Lipid Phase Separation Research Articles

Kinetic Process of Formation and Reconstruction of Small Unilamellar Vesicles Consisting of Long- and Short-Chain Lipids

Whereas the phase separation of normal phospholipids induces formation of microdomains on the surface of spherical vesicles, the separation of a long- and short-chain lipids can induce perforation of small unilamellar vesicles (SUVs) and transformation into bilayered micelles (bicelles) because the edges of the bilayers are stabilized by the short-chain lipid microdomain. In this study, the effect of the phase separation of lipids on the transformation behavior of SUVs consisting of a mixture of long- and short-chain lipids was investigated using small-angle neutron scattering. At the temperature jump from below to above the chain melting temperature of the long-chain lipid, T(c), bicelles fused together and transformed into SUVs when their size reached a critical radius. In contrast, a sequential transformation of small SUVs to small bicelles, small bicelles to large bicelles, and large bicelles to large SUVs occurred when the temperature jumped from a value far above T(c) to one slightly above T(c). To the best of my knowledge, this is the first report of such reconstruction of vesicles. By considering the bending energy of the membrane, the line tension of the rim, and the perforation due to the phase separation, the mechanism of the transformation processes was clarified.

Glycans pattern the phase behaviour of lipid membranes

Hydrated networks of glycans (polysaccharides)--in the form of cell walls, periplasms or gel-like matrices--are ubiquitously present adjacent to cellular plasma membranes. Yet, despite their abundance, the function of glycans in the extracellular milieu is largely unknown. Here we show that the spatial configuration of glycans controls the phase behaviour of multiphase model lipid membranes: inhomogeneous glycan networks stabilize large lipid domains at the characteristic length scale of the network, whereas homogeneous networks suppress macroscopic lipid phase separation. We also find that glycan-patterned phase separation is thermally reversible--thus indicating that the effect is thermodynamic rather than kinetic--and that phase patterning probably results from a preferential interaction of glycans with ordered lipid phases. These findings have implications for membrane-mediated transport processes, potentially rationalize long-standing observations that differentiate the behaviour of native and model membranes and may indicate an intimate coupling between cellular lipidomes and glycomes.

Effects of surfactin on membrane models displaying lipid phase separation

Surfactin, a bacterial amphiphilic lipopeptide is attracting more and more attention in view of its bioactive properties which are in relation with its ability to interact with lipids of biological membranes. In this work, we investigated the effect of surfactin on membrane structure using model of membranes, vesicles as well as supported bilayers, presenting coexistence of fluid-disordered (DOPC) and gel (DPPC) phases. A range of complementary methods was used including AFM, ellipsometry, dynamic light scattering, fluorescence measurements of Laurdan, DPH, calcein release, and octadecylrhodamine B dequenching. Our findings demonstrated that surfactin concentration is critical for its effect on the membrane. The results suggest that the presence of rigid domains can play an essential role in the first step of surfactin insertion and that surfactin interacts both with the membrane polar heads and the acyl chain region. A mechanism for the surfactin lipid membrane interaction, consisting of three sequential structural and morphological changes, is proposed. At concentrations below the CMC, surfactin inserted at the boundary between gel and fluid lipid domains, inhibited phase separation and stiffened the bilayer without global morphological change of liposomes. At concentrations close to CMC, surfactin solubilized the fluid phospholipid phase and increased order in the remainder of the lipid bilayer. At higher surfactin concentrations, both the fluid and the rigid bilayer structures were dissolved into mixed micelles and other structures presenting a wide size distribution.

Endosomal Sorting Complex Required for Transport (ESCRT) Complexes Induce Phase-separated Microdomains in Supported Lipid Bilayers

The endosomal sorting complex required for transport (ESCRT) system traffics ubiquitinated cargo to lysosomes via an unusual membrane budding reaction that is directed away from the cytosol. Here, we show that human ESCRT-II self-assembles into clusters of 10-100 molecules on supported lipid bilayers. The ESCRT-II clusters are functional in that they bind to ubiquitin and the ESCRT-III subunit VPS20 at nanomolar concentrations on membranes with the same stoichiometries observed in solution and in crystals. The clusters only form when cholesterol is included in the lipid mixture at >10 mol %. The clusters induce the formation of ordered membrane domains that exclude the dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbo-cyanine perchlorate. These results show that ESCRT complexes are capable of inducing lateral lipid phase separation under conditions where the lipids themselves do not spontaneously phase-separate. This property could facilitate ESCRT-mediated membrane budding.

Time-Resolved Surface-Enhanced Ellipsometric Contrast Imaging for Label-Free Analysis of Biomolecular Recognition Reactions on Glycolipid Domains

We have applied surface-enhanced ellipsometry contrast (SEEC) imaging for time-resolved label-free visualization of biomolecular recognition events on spatially heterogeneous supported lipid bilayers (SLB). Using a conventional inverted microscope equipped with total internal reflection (TIR) illumination, biomolecular binding events were monitored with a lateral resolution near the optical diffraction limit at an acquisition rate of ~1 Hz with a sensitivity in terms of surface coverage of ~1 ng/cm(2). Despite the significant improvement in spatial resolution compared to alternative label-free surface-based imaging technologies, the sensitivity remains competitive with surface plasmon resonance (SPR) imaging and imaging ellipsometry. The potential of the technique to discriminate local differences in protein binding kinetics was demonstrated by time-resolved imaging of anti-GalCer antibodies binding to phase-separated lipid bilayers consisting of phosphatidylcholine (POPC) and galactosylceramide (GalCer). A higher antibody binding capacity was observed on the GalCer-diluted fluid region in comparison to the GalCer-rich gel phase domains. This observation is tentatively attributed to differences in the presentation of the GalCer epitope in the two phases, resulting in differences in availability of the ligand for antibody binding. The complementary information obtained by swiftly switching between SEEC and fluorescence (including TIR fluorescence) imaging modes was used to support the data interpretation. The simplicity and generic applicability of the concept is discussed in terms of microfluidic applications.

Monitoring Lipid Anchor Organization in Cell Membranes by PIE-FCCS

This study examines the dynamic co-localization of lipid-anchored fluorescent proteins in living cells using pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS) and fluorescence lifetime analysis. Specifically, we look at the pairwise co-localization of anchors from lymphocyte cell kinase (LCK: myristoyl, palmitoyl, palmitoyl), RhoA (geranylgeranyl), and K-Ras (farnesyl) proteins in different cell types. In Jurkat cells, a density-dependent increase in cross-correlation among RhoA anchors is observed, while LCK anchors exhibit a more moderate increase and broader distribution. No correlation was detected among K-Ras anchors or between any of the different anchor types studied. Fluorescence lifetime data reveal no significant Förster resonance energy transfer in any of the data. In COS 7 cells, minimal correlation was detected among LCK or RhoA anchors. Taken together, these observations suggest that some lipid anchors take part in anchor-specific co-clustering with other existing clusters of native proteins and lipids in the membrane. Importantly, these observations do not support a simple interpretation of lipid anchor-mediated organization driven by partitioning based on binary lipid phase separation.

Abstract 2750: Short-chain lipids catalyse intracellular drug accumulation by a transient molecular gateway that allows plasma membrane traversal

Abstract BACKGROUND: The lipid composition of the plasma membrane provides an effective barrier towards exogenous, potentially cytotoxic molecules. As a consequence, membrane traversal of most therapeutic drugs is sub-optimal and remains a major limitation for clinical efficacy. We previously identified a class of short-chain sphingolipids that facilitate the accumulation of amphiphilic compounds, including the cytostatic drug doxorubicin, within tumor cells, leading to improved therapy response. AIM: In order to elucidate the mechanism of action of short-chain lipids, both in vitro and model membrane studies and in silico molecular dynamics simulation experiments were designed. RESULTS: We demonstrate that short-chain sphingolipids act at the level of the plasma membrane, however, independently of lipid microdomain (raft) formation or membrane proteins. In artificial lipid membranes of well-defined compositions short-chain sphingolipids enhanced doxorubicin-membrane traversal similar to cell membranes; N-octanoyl-glucosylceramide decreased the translocation half-time of doxorubicin over liquid ordered lipid membranes by a factor 1.93 (p < 0.05). In case of liquid disordered membranes, a comparable effect was observed (factor 1.91; p < 0.05); lipid phase separation (raft formation) did not affect enhanced doxorubicin translocation. Molecular dynamics simulations revealed that the short-chain lipids act by assembling a transient molecular gateway for doxorubicin. Accordingly, the free energy barrier for the hydrophilic head group of the drug to dehydrate and translocate to the opposite side of the membrane is reduced by two-fold (p < 0.05). CONCLUSION: Short-chain lipids catalyse the transbilayer movement of amphiphilic cytostatic drugs to achieve higher intratumoral drug accumulation, and their action is explained by the formation of a transient lipid gateway in the plasma membrane of the tumor cell, thus facilitating drug translocation and subsequent intracellular accumulation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2750. doi:1538-7445.AM2012-2750

Floret-Shaped Solid Domains on Giant Fluid Lipid Vesicles Induced by pH

Lateral lipid phase separation of titratable PS or PA lipids and their assembly in domains induced by changes in pH are significant in liposome-based drug delivery: environmentally responsive lipid heterogeneities can be tuned to alter collective membrane properties such as permeability (altering drug release) and surface topography (altering drug carrier reactivity) impacting, therefore, the therapeutic outcomes. At the micrometer scale fluorescence microscopy on giant unilamellar fluid vesicles (GUVs) shows that lowering pH (from 7.0 to 5.0) promotes condensation of titratable PS or PA lipids into beautiful floret-shaped domains in which lipids are tightly packed via hydrogen-bonding and van der Waals interactions. The order of lipid packing within domains increases radially toward the domain center. Lowering pH enhances the lipid packing order, and at pH 5.0 domains appear to be entirely in the solid (gel) phase. Domains phenomenologically comprise a circular "core" cap beyond which interfacial instabilities emerge resembling leaf-like stripes. At pH 5.0 stripes are of almost vanishing Gaussian curvature independent of GUVs' preparation path and in agreement with a general condensation mechanism. Increasing incompressibility of domains is strongly correlated with a larger number of thinner stripes per domain and increasing relative rigidity of domains with smaller core cap areas. Line tension drives domain ripening; however, the final domain shape is a result of enhanced incompressibility and rigidity maximized by domain coupling across the bilayer. Introduction of a transmembrane osmotic gradient (hyperosmotic on the outer lipid leaflet) allows the domain condensation process to reach its maximum extent which, however, is limited by the minimal expansivity of the continuous fluid membrane.

Tuning Membrane Phase Separation Using Nonlipid Amphiphiles

Lipid phase separation may be a mechanism by which lipids participate in sorting membrane proteins and facilitate membrane-mediated biochemical signaling in cells. To provide new tools for membrane lipid phase manipulation that avoid direct effects on protein activity and lipid composition, we studied phase separation in binary and ternary lipid mixtures under the influence of three nonlipid amphiphiles, vitamin E (VE), Triton-X (TX)-100, and benzyl alcohol (BA). Mechanisms of additive-induced phase separation were elucidated using coarse-grained molecular dynamics simulations of these additives in a liquid bilayer made from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC). From simulations, the additive's partitioning preference, changes in membrane thickness, and alterations in lipid order were quantified. Simulations showed that VE favored the DPPC phase but partitioned predominantly to the domain boundaries and lowered the tendency for domain formation, and therefore acted as a linactant. This simulated behavior was consistent with experimental observations in which VE promoted lipid mixing and dispersed domains in both gel/liquid and liquid-ordered/liquid-disordered systems. From simulation, BA partitioned predominantly to the DUPC phase, decreased lipid order there, and thinned the membrane. These actions explain why, experimentally, BA promoted phase separation in both binary and ternary lipid mixtures. In contrast, TX, a popular detergent used to isolate raft membranes in cells, exhibited equal preference for both phases, as demonstrated by simulations, but nonetheless, was a strong domain promoter in all lipid mixtures. Further analysis showed that TX increased membrane thickness of the DPPC phase to a greater extent than the DUPC phase and thus increased hydrophobic mismatch, which may explain experimental observation of phase separation in the presence of TX. In summary, these nonlipid amphiphiles provide new tools to tune domain formation in model vesicle systems and could provide the means to form or disperse membrane lipid domains in cells, in addition to the well-known methods involving cholesterol enrichment and sequestration.

Lateral diffusion in equimolar mixtures of natural sphingomyelins with dioleoylphosphatidylcholine

Cellular membranes of mammals are composed of a complex assembly of diverse phospholipids. Sphingomyelin (SM) and phosphatidylcholine (PC) are important lipids of eukaryotic cellular membranes and neuronal tissues, and presumably participate in the formation of membrane domains, known as “rafts,” through intermolecular interaction and lateral microphase decomposition. In these two-dimensional membrane systems, lateral diffusion of lipids is an essential dynamic factor, which might even be indicative of lipid phase separation process. Here, we used pulsed field gradient nuclear magnetic resonance to study lateral diffusion of lipid components in macroscopically oriented bilayers composed of equimolar mixtures of natural SMs of egg yolk, bovine brain, bovine milk and dipalmitoylphosphatidylcholine (DPPC) with dioleoylphosphatidylcholine (DOPC). In addition, differential scanning calorimetry was used as a complementary technique to characterize the phase state of the lipid bilayers. In fully liquid bilayers, the lateral diffusion coefficients in both DOPC/DPPC and DOPC/SM systems exhibit mean values of the pure bilayers. For DOPC/SM bilayer system, this behavior can be explained by a model where most SM molecules form short-lived lateral domains with preferential SM–SM interactions occurring within them. However, for bilayers in the presence of their low-temperature gel phase, lateral diffusion becomes complicated and cannot simply be understood solely by a simple change in the liquid phase decomposition.

Effect of Mechanical Stresses on Lipid Membrane Phase Properties

We currently investigate how membrane tension modifies the miscibility properties of model lipid bilayers, namely giant unilamellar vesicles. We use fluorescence microscopy to observe coexisting liquid phases within temperature-controlled vesicles under surface tension induced by micropipette aspiration. We measure shifts in liquid/liquid miscibility temperatures as a function of applied tension.We compare our experimental data to the theoretical prediction of [1], by which a surface tension of ∼1mN/m should cause a decrease in miscibility temperature of ∼1K. This prediction may explain why Giant Plasma Membrane Vesicles (GPMVs) show large-scale liquid/liquid phase coexistence [2,3] whereas undisturbed cell membranes do not. GPMV's, which are detached from the cytoskeleton, are hypothesized to have lower surface tensions than cell membranes.[1] Uline, Schick and Szleifer. Changes in phase behavior of lipid bilayers under tension. Submitted.[2] Baumgart, Hammond, Sengupta, Hess, Holowka, Baird and Webb. Large-scale fluid/fluid phase separation of proteins and lipids in giant plasma membrane vesicles. Proc. Natl. Acad. Sci. USA, 104:3165-3170, 2007.[3] Veatch, Cicuta, Sengupta, Honerkamp-Smith, Holowka and Baird. Critical fluctuations in plasma membrane vesicles. ACS Chem Biol., 3:287-293, 2008.

Lipid Bilayer Membrane Shape Remodeling by Lipid-Modified Ras Proteins

Ras proteins are small GTP-hydrohydrolyzing enzymes that operate as molecular switches in signal transduction pathways and are present in a mutant, activated state in many human tumors. It has been shown that the lipid-modified C-terminus drive lateral segregation of Ras proteins into membrane sub-structure on the plasma membrane. Such transient and dynamic molecular assembly, so-call nanocluster, is emerging as a crucial mechanism by which cells achieve high-fidelity signal transmission. However, little is known about the underlying force driving the formation of nanoclusters on the plasma membrane and their influence on the membrane structure. To investigate the interaction between nanocluster and the membrane, we carried out extensive semi-atomistic molecular dynamics simulations of the C-terminal membrane-targeting motif of H-ras (tH) in a phase separated lipid bilayer composed of 2000 DPPC, DliPC and cholesterol molecules. We found out that at ambient temperature approximately 30–40% of tH molecules assemble into clusters of 4-9 proteins, and preferentially localize at the interface between the liquid-order and liquid-disorder phases of the membrane. In the nanoclusters, tH backbones preferably exhibits an extended conformation and align linearly at the phase interface. With such molecular-level organization, tH molecule act as a linactant at the interface and reduce line tension of the anchor-containing monolayer and induces shape transition of the overall membrane due to the tendency of the system to minimize the free energy. The resultant geometry of the membrane is determined by the different elastic bending modulus of different phases as well as the phase boundary line tension, which is strongly affected by the localization of the nanoclusters. Our findings were explained based on the domain-induced budding model and shed light on the interaction between membrane-bounded proteins and membrane domains, such as lipid rafts.

The Molecular Basis of Cluster Formation by Membrane-Bound Lipidated Ras

Recent studies found that stimulus dependent assembly and disassembly of clusters of lipid-anchored signaling proteins on the plasma membrane is a crucial mechanism by which cells achieve high-fidelity signal transmission. The best characterized examples of lipid-protein assemblies are Ras nanoclusters at the inner leaflet of the plasma membrane. Independent of expression level, 30-40% of Ras proteins assemble into 6-8 proteins per cluster. However, little is known about the physical forces underlying the domain-specific localization and clustering of these proteins. To address this issue, we carried out extensive semi-atomistic molecular dynamics (MD) simulations of the C-terminal membrane-targeting motif of H-ras (tH) in a phase-separated bilayer composed of 2000 DPPC, DLiPC, and cholesterol molecules mixed in a 5:3:2 molar ratio. We found that 4-10 tH molecules assemble into dynamic clusters whose stability varies with the extent of lipid phase separation. At ambient temperatures, the calculated cluster size distributions and the clustered fraction agree remarkably well with the available experimental data. Clusters, but not monomers or dimers, segregate to the interface between the liquid ordered and liquid disordered phases. The segregation is driven by the preferential interaction of the saturated palmitoyls in tH clusters with the similarly saturated DPPC, and of the polyunsaturated farnesyl with the unsaturated DLiPC lipids. This was confirmed by additional simulations in which individual lipid modifications were systematically removed through a process of de-palmitoylation and de-farnesylation. The preferential localization of the tH clusters at the domain boundaries resulted in a significant reduction in the line tension and changes in membrane curvature. Initial results on the full-length H-ras suggest that the same fundamental forces drive its clustering, but steric effects modulate the size and distribution of the clusters as well as the elastic properties of the bilayer.

High Affinity Immobilization of Giant Unilamellar Vesicles (GUVs) Induces Redistribution of Lipid Domains

Fluorescence microscopy imaging of giant unilamellar vesicles (GUVs) is a popular tool for the study of lipid phase separation. Studies with GUVs frequently require immobilization of the vesicles, and several methods are available for that effect. One of the most common methods of immobilization requires the inclusion of a very small fraction of biotin labeled lipids in the lipid mixture. Following vesicle formation, the sample is added to avidin coated imaging chambers and the interaction between biotin and avidin ensures that GUVs remain fully immobile during data acquisition. Here, we analyze the effect of biotinylated lipids with different structures on the distribution of lipid domains within the GUVs. We show that the different biotinylated lipids present differential affinities for liquid disordered, liquid ordered and gel lipid phases, and as a consequence the distribution of lipid domains inside GUVs was dramatically affected. Additionally, even in the absence of immobilization, interaction of the GUVs with the coverslip solid surface induced changes in lipid domain distribution. A very common methodology in GUV imaging is the acquisition of data only from the top hemisphere. Taking our results into account, it is clear that this creates a bias relatively to the total composition of the vesicles. Thus, in order to correctly quantify lipid phase fractions in GUVs, it is absolutely essential to acquire data from the entire vesicle.

Controlled Protein Confinement in Phase-Separated Membranes Tethered onto Micro-Patterned Supports

Phase-separation of lipid membranes into liquid-disordered (ld) and liquid-ordered (lo) domains has been recognized as a key principle for the functional organization of the plasma membrane. In classic model systems such as GUVs, the spatial organization of phase separated membranes is a stochastic, time-dependent process, which depends on the lipid composition and often leads to a complete coalescence of the lipid phases. We have here established an approach for a spatial control of lipid phase separation in tethered polymer-supported membranes (PSM). On a dense poly(ethylene glycol) polymer brush functionalized with hydrophobic tethers, contiguous, highly fluid PSM were obtained by means of fusion of SUVs.1 Free diffusion of lipids and reconstituted transmembrane proteins in these PSM was confirmed by FRAP, FCS and single molecule tracking. Strikingly, phase separation of ternary lipid mixtures (DOPC/SM/cholesterol) in PSM into ld and lo phases was dependent on the properties of the anchoring group. We exploited these features for assembly of lo domains into defined structures using micropatterned tethers. Within isolated micropatterns, ld and lo phases self-assembled into stable, reproducible membrane architectures. By binary micro-patterning of different tethering groups into complementary areas, ternary lipids mixtures separated into lo and ld phases controlled by the geometry of the underlying tethers. Transmembrane proteins reconstituted in these phase-separated PSM strictly partitioned into the ld phase. Hence, the lo phase could be used for confining transmembrane proteins into microscopic and submicroscopic domains. The permeability of these barriers for lipids and proteins and thus their exchange between adjacent ld compartments can be globally and locally controlled by the temperature. These features have been exploited for probing interactions and diffusion of a transmembrane receptor in the context of ld and lo phases.1) Roder, F.; et al. Anal Chem 2011, 83, 6792-6799.

Electro-optical BLM chips enabling dynamic imaging of ordered lipid domains

Studies of lipid rafts, ordered microdomains of sphingolipids and cholesterol within cell membranes, are essential in probing the relationships between membrane organization and cellular function. While in vitro studies of lipid phase separation are commonly performed using spherical vesicles as model membranes, the utility of these models is limited by a number of factors. Here we present a microfluidic device that supports simultaneous electrical measurements and confocal imaging of on-chip bilayer lipid membranes (BLMs), enabling real-time multi-domain imaging of membrane organization. The chips further support closed microfluidic access to both sides of the membrane, allowing the membrane boundary conditions to be rapidly changed and providing a mechanism for dynamically adjusting membrane curvature through application of a transmembrane pressure gradient. Here we demonstrate the platform through the study of dynamic generation and dissolution of ordered lipid domains as membrane components are transported to and from the supporting annulus containing solvated lipids and cholesterol.

Inter-leaflet interaction and asymmetry in phase separated lipid bilayers: molecular dynamics simulations

In order to investigate experimentally inaccessible, molecular-level detail regarding interleaflet interaction in membranes, we have run an extensive series of coarse-grained molecular dynamics sim...

Fluorescent probe partitioning in GUVs of binary phospholipid mixtures: Implications for interpreting phase behavior

The phase behavior of membrane lipids is known to influence the organization and function of many integral proteins. Giant unilamellar vesicles (GUVs) provide a very useful model system in which to examine the details of lipid phase separation using fluorescence imaging. The visualization of domains in GUVs of binary and ternary lipid mixtures requires fluorescent probes with partitioning preference for one of the phases present. To avoid possible pitfalls when interpreting the phase behavior of these lipid mixtures, sufficiently thorough characterization of the fluorescent probes used in these studies is needed. It is now evident that fluorescent probes display different partitioning preferences between lipid phases, depending on the specific lipid host system. Here, we demonstrate the benefit of using a panel of fluorescent probes and confocal fluorescence microscopy to examine phase separation in GUVs of binary mixtures of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). Patch and fibril gel phase domains were found to co-exist with liquid disordered (ld) domains on the surface of GUVs composed of 40:60mol% DOPC/DPPC, over a wide range of temperatures (14–25°C). The fluorescent lipid, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl (NBD-DPPE), proved to be the most effective probe for visualization of fibril domains. In the presence of LissamineTM rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (Rh-DPPE) we were unable to detect fibril domains. This fluorophore also affected the partitioning behavior of other fluorescent probes. Overall, we show that the selection of different fluorescent probes as lipid phase reporters can result in very different interpretation of the phase behavior of DOPC/DPPC mixtures.

Emulsifying Property and Antioxidative Activity of Cuttlefish Skin Gelatin Modified with Oxidized Linoleic Acid and Oxidized Tannic Acid

Cuttlefish skin gelatins modified with oxidized linoleic acid (OLA) and oxidized tannic acid (OTA) were characterized and determined for emulsifying properties and antioxidative activity. Modification of gelatin with 5% OTA increased the total phenolic content and 1,1-diphenyl-2-picrylhydrazyl, 2,2′-azinobis(3-ethylbenzothiazoline-6-sulphonic acid) radical scavenging activity and ferric reducing antioxidant power of gelatin–OTA. Incorporation of OLA into gelatin (OLA-to-free amino group molar ratio of 10:1) increased surface hydrophobicity of gelatin from 17.39 to 32.38 and reduce surface tension at air/water interface of gelatin solution from 53 to 32 mN/m. Gelatin–OLA had the increase in emulsion activity index, compared with gelatin without modification and was capable of producing a fine emulsion (d32 = 0.79 μm, d43 = 0.82 μm). Modification of gelatin–OLA complex with OTA at different concentrations (2.5%, 5%, and 10%) increased antioxidative activity but decrease emulsifying properties. However, gelatin–OLA modified with 5% OTA had higher emulsifying properties than the commercial gelatin (bovine gelatin). The presence of an alkyl group and a hydroxyl group in gelatin after modification with OLA and OTA, respectively, was revealed by Fourier transform infrared study. Coincidental decrease in free amino group was also noticeable in modified gelatin. Menhaden oil-in-water emulsion stabilized by gelatin modified with OLA and 5% OTA was more resistant to lipid oxidation and phase separation as evidenced by the lower thiobarbituric acid reactive substances value and smaller oil droplet size, compared with that stabilized by commercial bovine gelatin. Thus, the modification of gelatin by both OLA and OTA was able to improve antioxidative and emulsifying properties of cuttlefish skin gelatin.

Formation of Supported Lipid Bilayers at Surfaces with Controlled Curvatures: Influence of Lipid Charge

We have developed and characterized novel biomimetic membranes, formed at nanostructured sensor substrates with controlled curvatures, motivated by the many biological processes that involve membrane curvature. Model systems with convex nanostructures, with radii of curvatures (ROCs) of 70, 75, and 95 nm, were fabricated utilizing colloidal assembly and used as substrates for supported lipid bilayers (SLBs). The SLBs were formed via vesicle adsorption and rupture, and the vesicle deposition pathway was studied by means of quartz crystal microbalance with dissipation (QCM-D) and fluorescence microscopy. SLBs conforming to the underlying nanostructured surfaces, which exhibit increased surface area with decreased ROC, were confirmed from excess mass, monitored by QCM-D, and excess total fluorescence intensities. The formation of SLBs at the nanostructured surfaces was possible, however, depending on the ROC of the structures and the lipid vesicle charge the quality varied. The presence of nanostructures was shown to impair vesicle rupture and SLB formation was progressively hindered at surfaces with structures of decreasing ROCs. The introduction of a fraction of the positively charged lipid POEPC in the lipid vesicle membrane allowed for good quality and conformal bilayers at all surfaces. Alternatively, for vesicles formed from lipid mixtures with a fraction of the negatively charged lipid POPS, SLB formation was not at all possible at surfaces with the lowest ROC. Interestingly, the vesicle adsorption rate and the SLB formation were faster at surfaces with nanostructures of progressively smaller ROCs at high ratios of POPS in the vesicles. Development of templated SLBs with controlled curvatures provides a new experimental platform, especially at the nanoscale, at which membrane events such as lipid sorting, phase separation, and protein binding can be studied.