Francisco Monroy

Ultrathin Shell Double Emulsion Templated Giant Unilamellar Lipid Vesicles with Controlled Microdomain Formation

A microfluidic approach is reported for the high-throughput, continuous production of giant unilamellar vesicles (GUVs) using water-in-oil-in-water double emulsion drops as templates. Importantly, these emulsion drops have ultrathin shells; this minimizes the amount of residual solvent that remains trapped within the GUV membrane, overcoming a major limitation of typical microfluidic approaches for GUV fabrication. This approach enables the formation of microdomains, characterized by different lipid compositions and structures within the GUV membranes. This work therefore demonstrates a straightforward and versatile approach to GUV fabrication with precise control over the GUV size, lipid composition and the formation of microdomains within the GUV membrane.

Dilational viscoelasticity of surfactant monolayers

Abstract The dilational viscoelasticity of the surface of cationic surfactant solutions was studied using an excited capillary waves technique. It was found that the dilational viscosity is negative in some cases, depending on the surfactant chain length, bulk concentration and frequency of the waves. It was checked that the response of the monolayer is linear, thus excluding non-linear rheological behavior or surface modes coupling. No satisfactory model was found to explain this behavior.

Stiffening Effect of Cholesterol on Disordered Lipid Phases: A Combined Neutron Spin Echo + Dynamic Light Scattering Analysis of the Bending Elasticity of Large Unilamellar Vesicles

In this study, the center-of-mass diffusion and shape fluctuations of large unilamellar 1-palmitoyl-2-oleyl-sn-glycero-phosphatidylcholine vesicles prepared by extrusion are studied by means of neutron spin echo in combination with dynamic light scattering. The intermediate scattering functions were measured for several different values of the momentum transfer, q, and for different cholesterol contents in the membrane. The combined analysis of neutron spin echo and dynamic light scattering data allows calculation of the bending elastic constant, kappa, of the vesicle bilayer. A stiffening effect monitored as an increase of kappa with increasing cholesterol molar ratio is demonstrated by these measurements.

DILATATIONAL RHEOLOGY OF INSOLUBLE POLYMER MONOLAYERS : POLY(VINYLACETATE)

The dilatational rheology of the poly~vinylacetate! monolayer onto an aqueous subphase with pH52.0 has been studied between 1 °C and 25 °C. The combination of several techniques, relaxation after a step compression, oscillatory barrier experiments, electrocapillary waves, and surface light scattering ~SLS! by thermal capillary waves, has allowed us to explore a broad frequency range. The relaxation experiments show multiexponential decay curves, whose complexity increases with decreasing the temperature. A regularization technique has been used to obtain the relaxation spectra from the relaxation curves and the dilatational viscoelastic parameters have been calculated from the spectra. The oscillatory barrier experiments confirm the results obtained from the step compression experiments. The dilatational viscosity increases very steeply in the frequency range 0.1–0.001 Hz. The shapes of the relaxation spectra follow the qualitative trends predicted a model recently proposed by Noskov @Colloid Polym. Sci. 273, 263 ~1995!#. The temperature dependence of the fundamental relaxation time follows a Williams-Landel-Ferry equation above 14 °C. These results correspond to the many-chain dynamics regime. The kilohertz region has been explored by the SLS technique. These results are compatible with the existence of a single Maxwell mode, with a relaxation time that has an Arrhenius-type temperature dependence. In the intermediate-frequency regime ~10 Hz to 2 kHz! a further Maxwell process is found. It might correspond to the dynamics of loops and tails out of the surface plane. @S1063-651X~98!08012-X#

Shear rheology of lipid monolayers and insights on membrane fluidity.

The concept of membrane fluidity usually refers to a high molecular mobility inside the lipid bilayer which enables lateral diffusion of embedded proteins. Fluids have the ability to flow under an applied shear stress whereas solids resist shear deformations. Biological membranes require both properties for their function: high lateral fluidity and structural rigidity. Consequently, an adequate account must include, in addition to viscosity, the possibility for a nonzero shear modulus. This knowledge is still lacking as measurements of membrane shear properties have remained incomplete so far. In the present contribution we report a surface shear rheology study of different lipid monolayers that model distinct biologically relevant situations. The results evidence a large variety of mechanical behavior under lateral shear flow.

Polymersomes: smart vesicles of tunable rigidity and permeability

We report an experimental study on the mechanical and permeability properties of giant polymersomes made of diblock (PBD–PEO) and triblock (PEO–PPO–PEO) copolymers. These polymer amphiphiles bear the architecture and macromolecular dimensions adequate for assembling stable flat bilayers with a different hydrophobicity. In the highly hydrophobic case (PBD–PEO) an extremely compact membrane is formed, resulting in rigid polymersomes which represent a permeability barrier against solute transport across. In the case of water soluble PEO–PPO–PEO triblock copolymers, the bilayer structure is less stable in favour of the micellar state; therefore giant vesicles can be solely formed at large PPO contents. These cases (Pluronics® L121 and its mixtures with P85 and P105) are characterised by a much lower chain entangling than highly hydrophobic membranes, their polymersomes being softer than those based on PBD–PEO. Pluronic-based polymersomes are also found to be highly permeable to hydrophilic solutes, even remaining undamaged in the case of an extreme osmotic shock. This high permeability together with their high flexibility endows Pluronics polymersomes smart core/shell properties ideal to catch large biomolecules inside and able to resist under osmotic and mechanical stresses.

Polymer monolayers with a small viscoelastic linear regime: equilibrium and rheology of poly(octadecyl acrylate) and poly(vinyl stearate).

The equilibrium properties of monolayers of two polymers: poly(octadecyl acrylate) and poly(vinyl stearate) on water have been measured. The surface pressure (Pi) versus surface concentration (Gamma) curves indicate that the water-air interface is a poor solvent for both polymers. The thermal expansivity shows a sharp change near room temperature. This behavior is typical of a glass transition; this is the first time that such a plot is observed for Langmuir films. The Pi vs Gamma curves measured by the continuous compression method show strong anisotropy effects. They also show that the monolayer is brought into nonequilibrium states depending on the compression rate. Within the linear regime, the relaxation experiments were bimodal. The longest relaxation time strongly increases as T is decreased, which might be compatible with the high increase of viscosity in the glass transition. The oscillatory barrier experiments showed that the maximum strain of the linear regime is smaller than 3% for both monolayers. The Fourier-transform analysis of the oscillatory experiments beyond the linear regime points out the contribution of different harmonics in the response function. Oscillations in the nonlinear regime show hysteresis cycles. The results obtained indicate that some of the previously published data for these polymer monolayers correspond to nonequilibrium states.

Dilational rheology of Langmuir polymer monolayers: Poor-solvent conditions

The viscoelastic moduli (elasticity and dilational viscosity) of monolayers of P4HS have been studied over a broad frequency range (0.1 mHz–200 kHz) using a combination of relaxation and capillary waves techniques. The analysis of the surface pressure, the elasticity and the viscosity in the semidilute regime show that the air–water interface is a poor (near-Θ) solvent for the monolayer. The results of viscoelastic moduli show that there is a broad relaxation process in the low-frequency range (ω<1 Hz), and another very intense relaxation process centered in the kHz region. This behavior contrasts with the one previously found for PVAc, a polymer for which the interface is a good solvent. For PVAc the relaxation found at low frequencies is much narrower, and two processes are clearly distinguished at higher frequencies: one centered at 500 Hz and another one at 40 kHz.

Ceramide: from lateral segregation to mechanical stress.

Ceramide is a sphingolipid present in eukaryotic cells that laterally segregates into solid domains in model lipid membranes. Imaging has provided a wealth of structural information useful to understand some of the physical properties of these domains. In biological membranes, ceramide is formed on one of the membrane leaflets by enzymatic cleavage of sphyngomyelin. Ceramide, with a smaller head size than its parent compound sphyngomyelin, induces an asymmetric membrane tension and segregates into highly ordered domains that have a much high shear viscosity than that of the surrounding lipids. These physical properties, together with the rapid transmembrane flip-flop of the locally produced ceramide, trigger a sequence of membrane perturbations that could explain the molecular mechanism by which ceramide mediates different cell responses. In this review we will try to establish a connection between the physical membrane transformations in model systems known to occur upon ceramide formation and some physiologically relevant process in which ceramide is known to participate.

A combined action of pulmonary surfactant proteins SP-B and SP-C modulates permeability and dynamics of phospholipid membranes.

Proteins SP-B and SP-C are essential to promote formation of surface-active films at the respiratory interface, but their mechanism of action is still under investigation. In the present study we have analysed the effect of the proteins on the accessibility of native, quasi-native and model surfactant membranes to incorporation of the fluorescent probes Nile Red (permeable) and FM 1-43 (impermeable) into membranes. We have also analysed the effect of single or combined proteins on membrane permeation using the soluble fluorescent dye calcein. The fluorescence of FM 1-43 was always higher in membranes containing SP-B and/or SP-C than in protein-depleted membranes, in contrast with Nile Red which was very similar in all of the materials tested. SP-B and SP-C promoted probe partition with markedly different kinetics. On the other hand, physiological proportions of SP-B and SP-C caused giant oligolamellar vesicles to incorporate FM 1-43 from the external medium into apparently most of the membranes instantaneously. In contrast, oligolamellar pure lipid vesicles appeared to be mainly labelled in the outermost membrane layer. Pure lipidic vesicles were impermeable to calcein, whereas it permeated through membranes containing SP-B and/or SP-C. Vesicles containing only SP-B were stable, but prone to vesicle-vesicle interactions, whereas those containing only SP-C were extremely dynamic, undergoing frequent fluctuations and ruptures. Differential structural effects of proteins on vesicles were confirmed by electron microscopy. These results suggest that SP-B and SP-C have different contributions to inter- and intra-membrane lipid dynamics, and that their combined action could provide unique effects to modulate structure and dynamics of pulmonary surfactant membranes and films.