Adsorption and self-assembly properties of the plant based biosurfactant, Glycyrrhizic acid.

There is an increased interest in the use of natural surfactant as replacements for synthetic surfactants due to their biosustainable and biocompatible properties. A category of natural surfactants which are attracting much current interest is the triterpenoid saponins; surface active components found extensively in a wide range of plant species. A wide range of different saponin structures exist, depending upon the plant species they are extracted from; but regardless of the variation in structural details they are all highly surface active glycosides. Greater exploitation and application requires a characterisation and understanding of their basic adsorption and self-assembly properties. HYPOTHESIS: Glycyrrhizic acid, extracted from Licorice root, is a monodesmosidic triterpenoid saponin. It is widely used in cosmetic and pharmaceutical applications due to its anti-inflammatory properties, and is an ingredient in foods as a sweetener additive. It has an additional attraction due to its gel forming properties at relatively low concentrations. Although it has attracted much recent attention, many of its basic surface active characteristics, adsorption and self-assembly, remain relatively unexplored. How the structure of the Glycyrrhizic acid saponin affects its surface active properties and the impact of gelation on these properties are important considerations, and to investigate these are the focus of the study. EXPERIMENTS: In this paper the adsorption properties at the air-water interface and the self-assembly in solution have been investigated using by neutron reflectivity and small angle neutron scattering; in non-gelling and gelling conditions. FINDINGS: The adsorption isotherm is determined in water and in the presence of gelling additives, and compared with the adsorption behaviour of other saponins. Gelation has minimal impact on the adsorption; apart from producing a rougher surface with a surface texture on a macroscopic length scale. Globular micelles are formed in aqueous solution with modest anisotropy, and are compared with the structure of other saponin micelles. The addition of gelling agents results in only minimal micelle growth, and the solutions remain isotropic under applied shear flow.

Mixing Natural and Synthetic Surfactants: Co-Adsorption of Triterpenoid Saponins and Sodium Dodecyl Sulfate at the Air-Water Interface.

Saponins are highly surface active glycosides, derived from a wide range of plant species. Their ability to produce stable foams and emulsions has stimulated their applications in beverages, foods, and cosmetics. To explore a wider range of potential applications, their surface mixing properties with conventional surfactants have been investigated. The competitive adsorption of the triterpenoid saponin escin with an anionic surfactant sodium dodecyl sulfate, SDS, at the air-water interface has been studied by neutron reflectivity, NR, and surface tension. The NR measurements, at concentrations above the mixed critical micelle concentration, demonstrate the impact of the relative surface activities of the two components. The surface mixing is highly nonideal and can be described quantitatively by the pseudophase approximation with the inclusion of the quadratic and cubic terms in the excess free energy of mixing. Hence, the surface mixing is highly asymmetrical and reflects both the electrostatic and steric contributions to the intermolecular interactions. The relative importance of the steric contribution is reinforced by the observation that the micelle mixing is even more nonideal than the surface mixing. The mixing properties result in the surface adsorption being largely dominated by the SDS over the composition and concentration range explored. The results and their interpretation provide an important insight into the wider potential for mixing saponins with more conventional surfactants.

Surfactant/biosurfactant mixing: Adsorption of saponin/nonionic surfactant mixtures at the air-water interface.

Saponins are naturally occurring biosurfactants present in a wide range of plant species. They are highly surface active glycosides, and are used to stabilise foams and emulsions in foods, beverages and cosmetics. They have great potential for an even wider range of applications, especially when mixed with different synthetic surfactants. Understanding those mixing properties are key to the exploitation of saponins in that wider range of potential applications. The surface adsorption properties of the saponin, escin, with two conventional nonionic surfactants, polyethylene glycol surfactants, have been studied at the air-water interface using neutron reflectivity, NR, and surface tension, ST. Although the saponin and polyethylene glycol, CnEOm, surfactants are both nonionic the disparity in the relative surface activities and packing constraints result in non-ideal mixing. Comparison with the predictions of the pseudo phase approximation requires the inclusion of the quadratic, cubic and quartic terms in the expansion of the excess free energy of mixing to explain the variations in the surface composition. For escin/pentaethylene glycol monododecyl ether, C12EO5, the interaction is attractive and close to ideal. For escin/octaethylene glycol monododecyl ether, C12EO8, it is repulsive and close to the criteria for demixing. The differences in mixing behaviour are attributed to greater packing constraints imposed by the larger ethylene oxide headgroup of the C12EO8 compared to C12EO5.

Saponin Adsorption at the Air-Water Interface-Neutron Reflectivity and Surface Tension Study.

Saponins are a large group of glycosides present in many plant species. They exhibit high surface activity, which arises from a hydrophobic scaffold of triterpenoid or steroid groups and attached hydrophilic saccharide chains. The diversity of molecular structures, present in various plants, gives rise to a rich variety of physicochemical properties and biological activity and results in a wide range of applications in foods, cosmetics, medicine, and several other industrial sectors. Saponin surface activity is a key property in such applications and here the adsorption of three triterpenoid saponins, escin, tea saponins, and Quillaja saponin, is studied at the air-water interface by neutron reflectivity and surface tension. All these saponins form adsorption layers with very high surface visco-elasticity. The structure of the adsorbed layers has been determined from the neutron reflectivity data and is related to the molecular structure of the saponins. The results indicate that the structure of the saturated adsorption layers is governed by densely packed hydrophilic saccharide groups. The tight molecular packing and the strong hydrogen bonds between the neighboring saccharide groups are the main reasons for the unusual rheological properties of the saponin adsorption layers.

Neutron Scattering Studies of the Effects of Formulating Amphotericin B with Cholesteryl Sulfate on the Drug's Interactions with Phospholipid and Phospholipid-Sterol Membranes.

Langmuir surface pressure, small-angle neutron scattering (SANS), and neutron reflectivity (NR) studies have been performed to determine how formulation of the antifungal drug amphotericin B (AmB), with sodium cholesteryl sulfate (SCS)-as in Amphotec-affects its interactions with ergosterol-containing (model fungal cell) and cholesterol-containing (model mammalian cell) membranes. The effects of mixing AmB in 1:1 molar ratio with cholesteryl sulfate (yielding AmB-SCS micelles) are compared against those of free AmB, using monolayers and bilayers formed from palmitoyloleoylphosphatidylcholine (POPC) in the absence and presence of 30 mol % ergosterol or cholesterol, in all cases employing a 1:0.05 molar ratio of lipid:AmB. Analyses of the (bilayer) SANS and (monolayer) NR data indicate that the equilibrium changes in membrane structure induced in sterol-free and sterol-containing membranes are the same for free AmB and AmB-SCS. Stopped-flow SANS experiments, however, reveal that the structural changes to vesicle membranes occur far more rapidly following exposure to AmB-SCS vs free drug, with the kinetics of these changes varying with membrane composition. With POPC vesicles, the structural changes induced by AmB-SCS become apparent only after several minutes, and equilibrium is reached after ∼30 min. The corresponding onset of changes in POPC-ergosterol and POPC-cholesterol vesicles, however, occurs within ∼5 s, with equilibrium reached after 10 and 120 s, respectively. The rate of insertion of AmB into POPC-sterol membranes is thus increased through formulation as AmB-SCS. Moreover, the differences in monolayer surface pressure and SANS structure-change equilibration times suggest significant rearrangement of AmB within these membranes following insertion. The reduced times to equilibrium for the POPC-ergosterol vs POPC-cholesterol systems are consistent with the known differences in affinity of AmB for these two sterols, and the reduced time to equilibrium for AmB-SCS interaction with POPC-ergosterol membranes vs that for free AmB is consistent with the reduced host toxicity of Amphotec.

Adsorption of Bovine Serum Albumin (BSA) at the Oil/Water Interface: A Neutron Reflection Study.

The structure of the adsorbed protein layer at the oil/water interface is essential to the understanding of the role of proteins in emulsion stabilization, and it is important to glean the mechanistic events of protein adsorption at such buried interfaces. This article reports on a novel experimental methodology for probing protein adsorption at the buried oil/water interface. Neutron reflectivity was used with a carefully selected set of isotopic contrasts to study the adsorption of bovine serum albumin (BSA) at the hexadecane/water interface, and the results were compared to those for the air/water interface. The adsorption isotherm was determined at the isoelectric point, and the results showed that a higher degree of adsorption could be achieved at the more hydrophobic interface. The adsorbed BSA molecules formed a monolayer on the aqueous side of the interface. The molecules in this layer were partially denatured by the presence of oil, and once released from the spatial constraint by the globular framework they were free to establish more favorable interactions with the hydrophobic medium. Thus, a loose layer extending toward the oil phase was clearly observed, resulting in an overall broader interface. By analogy to the air/water interface, as the concentration of BSA increased to 1.0 mg mL(-1) a secondary layer extending toward the aqueous phase was observed, possibly resulting from the steric repulsion upon the saturation of the primary monolayer. Results clearly indicate a more compact arrangement of molecules at the oil/water interface: this must be caused by the loss of the globular structure as a consequence of the denaturing action of the hexadecane.

Solution pH and oligoamine molecular weight dependence of the transition from monolayer to multilayer adsorption at the air-water interface from sodium dodecyl sulfate/oligoamine mixtures.

Neutron reflectivity and surface tension have been used to investigate the solution pH and oligoamine molecular weight dependence of the adsorption of sodium dodecyl sulfate (SDS)/oligoamine mixtures at the air-water interface. For diethylenetriamine, triamine, or triethylenetetramine, tetramine mixed with SDS, there is monolayer adsorption at pH 7 and 10, and multilayer adsorption at pH 3. For the slightly higher molecular weight tetraethylenepentamine, pentamine, and pentaethylenehexamine, hexamine, the adsorption is in the form of a monolayer at pH 3 and multilayers at pH 7 and 10. Hence, there is a pH driven transition from monolayer to multilayer adsorption, which shifts from low pH to higher pH as the oligoamine molecular weight increases from tetramine to pentamine. This results from the relative balance between the electrostatic attraction between the SDS and amine nitrogen group which decreases as the charge density decreases with increasing pH, the ion-dipole interaction between the amine nitrogen and SDS sulfate group which is dominant at higher pH, and the hydrophobic interalkyl chain interaction between bound SDS molecules which changes with oligoamine molecular weight.

Kinetics of surfactant desorption at an air-solution interface.

The kinetics of re-equilibration of the anionic surfactant sodium dodecylbenzene sulfonate at the air-solution interface have been studied using neutron reflectivity. The experimental arrangement incorporates a novel flow cell in which the subphase can be exchanged (diluted) using a laminar flow while the surface region remains unaltered. The rate of the re-equilibration is relatively slow and occurs over many tens of minutes, which is comparable with the dilution time scale of approximately 10-30 min. A detailed mathematical model, in which the rate of the desorption is determined by transport through a near-surface diffusion layer into a diluted bulk solution below, is developed and provides a good description of the time-dependent adsorption data. A key parameter of the model is the ratio of the depth of the diffusion layer, H(c), to the depth of the fluid, H(f), and we find that this is related to the reduced Péclet number, Pe*, for the system, via H(c)/H(f) = C/Pe*(1/2). Although from a highly idealized experimental arrangement, the results provide an important insight into the "rinse mechanism", which is applicable to a wide variety of domestic and industrial circumstances.

Structural studies of surfactants at the oil-water interface by neutron reflectometery.

We report information regarding the structure at the interface between hexadecane and an aqueous solution of trimethyl tetradecyl ammonium bromide (C14TAB) with appropriate deuterium labeling. We also report the role of the headgroup size and the charge on structures at the oil-water interface by comparing the data for C14TAB to that for a deuterated trimethyl tetradecyl ammonium sulfate (C14TAS) solution at the oil-water interface. As the charge on the counterion increases, this results in a reduction in the limiting area per molecule and an increase in the adsorbed amount at the oil-water interface.

Protein density profile at the interface of water with oligo(ethylene glycol) self-assembled monolayers.

We determined the density profile of a high-molecular-weight globular protein (bovine serum albumin, BSA) solution at the methoxy tri(ethylene glycol)-terminated undecanethiol SAM/protein solution interface by neutron reflectivity measurements. Information about the interactions between oligo(ethylene glycol) (OEG)-terminated self-assembled monolayers (SAMs) and proteins is derived from the analysis of the structure of the solid-liquid interface. The fitting results reveal oscillations of the protein density around the bulk value with decaying amplitude on a length scale of 4 to 5 nm. The amplitude, phase, period, and decay length are found to vary only slightly with temperature and the ionic strength of the protein solution. Adsorption is reversible within the limits of detection, which suggests that the hydrated ethylene glycol surface inhibits the protein from unfolding and irreversible bonding. The insensitivity of BSA adsorption toward the ionic strength of the solution contrasts with observations in surface force experiments with a fibrinogen-coated AFM tip, where electrostatic repulsion dominates theprotein/OEG SAM interaction. As reported previously, irreversible BSA adsorption takes place below 283 K, which we interpret as indicative of the presence of dynamic effects in the protein resistance of short-chain OEG-terminated surfaces.

Neutron reflectivity study of alkylated azacrown ether at the air-liquid and the liquid-liquid interfaces.

We report the neutron reflectometry study of partially deuterated di-hexadecyl-diaza-18-crown-6 ether (d-ACE-16) at the air-water and the oil-water interfaces. At the air-water interface, the thickness of the monolayer is smaller than that for a fully stretched d-ACE-16 molecule, suggesting a tilt of the alkyl chains with respect to the normal. At the oil-water interface, the same molecules were found to form a more diffuse layer distribution stretching across both sides of the interface. On the oil side, the molecules are densely packed within a thickness of 17 A, the hydrophilic part of the molecule with the azacrown ether ring being immersed in the adjacent aqueous side of the interface. The latter consists of a thick 38 A layer comprising staggered, loosely adsorbed d-ACE-16 molecules. With increasing spread amount, the adsorbed layer density increases at the oil side until saturation at ca. 2.25 x 10(-6) mol m(-2), above which the layer collapses.

Effects of surface pressure on the structure of the monolayer formed at the air/water interface by a non-ionic surfactant.

The monolayer formed at an air/water interface by the synthetic non-ionic surfactant, 1,2-di-O-octadecyl-rac-glyceryl-3-(omega-methoxydodecakis (ethylene glycol)) (2C18E12) has been characterized using Langmuir trough measurements, Brewster angle microscopy (BAM), and neutron reflectometry. The BAM and reflectometry studies were performed at four different surface pressures (pi) in the range 15-40 mN/m. The BAM studies (which give information on the in-plane organisation of the surfactant layer) demonstrate that the 2C18E12 molecules are arranged on the water surface to form distinct, approximately circular, 5 microm diameter domains. As the surface pressure is increased these domains retain their size and shape but are made progressively more close-packed, such that the monolayer is made more or less complete at pi=40 mN/m. The neutron reflectometry measurements were made to determine the structure of the interfacial surfactant layer at pi=15, 28, 34 and 40 mN/m, providing information on the thickness of the 2C18E12 alkyl chains', head groups' and associated solvent distributions (measured along the surface normal), along with the separations between these distributions, and the effective interfacial area per molecule. Partial structure factor analyses of the reflectivity data show that the effective interfacial area occupied decreases from 217 A2 per 2C18E12 molecule at pi=15 mN/m down to 102 A2 at pi=40 mN/m. There are concomitant increases in the widths of the surfactant's alkyl chains' and head groups' distributions (modelled as Gaussians), with the former rising from 12 A (at pi=15 mN/m) up to 19 A (at pi=40 mN/m) and the latter rising from 13 A (at pi=15 mN/m) up to 24 A (at pi=40 mN/m). The compression of the monolayer is also shown to give rise to an increased surface roughness, some of which is due to the thermal roughness caused by capillary waves, but with a significant contribution also coming from the intrinsic/structural disorder in the monolayer. At all surface pressures studied, the alkyl chains and head groups of the 2C18E12 are found to exhibit a significant overlap, and this increases with increasing pi. Given the various trends noted on how the structure of the 2C18E12 monolayer changes as a function of pi, we extrapolate to consider the structure of the monolayer at pi>40 mN/m (making comparison with its single chain (CnEm) counterparts) and then relate these findings to the observations recorded on the structure and solute entrapment efficiency of 2C18E12 vesicles.

The structure of zwitterionic phosphocholine surfactant monolayers.

The structure of a zwitterionic phosphocholine (PC) surfactant monolayer adsorbed on the surface of water has been determined using neutron reflectivity in combination with H/D isotopic substitution. The most significant results of this study are the level of hydration of the PC headgroup and the lack of dehydration with increasing temperature and salt addition. The fraction of the alkyl chain (f(c)) immersed in water for all three chain isomers studied was found to be around 0.15, suggesting that the PC headgroup geometries influenced not only the headgroup hydration but also the degree of immersion of the alkyl chain in water. At the critical micelle concentration (CMC), the number of water molecules associated with the PC headgroup in C(m)PC (m = 12, 14, 16) was on order of 15. This value was significantly greater than that obtained for nonionic and ionic surfactants with similar limiting area per molecule at the CMC (A(cmc)). However, the fraction of the chain immersed in water for the ionic and nonionic surfactants was much greater. This suggests that the unique surface biocompatibility of PC surfactants arises from their strong affinity for water, and the relatively low fraction of mixing with the alkyl chain arises from the higher structural order within the PC monolayer. As surface coverage decreased, the number of water molecules associated with each PC headgroup increased, but f(c) remained constant for all the surfactants. This observation was consistent with the small variation in the thickness of the headgroup region, and the entire layer changed little with surfactant concentration. This is attributed to the role of PC headgroup geometries to maintain the conformational order within the layer as packing density varies. Further structural analysis based on a kinematic approach showed that, as the chain length was increased from C12 to C14 to C16 at the CMC, the angle of tilt for the alkyl chain increased from 40 degrees to 48 degrees to 53 degrees , respectively, whereas the thickness of the whole layer and that of the PC head region was largely constant. The almost vertical projection of the PC headgroup from these single alkyl chain surfactants is in sharp contrast to its strongly tilted conformation, as reported for dichain phospholipids such as dipalmitoyl glycerol phosphocholine (DPPC).

Adsorption of single chain zwitterionic phosphocholine surfactants: effects of length of alkyl chain and head group linker.

The adsorption of a range of single chain zwitterionic phosphocholine surfactants (C(n)P(m)C) at the air/liquid interface has been studied by a combination of surface tension and neutron reflectivity. The critical micellar concentration (CMC) for C(n)PC (or C(n)P(2)C), where n varied from 12, 14 to 16, was found to be 0.91, 0.14, and 1.2 x 10(-2) mM respectively, and followed the same trend as observed for other zwitterionic and non-ionic surfactants. The area per molecule at the CMC, A(cmc), for C(n)PC was found to remain constant between 50 and 53 A(2), indicating that the increase in the alkyl chain length had little effect on A(cmc) at the interface. The neutron reflection measurement also showed an almost constant layer thickness (tau) of 20+/-2 A from all the alkyl chain deuterated PC surfactants (dC(n)hPC) in null reflecting water (NRW), suggesting that the alkyl chains of the surfactant responded to changes in either chain length or solution concentration by varying their angle of tilt. In contrast, increasing the length of head group linker between P and N atoms in C(12)P(m)C, where m=2, 4, to 6, resulted in a much slower decrease of CMC from 0.91, 0.7, to 0.5 mM, consistent with a different contribution to the free energy of micellization. A(cmc) for C(12)P(m)C did not vary when m was increased from 2 to 4, and this observation together with the thickness of the head group region indicated an almost perpendicular projection of the head group in C(12)P(2)C and C(12)P(4)C. A further increase in m to 6 resulted in an A(cmc) of 70 A(2). This increase in A(cmc) however did not result in any change in either the total layer thickness or the fraction of the head group region submerged in the aqueous subphase, suggesting that the head group in C(12)P(6)C was bent away from the surface normal direction. Both increase in temperature from 25 to 40 degrees C and the addition of 0.1 M NaCl had little effect on the area per molecule or the thickness of C(12)P(m)C surfactant layer, showing that the C(12)P(m)C series behaved like C(n)P(2)C series. The main conclusion from this study is that for all the C(n)P(m)C surfactants studied, change in m or n has little effect on the total thickness, the thickness of the alkyl chain or that of the head group region.

Responsive brushes and gels as components of soft nanotechnology.

Progress in the development of generic molecular devices based on responsive polymers is discussed. Characterisation of specially synthesised polyelectrolyte gels, "grafted from" brushes and triblock copolymers is reported. A Landolt pH-oscillator, based on bromate/ sulfite/ferrocyanide, with a room temperature period of 20 min and a range of 3.1 < pH < 7.0, has been used to drive periodic oscillations in volume in a pH responsive hydrogel. The gel is coupled to the reaction and changes volume by a factor of at least 6. A continuously stirred, constant volume, tank reactor was set-up on an optical microscope and the reaction pH and gel size monitored. The cyclic force generation of this system has been measured directly in a modified JKR experiment. The responsive nature of polyelectrolyte brushes, grown by surface initiated ATRP, have been characterised by scanning force microscopy, neutron reflectometry and single molecule force measurements. Triblock copolymers, based on hydrophobic end-blocks and either polyacid or polybase mid-block, have been used to produce polymer gels where the deformation of the molecules can be followed directly by SAXS and a correlation between molecular shape change and macroscopic deformation has been established. The three systems studied allow both the macroscopic and a molecular response to be investigated independently for the crosslinked gels and the brushes. The triblock copolymers demonstrate that the individual response of the polyelectrolyte molecules scale-up to give the macroscopic response of the system in an oscillating chemical reaction.