolded polypeptide chains of the protein. We show such structure of the protein-surfactant complex can be stabilized at higher temperatures, which is not the case for pure protein.

Structural study of coacervation in protein-polyelectrolyte complexes.

Coacervation is a dense liquid-liquid phase separation and herein we report coacervation of protein bovine serum albumin (BSA) in the presence of polyelectrolyte sodium polystyrene sulfonate (NaPSS) under varying solution conditions. Small-angle neutron scattering (SANS) measurements have been performed on above protein-polyelectrolyte complexes to study the structural evolution of the process that leads to coacervation and the phase separated coacervate as a function of solution pH , protein-polyelectrolyte ratio and ionic strength. SANS study prior to phase separation on the BSA-NaPSS complex shows a fractal structure representing a necklace model of protein macromolecules randomly distributed along the polystyrene sulfonate chain. The fractal dimension of the complex decreases as pH is shifted away from the isoelectric point ( approximately 4.7) of BSA protein, which indicates the decrease in the compactness of the complex structure due to increase in the charge repulsion between the protein macromolecules bound to the polyelectrolyte. Concentration-dependence studies of the polyelectrolyte in the complex suggest coexistence of two populations of polyelectrolytes, first one fully saturated with proteins and another one free from proteins. Coacervation phase has been obtained through the turbidity measurement by varying pH of the aqueous solution containing protein and polyelectrolyte from neutral to acidic regime to get them to where the two components are oppositely charged. The spontaneous formation of coacervates is observed for pH values less than 4. SANS study on coacervates shows two length scales related to complex aggregations (mesh size and overall extent of the complex) hierarchically branched to form a larger network. The mesh size represents the distance between cross-linked points in the primary complex, which decreases with increase in ionic strength and remains the same on varying the protein-polyelectrolyte ratio. On the other hand, the overall extent of the complex shows a similar structure irrespective of varying ionic strength and protein-polyelectrolyte ratio. A large fraction ( approximately 50%) of protein-polyelectrolyte complexes is also found to be free in the supernatant after the coacervation.

New sources and instrumentation for neutrons in biology.

Neutron radiation offers significant advantages for the study of biological molecular structure and dynamics. A broad and significant effort towards instrumental and methodological development to facilitate biology experiments at neutron sources worldwide is reviewed.

A high pressure cell for small angle neutron scattering up to 500 MPa in combination with light scattering to investigate liquid samples.

We report on a high pressure cell to use with small angle neutron scattering (SANS) in a pressure range up to 500 MPa. The cell offers the new possibility to investigate liquid samples by a specially designed sample chamber, which allows changing of samples relatively easily. Since the cell construction uses sapphire as window material, also light scattering investigations can be performed simultaneously to the SANS measurements. In this article we describe the construction of a high pressure cell and we demonstrate the applicability of the construction for SANS in combination with dynamic light scattering showing data on the biological molecule lysozyme.

High hydrostatic pressure specifically affects molecular dynamics and shape of low-density lipoprotein particles.

Lipid composition of human low-density lipoprotein (LDL) and its physicochemical characteristics are relevant for proper functioning of lipid transport in the blood circulation. To explore dynamical and structural features of LDL particles with either a normal or a triglyceride-rich lipid composition we combined coherent and incoherent neutron scattering methods. The investigations were carried out under high hydrostatic pressure (HHP), which is a versatile tool to study the physicochemical behavior of biomolecules in solution at a molecular level. Within both neutron techniques we applied HHP to probe the shape and degree of freedom of the possible motions (within the time windows of 15 and 100 ps) and consequently the flexibility of LDL particles. We found that HHP does not change the types of motion in LDL, but influences the portion of motions participating. Contrary to our assumption that lipoprotein particles, like membranes, are highly sensitive to pressure we determined that LDL copes surprisingly well with high pressure conditions, although the lipid composition, particularly the triglyceride content of the particles, impacts the molecular dynamics and shape arrangement of LDL under pressure.

Investigation of Solid D 2 for UCN Sources.

Solid deuterium (sD2) will be used for the production of ultra-cold neutrons (UCN) in a new generation of UCN sources. Scattering cross sections of UCN in sD2 determine the source yield but until now have not been investigated. We report first results from transmission and scattering experiments with cold, very cold and ultra-cold neutrons on sD2 along with light transmission and Raman scattering studies showing the influence of the sD2 crystal properties.