Kinetic studies on the interactions between glycolipid biosurfactant assembled monolayers and various classes of immunoglobulins using surface plasmon resonance

Kinetic studies on the interactions between self-assembled monolayers of mannosylerythritol lipids (MELs), which are glycolipid biosurfactants abundantly produced by microorganisms, and various classes of immunoglobulins including human IgG, IgA, and IgM were performed using surface plasmon resonance (SPR). The effect of the MEL structure on the binding behavior of HIgG was examined. Assembled monolayers of MEL-A having two acetyl groups on the headgroup gave a high affinity (K_d = 1.7 × 10⁻⁶ M) toward HIgG, while those of MEL-B or MEL-C having only one acetyl group at C-6′ or C-4′ position gave little affinity. Our kinetic analysis revealed that the binding manner of HIgG, HIgA (K_d = 2.4 × 10⁻⁷ M), and HIgM (K_d = 2.2 × 10⁻⁷ M) to the assembled monolayers of MEL-A is not the monovalent mode but the bivalent mode, and both the first and second rate association constants (k_a1, k_a2) increase with an increase in the number of antibody binding sites, while those for dissociation (k_d1, k_d2) changed little. Moreover, we succeeded in directly observing great amounts of HIgG, HIgA, and HIgM bound to MEL-A monolayers using atomic force microscopy (AFM). Finally, we found that MEL-A assembled monolayer binds toward various IgG derived from mouse, pig, rabbit, horse, goat, rat, and bovine as well as human IgG (HIgG), and the only exception was sheep IgG. These results clearly demonstrate that MEL-A assembled monolayers would be useful as noble affinity ligand system for various immunoglobulins.     

Photooxidative mineralization of microorganisms-produced glycolipid biosurfactants by a titania-mediated advanced oxidation process

The photooxidative mineralizations of two microorganisms-produced glycolipid biosurfactants 4-O-(4′,6′-di-O-acetyl-2′,3′-di-O-alkanoyl-β-d-mannopyranosyl)-d-erythritol (MEL-A) and 1-O-(6′-O-acetyl-2′,3′-di-O-alkanoyl-β-d-mannopyranosyl)-d-erythritol (MEL-B) were examined by monitoring the temporal changes in UV absorption, the time profiles of CO₂ evolution and the changes in interfacial tension occurring by an advanced oxidation process in the presence of a metal-oxide (TiO₂). Features of their mineralization are compared to the photomineralization of the anionic dodecylbenzene sulfonate (DBS) surfactant carried out under otherwise identical conditions. The adsorption of surfactants on the TiO₂ surface and the positions of attack of the photogenerated OH radicals on the surfactants’ structure were assessed by molecular orbital (MO) calculations of partial charges and frontier orbital electron densities, respectively. The photodegradation of DBS was faster than the MELs as also evidenced by surface tension measurements, whereas the photomineralization of the anionic DBS surfactant was definitely slower than that of the MEL biosurfactants due to the hydrophobic alkyl chain in the DBS structure. Possible initial mechanistic stages of the photooxidation of MEL-A and MEL-B are proposed based on experimental data and comparison with MO calculations.     

Coacervate formation from natural glycolipid: one acetyl group on the headgroup triggers coacervate-to-vesicle transition

Coacervate (L3 phase) formation of the single component "natural" glycoliped biosurfactant, MEL-A, was observed for the first time by using an optical microscope, a confocal laser scanning microscope (CLSM), and a freeze-fracture electron microscope (FF-TEM). It was also found that only a slight decrease in spontaneous curvature resulting from the absence of one acetyl group on the headgroup induced a drastic morphological change in the 3D self-assembled structure from coacervates (L3 phase) to ordered vesicles (Lalpha phase).     

Thermodynamically stable vesicle formation from glycolipid biosurfactant sponge phase

Thermodynamically stable vesicle (L(alpha1)) formation from glycolipid biosurfactant sponge phase (L(3)) and its mechanism were investigated using a "natural" biocompatible mannosyl-erythritol lipid-A (MEL-A)/L-alpha-dilauroylphosphatidylcholine (DLPC) mixture by varying the composition. The trapping efficiency for calcein and turbidity measurements clearly indicated the existence of three regions: while the trapping efficiencies of the mixed MEL-A/DLPC assemblies at the compositions with X(DLPC)< or =0.1 or X(DLPC)> or =0.8 were almost zero, the mixed assemblies at the compositions with 0.1 or =0.8 were multilamellar vesicles (L(alpha)) with diameter from 2 to 10 microm. Meanwhile, dynamic light scattering (DLS) measurement revealed that the average size of the vesicles at the composition of X(DLPC)=0.3 was 633.2 nm, which is remarkably small compared to other compositions. Moreover, the mixed vesicle solution at the composition of X(DLPC)=0.3 was slightly bluish and turbid and kept its dispersion stability at 25 degrees C for more than 3 months, indicating the formation of a thermodynamically stable vesicle (L(alpha1)). These results exhibited the formation of a thermodynamically stable vesicle (L(alpha1)) with a high dispersibility from the MEL-A/DLPC mixture. The asymmetric distribution of MEL-A and DLPC in the two vesicle monolayers caused by the difference in geometrical structures is very likely to have changed their self-assembled structure from a sponge phase (L(3)) to a thermodynamically stable vesicle (L(alpha1)).     

Interfacial and self-assembly properties of bolaamphiphilic compounds derived from a multifunctional oil

The self-assembly characteristics in aqueous solutions of cationic bolaamphiphiles with systematic changes in their chemical structure is described with respect to their interfacial properties within water and at the air/water interface. Six cationic bolaamphiphiles were synthesized from multifunctional vernonia oil with the following variations: (a) two different alkyl chain lengths connecting the head groups, (b) polar ester or hydrogen bonding amide groups within the hydrophobic domain, and (c) an acetylcholine cationic head group with different conjugation sites to the alkyl chain. Surface tension measurements were used for determining critical aggregation concentration (CAC) values and air/water interfacial parameters such as 'effectiveness', surface excess concentration and area occupied by one molecule in the air/water interface. Fluorescent studies with pyrene were used to characterize CAC properties within the aqueous volume and transmission electron microscopy (TEM) for determining the aggregate structure's size, homogeneity and morphology. A bolaamphiphile molecular structure vs. interfacial property relationship was derived from this data which could be used to determine the molecular structure properties needed to generate interfacial forces to form either spherical vesicles or fibrous networks. The effects of the aliphatic chain length, head group orientation and functional groups within the hydrophobic domain on CAC, surface tension properties and self-aggregate morphology are described. Most bolaamphiphiles studied had CAC values in the 10-190 μM range, while two out of the six were found to assemble into MLM spherical vesicles with diameters ranging up to 120 nm suitable for drug delivery applications. Others formed a gelatinous network of fibers or multi-lamellar vesicles.     

Aqueous-phase behavior of natural glycolipid biosurfactant mannosylerythritol lipid A: sponge, cubic, and lamellar phases

The aqueous-phase behavior of mannosylerythritol lipid A (MEL-A), which is a glycolipid biosurfactant produced from vegetable oils by yeast strains of the genus Pseudozyma, was investigated using polarized optical microscopy, small-angle X-ray scattering (SAXS), and differential scanning calorimetry (DSC). MEL-A was found to self-assemble into a variety of distinctive lyotropic liquid crystals including sponge (L3), bicontinuous cubic (V2), and lamella (Lalpha) phases. On the basis of SAXS measurements, we determined the structure of the liquid crystals. The estimated lattice constant for Lalpha was 3.58 nm. DSC measurement revealed that the phase transition enthalpies from the liquid crystal to the fluid isotropic phase were in the range of 0.22-0.44 kJ/mol. Although the present MEL-A phase diagram closely resembled that obtained from relatively hydrophobic poly(oxyethylene) or fluorinated surfactants, the MEL-A L3 region was spread considerably over a wide temperature range (20-65 degrees C) compared to L3 of those surfactants: this is probably due to the unique structure which is molecularly engineered by microorganisms. In this paper, we clarify the aqueous phase diagram of the natural glycolipid biosurfactant MEL-A, and we suggest that the obtained lyotropic crystals are potentially useful as novel nanostructured biomaterials.     

Synthesis and aggregation behaviour of a new sultaine surfactant

The aggregation properties of a new sultaine surfactant have been studied in buffered aqueous solution at pH 7.4 under controlled condition of osmolarity. Spontaneously formed sultaine vesicles with a mean diameter of about 1 μm can be observed by optical microscopy. The phase behaviour of the surfactant has been investigated by differential scanning calorimetry (DSC) and Nile Red fluorescence. Two critical vesicular concentrations (CVC(1) and CVC(2)) have been fluorimetrically measured, by using pyrene and Nile Red as the fluorescent probes. The two populations of vesicles behave differently as a consequence of their size. The stability of extruded large unilamellar vesicles (LUV) formed slightly above the CVC(1) has been evaluated in the temperature range 25-75°C by following the rate of spontaneous release of entrapped 5(6)-carboxyfluorescein (CF). The stability of the same vesicles at 70°C has also been investigated under osmotic stress obtained by adding NaCl or sucrose to the bulk solution. At a sultaine concentration above the CVC(2) LUV tend to associate and form stable larger closely packed aggregates as suggested by Dynamic Laser Light Scattering and rheological measurements.     

The surface and solution properties of dihexadecyl dimethylammonium bromide

The surface adsorption behavior and solution aggregate microstructure of the dichain cationic surfactant dihexadecyl dimethylammonium bromide (DHDAB) have been studied using small angle neutron scattering (SANS), light scattering, neutron reflectivity (NR), and surface tension (ST). Using a combination of surface tension and neutron reflectivity, the DHDAB equilibrium surface excess at saturation adsorption has been measured as 2.60 +/- 0.05 x 10 (-10) mol.cm (-2). The values obtained by both methods are in good agreement and are consistent with the values reported for other dialkyl chain surfactants. The critical aggregation concentration (CAC) values obtained from both methods (NR and ST) are also in good agreement, with a mean value for the CAC of 4 +/- 2 x 10 (-5) M. The surface equilibrium is relatively slow, and this is attributed to monomer depletion in the near surface region, as a consequence of the long monomer residence times in the surfactant aggregates. The solution aggregate morphology has been determined using a combination of SANS, dynamic light scattering (DLS), cryogenic transmission electron microscopy (CryoTEM), and ultrasmall angle neutron scattering (USANS). Within the concentration range 1.5-80 mM, the aggregates are in the form of bilamellar vesicles with a lamellar " d-spacing" of the order of 900 A. The vesicles are relatively polydisperse with a particle size in the range 2000-4000 A. Above 80 mM, the bilamellar vesicles coexist with an additional L beta lamellar phase.     

CHLOROPHYLL PHOTOSENSITIZED ELECTRON TRANSFER IN PHOSPHOLIPID VESICLE SYSTEMS: COMPARISON OF INSIDE-OUTSIDE ASYMMETRY BETWEEN LARGE AND SMALL UNILAMELLAR VESICLES*

Abstract— We have determined the chlorophyll triplet quenching efficiencies, the chlorophyll cation radical yields and the conversion efficiencies of chlorophyll triplet to radical in large and small unilamellar phosphatidylcholine vesicles (LUV and SUV, respectively) in the presence of electrically-charged electron acceptors (ferricyanide and oxidized cytochrome c) located in either the inner or outer aqueous compartments of the vesicles. Both types of vesicles displayed inside-outside asymmetry, although the properties were reversed. Triplet quenching in SUV was more efficient when ferricyanide was located within the vesicle interior, whereas the reverse was true in LUV. When ferricyanide was located on the outside of the vesicles, the extent of triplet quenching in LUV was about two times that in SUV and the amount of cation radical formed in LUV was about two times that in SUV. Under these conditions, the conversion efficiencies of chlorophyll triplet to radical were 12.2% for LUV and 8.5% for SUV. With cytochrome c as an electron acceptor in negatively charged vesicles (25 mol per cent dixhexadecylphosphate incorporated) similar results were obtained. Again, the triplet quenching and radical yield inside-outside asymmetry properties were reversed between the two types of vesicles, and radical formation efficiencies when cyt c was located outside the vesicles were higher in LUV (11.7%) than in SUV (4.2%). We conclude that the inside-outside asymmetric photochemical behavior of unilamellar phosphatidylcholine vesicles is influenced by factors in addition to the difference in radius of curvature between the inside and outside surfaces. It is suggested that transmembrane electrostatic potentials may be involved. Furthermore, in the present system the properties of LUV were more favorable to photochemical electron transfer product formation than those of SUV.     

Multilayer vesicles and vesicle clusters formed by the fullerene-based surfactant C60(CH3)5K

The self-assembly behavior of a fullerene-based surfactant, C60(CH3)5K, in water was studied using a combination of static and dynamic light scattering, as well as transmission electron microscopy, and compared to that of the compound C60(C6H5)5K. Both fullerene surfactant systems spontaneously assemble into large vesicles consisting of closed spherical shells formed by bilayers, with critical aggregation concentrations (CAC) lower than 10(-6) g ml(-1). At low concentrations, the aggregate sizes of C60(CH3)5K (radius R approximately 26.8 nm) and C60(C6H5)5K (R approximately 17.0 nm) were found to be substantially different from each other, showing that the change of the substituents surrounding the polar cyclopentadienide head group makes it possible to control the size of the resulting aggregates. Furthermore, the C60(CH3)5K vesicles were found to exist in two qualitatively different types of aggregation with a critical reaggregation concentration (CRC) located at 3.30 x 10(-6) g ml(-1). Above the CRC, larger aggregates were observed (R approximately 37.6 nm), showing a more complex form of supramolecular aggregation, e.g., in terms of multi-bilayer vesicles and/or of clusters of bilayer vesicles.     

Monte carlo simulations of self-assembled surfactant aggregates

Monte Carlo simulations provide some insight into the self-assembly of amphiphiles in aqueous environment. A rather simple solvent-free model, with only two adjustable parameters in the effective pair potential, allows one to describe the formation of micelles, stable curved membranes, and metastable vesicles. Characteristic features of the self-assembled aggregates, such as the distribution of the micelle size and the value of the curvature elastic constant for membranes, can be obtained from simulated data. The capability of the simple approach was demonstrated for a surfactant model with three spherical segments. The extension of the simulation to molecules with more segments and branched amphiphiles is straightforward.     

Effect of alpha-lactalbumin on aerosol-OT phase structures in oil/water mixtures

The ability of water-soluble, globular proteins to tune surfactant/oil/water self-assemblies has potential for the formation of biocompatible microemulsions and also plays a role in protein function at biological interfaces. In this work, we examined the effect of the protein alpha-lactalbumin on Aerosol-OT (AOT) phase structures in equivolume mixtures of oil and 0.1 M brine. In this pseudo-ternary system, surfactants are free to move to either oil or water phase to adopt phase structures close to the spontaneous curvature of the surfactants. Using small-angle X-ray scattering, we observed that addition of this protein changed the spontaneous curvature of the surfactant monolayer substantially. In the absence of protein, AOT adopted a negative spontaneous curvature to form spherical w/o microemulsion droplets. When less than 1 wt % of alpha-lactalbumin was added into the system, the w/o droplets became nonspherical and larger in volume, corresponding to an increase in water uptake into the droplets. As the protein-to-surfactant ratio increased, protein, surfactant, and oil increasingly partitioned toward the aqueous phase. There the protein triggered the formation of o/w microemulsions with a positive spontaneous curvature. These protein-containing structures exhibited significant interparticle attraction. We also compared the influence of two oil types, isooctane and cyclohexane, on the protein/surfactant interactions. We propose that the more negative natural curvature of the AOT/cyclohexane monolayer in the absence of protein prevented protein incorporation within organic phase structures and consequently pushed the system self-assembly toward aqueous aggregate formation.     

Evidence for vesicle formation from 1:1 nonionic surfactant span 60 and fatty alcohol mixtures in aqueous ethanol: potential delivery vehicle composition

A study of the self-organization of nonionic surfactant span 60 (sorbitan mono stearate) in presence of fatty alcohol (stearyl, cetyl and lauryl) is presented. When ethanolic solution of the surfactant–fatty alcohol (1:1) mixture is added in water spontaneous large unilamellar vesicles (LUV) are formed which may potentially be useful vehicles for drug delivery purposes. Vesicular suspension has been characterized by transmission electron microscopy, dynamic light scattering, confocal laser scanning microscopy, dye entrapment and release studies. Surface tension measurement indicates the suitability of fatty alcohols towards spontaneous vesicle formation from span 60.     

Counterion-controlled transition of a cationic gemini from submicroscopic to giant vesicles

While much is known about the self-assembly of lipids on nanoscale, our understanding of their biologically relevant mesoscale organization remains incomplete. Here, we show for a cationic gemini lipid a sharp and reversible transition from small vesicles with an average diameter of approximately 40 nm to giant vesicles (GVs) with an average diameter of approximately 11 microm. This transition is dependent on proper [NaCl] and specific temperature. Below this transition and in the vicinity of the air/water interface, a series of mesoscale morphological transitions was observed, revealing complex structures resembling biological membranes. On the basis of microscopy experiments, a tentative [NaCl] versus temperature shape/size phase diagram was constructed. To explain this unprecedented transition, we propose a novel mechanism whereby a specific interaction of Cl(-) counterion with the cationic gemini surfactant initiates the formation of a commensurate solute counterion lattice with low spontaneous curvature. In keeping with the high bending rigidity of NaCl crystal, this tightly associated ionic lattice enslaves membrane curvature and the mesoscale 3-D organization of this lipid.     

Hydrotropic salt promotes anionic surfactant self-assembly into vesicles and ultralong fibers

Molecular self-assembly has become a versatile approach to create complex and functional nanoarchitectures. In this work, the self-assembly behavior of an anionic surfactant (sodium dodecylbenzene sulfonate, SDBS) and a hydrotropic salt (benzylamine hydrochloride, BzCl) in aqueous solution is investigated. Benzylamine hydrochloride is found to facilitate close packing of surfactants in the aggregates, inducing the structural transformation from SDBS micelles into unilamellar vesicles, and multilamellar vesicles. The multilamellar vesicles can transform into macroscale fibers, which are long enough to be visualized by the naked eye. Particularly, these fibers are robust enough to be conveniently separated from the surfactant solution. The combined effect of non-covalent interactions (e.g., hydrophobic effect, electrostatic attractions, and π-π interactions) is supposed to be responsible for the robustness of these self-assembled aggregates, in which π-π interactions provide the directional driving force for one-dimensional fiber formation.     

Bending moduli and spontaneous curvature of the monolayer in a surfactant bilayer

We developed a method to evaluate the mechanical properties of the monolayers in symmetric surfactant bilayers using self-consistent field theory. A specific boundary condition is used to impose the same curvature onto the two opposing monolayers at the surfactant chemical potential equal to that of the corresponding homogeneously curved bilayer. Typically, the spontaneous monolayer curvature not equal 0 and its value depend on the surfactant architecture. This is of importance for the thermodynamics and topology of lamellar surfactant phases. Furthermore, it may be relevant in processes involving biological membranes, for example, the fusion and budding of vesicles and the incorporation of proteins in lipid bilayers.     

Experimental observations on the scaling of adsorption isotherms for nonionic surfactants at a hydrophobic solid-water interface

The self-assembly of nonionic surfactants in bulk solution and on hydrophobic surfaces is driven by the same intermolecular interactions, yet their relationship is not clear. While there are abundant experimental and theoretical studies for self-assembly in bulk solution and at the air-water interface, there are only few systematic studies for hydrophobic solid-water interfaces. In this work, we have used optical reflectometry to measure adsorption isotherms of seven different nonionic alkyl polyethoxylate surfactants (CH3(CH2)I-1(OCH2CH2)JOH, referred to as CIEJ surfactants, with I = 10-14 and J = 3-8), on hydrophobic, chemically homogeneous self-assembled monolayers of octadecyltrichlorosilane. Systematic changes in the adsorption isotherms are observed for variations in the surfactant molecular structure. The maximum surface excess concentration decreases (and minimum area/molecule increases) with the square root of the number of ethoxylate units in the surfactant (J). The adsorption isotherms of all surfactants collapse onto the same curve when the bulk and surface excess concentrations are rescaled by the bulk critical aggregation concentration (CAC) and the maximum surface excess concentration. In an accompanying paper we compare these experimental results with the predictions of a unified model developed for self-assembly of nonionic surfactants in bulk solution and on interfaces.     

Membrane dynamics of the amphiphilic siderophore, acinetoferrin

Acinetobacter haemolyticus is an antibiotic resistant, pathogenic bacterium responsible for an increasing number of hospital infections. Acinetoferrin (Af), the amphiphilic siderophore isolated from this organism, contains two unusual trans-2-octenoyl hydrocarbon chains reminiscent of a phospholipid structural motif. Here, we have investigated the membrane affinity of Af and its iron complex, Fe-Af, using small and large unilamellar phospholipid vesicles (SUV and LUV) as model membranes. Af shows a high membrane affinity with a partition coefficient, K(x)= 6.8 x 10(5). Membrane partitioning and trans-membrane flip-flop of Fe-Af have also been studied via fluorescence quenching of specifically labeled vesicle leaflets and (1)H NMR line-broadening techniques. Fe-Af is found to rapidly redistribute between lipid and aqueous phases with dissociation/partitioning rates of k(off) = 29 s(-1) and k(on) = 2.4 x 10(4) M(-1) s(-1), respectively. Upon binding iron, the membrane affinity of Af is reduced 30-fold to K'(x) = 2.2 x 10(4) for Fe-Af. In addition, trans-membrane flip-flop of Fe-Af occurs with a rate constant, k(p) = 1.2 x 10(-3) s(-1), with egg-PC LUV and a half-life time around 10 min with DMPC SUV. These properties are due to the phospholipid-like conformation of Af and the more extended conformation of Fe-Af that is enforced by iron binding. Remarkable similarities and differences between Af and another amphiphilic siderophore, marinobactin E, are discussed. The potential biological implications of Af and Fe-Af are also addressed. Our approaches using inner- and outer-leaflet-labeled fluorescent vesicles and (1)H NMR line-broadening techniques to discern Af-mediated membrane partitioning and trans-membrane diffusion are amenable to similar studies for other paramagnetic amphiphiles.     

Sugar-derived tricatenar catanionic surfactant: synthesis, self-assembly properties, and hydrophilic probe encapsulation by vesicles

A new sugar-derived tricatenar catanionic surfactant (TriCat) was developed to obtain stable vesicles that could be exploited for drug encapsulation. The presence of the sugar moiety led to the formation of highly hydrophilic stoichiometric catanionic surfactant systems. The three hydrophobic chains permitted vesicles to form spontaneously. The self-assembly properties (morphology, size, and stability) of TriCat were examined in water and in buffer solution. Encapsulation studies of a hydrophilic probe, arbutin, commonly used in cosmetics for its whitening properties, were performed to check the impermeability of the vesicle bilayer. The enhancement of hydrophobic forces by the three chains of TriCat prevented surfactant equilibrium between the bilayer and the solution and enabled the probe to be retained in the aqueous cavity of the vesicles for at least 30 h. Thus, the present study suggests that this tricatenar catanionic surfactant could be a promising delivery system for hydrophilic drugs.     

Lamellar gels and spontaneous vesicles in catanionic surfactant mixtures

Caillé analysis of the small-angle X-ray line shape of the lamellar phase of 7:3 wt/wt cetyltrimethylammonium tosylate (CTAT)/sodium dodecylbenzene sulfonate (SDBS) bilayers shows that the bending elastic constant is kappa = (0.62 +/- 0.09)k(B)T. From this and previous results, the Gaussian curvature constant is kappa = (-0.9 +/- 0.2)k(B)T. For 13:7 wt/wt CTAT/SDBS bilayers, the measured bending elasticity decreases with increasing water dilution, in good agreement with predictions based on renormalization theory, giving kappa(o) = 0.28k(B)T. These results show that surfactant mixing is sufficient to make kappa approximately k(B)T, which promotes strong, Helfrich-type repulsion between bilayers that can dominate the van der Waals attraction. These are necessary conditions for spontaneous vesicles to be equilibrium structures. The measurements of the bending elasticity are confirmed by the transition of the lamellar phase of CTAT/SDBS from a turbid, viscoelastic gel to a translucent fluid as the water fraction is decreased below 40 wt %. Freeze-fracture electron microscopy shows that the gel is characterized by spherulite defects made possible by spontaneous bilayer curvature and low bending elasticity. This lamellar gel phase is common to a number of catanionic surfactant mixtures, suggesting that low bending elasticity and spontaneous curvature are typical of these mixtures that form spontaneous vesicles.