Lipid diffusion in giant unilamellar vesicles is more than 2 times faster than in supported phospholipid bilayers under identical conditions

The lateral diffusion coefficients of a BODIPY tail-labeled lipid in two model systems, namely, free-standing giant unilamellar vesicles (GUVs) and supported phospholipid bilayers (SPBs), were determined by fluorescence correlation spectroscopy (FCS) using the Z-scan approach. For the first time, the performed measurements on 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bilayers maintain exactly the same experimental conditions for both systems, which allows for a quantitative comparison of lipid diffusion in these two commonly used model membranes. The results obtained revealed that the lipid mobility in free-standing bilayers (D=7.8+/-0.8 microm2 s-1) is significantly higher than in the bilayer created on the solid support (mica) (D=3.1+/-0.3 microm2 s-1).     

Surface-dependent transitions during self-assembly of phospholipid membranes on mica, silica, and glass

Formation of supported membranes by exposure of solid surfaces to phospholipid vesicles is a much-used technique in membrane research. Freshly cleaved mica, because of its superior flatness, is a preferred support, and we used ellipsometry to study membrane formation kinetics on mica. Neutral dioleoyl-phosphatidylcholine (DOPC) and negatively charged dioleoyl-phosphatidylserine/dioleoyl-phosphatidylcholine (20% DOPS/80% DOPC) vesicles were prepared by sonication. Results were compared with membrane formation on silica and glass, and the influence of stirring, buffer, and calcium was assessed. Without calcium, DOPC vesicles had a low affinity (Kd approximately 30 microM) for mica, and DOPS/DOPC vesicles hardly adsorbed. Addition of calcium promptly caused condensation of the adhering vesicles, with either loss of excess lipid or rapid additional lipid adsorption up to full surface coverage. Vesicle-mica interactions dominate the adsorption process, but vesicle-vesicle interactions also seem to be required for the condensation process. Membranes on mica proved unstable in Tris-HCl buffer. For glass, transport-limited adsorption of DOPC and DOPS/DOPC vesicles with immediate condensation into bilayers was observed, with and without calcium. For silica, vesicle adsorption was also rapid, even in the absence of calcium, but the transition to condensed layers required a critical surface coverage of about 50% of bilayer mass, indicating vesicle-vesicle interaction. For all three surfaces, additional adsorption of DOPC (but not DOPS/DOPC) vesicles to condensed membranes was observed. DOPC membranes on mica were rapidly degraded by phospholipase A2 (PLA2), which pleads against the role of membrane defects as initial PLA2 targets. During degradation, layer thickness remained unchanged while layer density decreased, in accordance with recent atomic force microscopy measurements of gel-phase phospholipid degradation by PLA2.     

Nanometre-sized molecular oxygen sensors prepared from polymer stabilized phospholipid vesicles

Nanometre-sized, chemically-stabilized phospholipid vesicle sensors have been developed for detection of dissolved molecular oxygen. Sensors were prepared by forming 150 nm phospholipid vesicles from 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or DOPC doped with small (<1%) mole percentages of 1,2-dioleoyl-sn-glycero-3-phosphoethanol amine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (NBD-PE). Sensors were stabilized via cross-linking polymerization of hydrophobic methacrylate monomers partitioned into the hydrophobic interior of the DOPC bilayer. The resultant unilamellar, nanometre-sized, polymer-lipid vesicles are spherical, biocompatible and protect sensing components that are loaded into the aqueous interior of the vesicle from interfering species in the exterior environment. For O(2) detection, the oxygen-sensitive fluorescent dye, tris(1,10-phenanthroline)ruthenium(II) chloride (Ru(phen)(3)) was encapsulated into the aqueous interior of the polymerized phospholipid vesicle. NBD-PE was introduced into the phospholipid bilayer of the sensor as a reference dye, allowing ratiometric sensors to be constructed. The resultant sensors show high sensitivity, excellent reversibility and excellent linearity over a physiological range of dissolved oxygen concentrations. These results suggest that polymerized phospholipid vesicle sensors can be used for monitoring intracellular O(2) dynamics.     

Dynamics of water-in-oil nanoemulsions revealed by fluorescence lifetime correlation spectroscopy

The size, diffusional properties, and dynamics of reverse water-in-oil nanoemulsions, or reverse micelles (RMs), have been widely investigated because of interest in this system as a model for biological compartmentalization. Here, we have employed fluorescence lifetime correlation spectroscopy (FLCS) to reveal the dynamics and sizes of aerosol-OT (AOT)/isooctane RMs using a fluorescent xanthene derivative called Tokyo Green II (TG-II). The dye undergoes a partition and a shift in its tautomeric equilibrium such that the TG-II anion remains in the inner micellar aqueous core, and the neutral quinoid form lies in the interfacial region. By applying FLCS, we specifically obtained the lifetime filtered autocorrelation curves of the anionic TG-II, which shows a characteristic lifetime of approximately 4 ns. Analysis of the FLCS curves provides the diffusion coefficient and hydrodynamic radius of the RMs as well as micelle dynamics in the same experiment. The FLCS curves show dynamics in the microsecond time range, which represents an interconversion rate that changes the distribution of the TG-II neutral and anionic forms in the hydrophobic interface and the water core.     

Formation of substrate-supported membranes from mixtures of long- and short-chain phospholipids

We studied the formation of substrate-supported planar phospholipid bilayers (SPBs) on glass and silica from mixtures of long- and short-chain phospholipids to assess the effects of detergent additives on SPB formation. 1,2-Hexyanoyl-sn-glycero-3-phosphocholine (DHPC-C6) and 1,2-heptanoyl-sn-glycero-3-phosphocholine (DHPC-C7) were chosen as short-chain phospholipids. 1-Palmitoyl-2-oleol-sn-glycero-3-phosphocholine (POPC) was used as a model long-chain phospholipid. Kinetic studies by quartz crystal microbalance with dissipation monitoring (QCM-D) showed that the presence of short-chain phospholipids significantly accelerated the formation of SPBs. Rapid rinsing with a buffer solution did not change the adsorbed mass on the surface if POPC/DHPC-C6 mixtures were used below the critical micelle concentration (cmc) of DHPC-C6, indicating that an SPB composed of POPC molecules remained on the surface. Fluorescence microscopy observation showed homogeneous SPBs, and the fluorescence recovery after photobleaching (FRAP) measurements gave a diffusion coefficient comparable to that for SPBs formed from POPC vesicles. However, mixtures of POPC/DHPC-C7 resulted in a smaller mass of lipid adsorption on the substrate. FRAP measurements also yielded significantly smaller diffusion coefficients, suggesting the presence of defects. The different behaviors for DHPC-C6 and DHPC-C7 point to the dual roles of detergents to enhance the formation of SPBs and to destabilize them, depending on their structures and aggregation properties.     

Formation and diffusivity characterization of supported lipid bilayers with complex lipid compositions

The moving edge of a hydrodynamically manipulated supported lipid bilayer (SLB) can be used to catalyze SLB formation of adsorbed lipid vesicles that do not undergo spontaneous SLB formation upon adsorption on SiO(2). By removing the lipid reservoir of an initially formed SLB, we show how a hydrodynamically moved SLB patch composed of POPC can be used to form isolated SLBs with compositions that to at least 95% represent that of the adsorbed lipid vesicles. The concept is used to investigate the diffusivity of lissamine rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (rhodamine-DHPE) in SLBs made from complex lipid compositions, revealing a decrease in diffusivity by a factor of 2 when the cholesterol content was increased from 0% to 50%. We also demonstrate how the concept can be used to induce stationary domains in SLBs containing 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and cholesterol (39:21:40 mol %, respectively). Because the method serves as a means to form SLBs with lipid compositions that hamper SLB formation via spontaneous rupture of adsorbed lipid vesicles, it opens up the possibility for new biophysical investigations of SLBs with more nativelike compositions.     

Ordering transitions in nematic liquid crystals induced by vesicles captured through ligand-receptor interactions

We report that phospholipid vesicles incorporating ligands, when captured from solution onto surfaces presenting receptors for these ligands, can trigger surface-induced orientational ordering transitions in nematic phases of 4'-pentyl-4-cyanobiphenyl (5CB). Specifically, whereas avidin-functionalized surfaces incubated against vesicles composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) were observed to cause the liquid crystal (LC) to adopt a parallel orientation at the surface, the same surfaces incubated against biotinylated vesicles (DOPC and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(biotinyl) (biotin-DOPE)) caused the homeotropic (perpendicular) ordering of the LC. The use of a combination of atomic force microscopy (AFM), ellipsometry and quantitative fluorimetry, performed as a function of vesicle composition and vesicle concentration in solution, revealed the capture of intact vesicles containing 1% biotin-DOPE from buffer at the avidin-functionalized surfaces. Subsequent exposure to water prior to contact with the LC, however, resulted in the rupture of the majority of vesicles into interfacial multilayer assemblies with a maximum phospholipid loading set by random close packing of the intact vesicles initially captured on the surface (5.1 ± 0.2 phospholipid molecules/nm(2)). At high concentrations of biotinylated lipid (>10% biotin-DOPE) in the vesicles, the limiting lipid loading was measured to be 4.0 ± 0.3 phospholipid molecules/nm(2), consistent with the maximum phospholipid loading set by the spontaneous formation of a bilayer during incubation with the biotinylated vesicles. We measured the homeotropic ordering of the LC on the surfaces independently of the initial morphology of the phospholipid assembly captured on the surface (intact vesicle, planar multilayer). We interpret this result to infer the reorganization of the phospholipid bilayers either prior to or upon contact with the LCs such that interactions of the acyl chains of the phospholipid and the LC dominate the ordering of the LC, a conclusion that is further supported by quantitative measurements of the orientation of the LC as a function of the phospholipid surface density (>1.8 molecules/nm(2) is required to cause the homeotropic ordering of the LC). These results and others presented herein provide fundamental insights into the interactions of phospholipid-decorated interfaces with LCs and thereby provide guidance for the design of surfaces on which phospholipid assemblies captured through ligand-receptor recognition can be reported via ordering transitions in LCs.     

Donor-donor energy migration (DDEM) as a tool for studying aggregation in lipid phases

A BODIPY-labelled sulfatide (N-(BODIPY-FL-pentanoyl)-galactosylcerebroside-sulfate, hereafter abbreviated as BD-Sulfatide) was solubilised at different concentrations in lipid vesicles of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). Time-correlated single photon counting experiments show that the fluorescence relaxation is mono-exponential (with a lifetime of 6.5 ns) at molar ratios of BD-Sulfatide: DOPC that are less than 1:100. The fluorescence steady-state anisotropy decreases monotonously at molar ratios smaller than 1:1000, which is compatible with donor-donor energy migration (DDEM) among the BODIPY groups. A model that assumes DDEM across the lipid bilayers, as well as in their planes, was used to analyse the time-resolved fluorescence anisotropy. Only two parameters appear in the model namely: the bilayer thickness (d) and the average number density (C2) distribution of BD-Sulfatide in the lipid bilayers. The extracted d-values vary between 35 and 40 A, which is about the reported thickness of a bilayer of DOPC (38 A). Hence, the BODIPY groups are preferentially located in the water-lipid interface. At low concentration the experimental C2-values and those independently calculated are in good agreement, while the experimental values gradually become lower with increasing BD-Sulfatide concentration. These results are compatible with an aggregation of the sulfatides and self-quenching of BODIPY, which is clearly established at higher concentrations of the BD-Sulfatide.     

Role of lipid charge in organization of water/lipid bilayer interface: insights via computer simulations

Anionic unsaturated lipid bilayers represent suitable model systems that mimic real cell membranes: they are fluid and possess a negative surface charge. Understanding of detailed molecular organization of water-lipid interfaces in such systems may provide an important insight into the mechanisms of proteins' binding to membranes. Molecular dynamics (MD) of full-atom hydrated lipid bilayers is one of the most powerful tools to address this problem in silico. Unfortunately, wide application of computational methods for such systems is limited by serious technical problems. They are mainly related to correct treatment of long-range electrostatic effects. In this study a physically reliable model of an anionic unsaturated bilayer of 1,2-dioleoyl-sn-glycero-3-phosphoserine (DOPS) was elaborated and subjected to long-term MD simulations. Electrostatic interactions were treated with two different algorithms: spherical cutoff function and particle-mesh Ewald summation (PME). To understand the role of lipid charge in the system behavior, similar calculations were also carried out for zwitterionic bilayer composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). It was shown that, for the charged DOPS bilayer, the PME protocol performs much better than the cutoff scheme. In the last case a number of artifacts in the structural organization of the bilayer were observed. All of them were attributed to inadequate treatment of electrostatic interactions of lipid headgroups with counterions. Electrostatic properties, along with structural and dynamic parameters, of both lipid bilayers were investigated. Comparative analysis of the MD data reveals that the water-lipid interface of the DOPC bilayer is looser than that for DOPS. This makes possible deeper penetration of water molecules inside the zwitterionic (DOPC) bilayer, where they strongly interact with carbonyls of lipids. This can lead to thickening of the membrane interface in zwitterionic as compared to negatively charged bilayers.     

Lateral organization of a membrane protein in a supported binary lipid domain: direct observation of the organization of bacterial light-harvesting complex 2 by total internal reflection fluorescence microscopy

A unique method is described for directly observing the lateral organization of a membrane protein (bacterial light-harvesting complex LH2) in a supported lipid bilayer using total internal reflection fluorescence (TIRF) microscopy. The supported lipid bilayer consisted of anionic 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1'-glycerol)] (DOPG) and 1,2-distearoly-sn-3-[phospho-rac-(1'-glycerol)] (DSPG) and was formed through the rupture of a giant vesicle on a positively charged coverslip. TIRF microscopy revealed that the bilayer was composed of phase-separated domains. When a suspension of cationic phospholipid (1,2-dioleoyl-sn-glycero-3-ethylphosphocholine: EDOPC) vesicles (approximately 400 nm in diameter), containing LH2 complexes (EDOPC/LH2 = 1000/1), was put into contact with the supported lipid bilayer, the cationic vesicles immediately began to fuse and did so specifically with the fluid phase (DOPG-rich domain) of the supported bilayer. Fluorescence from the incorporated LH2 complexes gradually (over approximately 20 min) spread from the domain boundary into the gel domain (DSPG-rich domain). Similar diffusion into the domain-structured supported lipid membrane was observed when the fluorescent lipid (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-lissamine-rhodamine B sulfonyl: N-Rh-DOPE) was incorporated into the vesicles instead of LH2. These results indicate that vesicles containing LH2 and lipids preferentially fuse with the fluid domain, after which they laterally diffuse into the gel domain. This report describes for first time the lateral organization of a membrane protein, LH2, via vesicle fusion and subsequent lateral diffusion of the LH2 from the fluid to the gel domains in the supported lipid bilayer. The biological implications and applications of the present study are briefly discussed.     

Cholesterol slows down the lateral mobility of an oxidized phospholipid in a supported lipid bilayer

We investigated the mobility and phase-partitioning of the fluorescent oxidized phospholipid analogue 1-palmitoyl-2-glutaroyl-sn-glycero-3-phospho-N-Alexa647-ethanolamine (PGPE-Alexa647) in supported lipid bilayers. Compared to the conventional phospholipid dihexadecanoylphosphoethanolamine (DHPE)-Bodipy we found consistently higher diffusion constants. The effect became dramatic when immobile obstacles were inserted into the bilayer, which essentially blocked the diffusion of DHPE-Bodipy but hardly influenced the movements of PGPE-Alexa647. In a supported lipid bilayer made of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), the differences in probe mobility leveled off with increasing cholesterol content. Using coarse-grained molecular dynamics simulations, we could ascribe this effect to increased interactions between the oxidized phospholipid and the membrane matrix, concomitant with a translation in the headgroup position of the oxidized phospholipid: at zero cholesterol content, its headgroup is shifted to the outside of the DOPC headgroup region, whereas increasing cholesterol concentrations pulls the headgroup into the bilayer plane.     

Absence of ethanol-induced interdigitation in supported phospholipid bilayers on silica surfaces

Membranes prepared by the adsorption of phospholipid vesicles on solid supports are much-used model systems in biomedical research. However, there is accumulating evidence that such membranes may not always be equivalent to the free-standing cellular membranes that they are modeling. In the present study, sonicated DOPC/DOPS (80/20 mol %) vesicles were adsorbed on hydrophilic silica surfaces, a system that has been demonstrated to produce confluent bilayers. In addition, pure DOPC and DLPC membranes were studied. It is demonstrated that ethanol-induced membrane interdigitation, as demonstrated for free-standing bilayers, does not occur in these supported membranes.     

Structure and mobility of lipid membranes on a thermoplastic substrate

Supported lipid membranes constitute one of the most important model systems for cell membranes. The properties of lipid membranes supported by the hydrophobic solid polymer cyclic olefin copolymer (COC) were investigated. Lipid layers consisting of varying amounts of 1,2-dioleoyl-3-trimethylammonium propane (DOTAP, cationic) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC, neutral) prepared by vesicle fusion and solvent exchange were compared. All lipid mixtures coated the COC surface homogeneously forming a fluid membrane as verified by fluorescence microscopy and fluorescence recovery after photobleaching (FRAP). The exact structure of the supported membranes was determined by synchrotron reflectivity experiments using a microfluidic chamber. The X-ray data are in agreement with a compressed (head-to-head distance = 29 angstroms) and less densely packed bilayer.     

Layer by layer surface engineering of poly (lactide-co-glycolide) nanoparticles: A versatile tool for nanoparticle engineering for targeted drug delivery

Recent work regarding the Layer by Layer (LbL) engineering of poly(lactide-co-glycolide) nanoparticles (PLGA NPs) is reviewed here.The LbL engineering of PLGA NPs is applied as a means of generating advanced drug delivery devices with tailored recognition,protection,cargo and release properties.LbL in combination with covalent chemistry is used to attach PEG and folic acid to control cell uptake and direct it towards cancer cells.LbL coatings composed of chitosan and alginate show low protein interactions and can be used as an alternative to Pegylation.The assembly on top of LbL coatings of lipid layers composed of variable percentages of 1,2-dioleoyl-sn-glycero-3-choline (DOPC) and 1,2-dioleoyl-sn-glycero-3-phosphoL-serine (DOPS) increases NP uptake and directs the NPs towards the endoplasmic reticulum.The antibody anti-TNF-α is encapsulated forming a complex with alginate that is assembled LbL on top of PLGA NPs.The antibody is released in cell culture following first order kinetics.The release kinetics of encapsulated molecules inside PLGA NPs are studied when the PLGA NPs are coated via LbL with different polyelectrolytes.The intracellular release of encapsulated Doxorubicin is studied in the HepG2 cell line by means of Fluorescence Lifetime Imaging.     

Arrays of lipid bilayers and liposomes on patterned polyelectrolyte templates

This paper presents novel methods to produce arrays of lipid bilayers and liposomes on patterned polyelectrolyte multilayers. We created the arrays by exposing patterns of poly(dimethyldiallylammonium chloride) (PDAC), polyethylene glycol (m-dPEG) acid, and poly(allylamine hydrochloride) (PAH) on polyelectrolyte multilayers (PEMs) to liposomes of various compositions. The resulting interfaces were characterized by total internal reflection fluorescence microscopy (TIRFM), fluorescence recovery after pattern photobleaching (FRAPP), quartz crystal microbalance (QCM), and fluorescence microscopy. Liposomes composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phosphate (monosodium salt) (DOPA) were found to preferentially adsorb on PDAC and PAH surfaces. On the other hand, liposome adsorption on sulfonated poly(styrene) (SPS) surfaces was minimal, due to electrostatic repulsion between the negatively charged liposomes and the SPS-coated surface. Surfaces coated with m-dPEG acid were also found to resist liposome adsorption. We exploited these results to create arrays of lipid bilayers by exposing PDAC, PAH and m-dPEG patterned substrates to DOPA/DOPC vesicles of various compositions. The patterned substrates were created by stamping PDAC (or PAH) on SPS-topped multilayers, and m-dPEG acid on PDAC-topped multilayers, respectively. This technique can be used to produce functional biomimetic interfaces for potential applications in biosensors and biocatalysis, for creating arrays that could be used for high-throughput screening of compounds that interact with cell membranes, and for probing, and possibly controlling, interactions between living cells and synthetic membranes.     

Dynamics imaging of lipid phases and lipid-marker interactions in model biomembranes

Biomembranes are complex systems that regulate numerous biological processes. Lipid phases that constitute these membranes influence their properties and transport characteristics. Here, we demonstrate the potential of short-range dynamics imaging (excited-state lifetime, rotational diffusion, and order parameter) as a sensitive probe of lipid phases in giant unilamellar vesicles (GUVs). Liquid-disordered and gel phases were labeled with Bodipy-PC at room temperature. Two-photon fluorescence lifetime imaging microscopy of single-phase GUVs reveals more heterogeneity in fluorescence lifetimes of Bodipy in the gel phase (DPPC: 3.8+/-0.6 ns) as compared with the fluid phase (DOPC: 5.2+/-0.2 ns). The phase-specificity of excited-state lifetime of Bodipy-PC is attributed to the stacking of ordered lipid molecules that possibly enhances homo-FRET. Fluorescence polarization anisotropy imaging also reveals distinctive molecular order that is phase specific. The results are compared with DiI-C12-labeled fluid GUVs to investigate the sensitivity of our fluorescence dynamics assay to different lipid-marker interactions. Our results provide a molecular perspective of lipid phase dynamics and the nature of their microenvironments that will ultimately help our understanding of the structure-function relationship of biomembranes in vivo. Furthermore, these ultrafast excited-state dynamics will be used for molecular dynamics simulation of lipid-lipid, lipid-marker and lipid-protein interactions.     

巨型囊泡的侧向相分离与出芽

用电形成法制备含三组分二油酰磷脂酰胆碱(DOPC)/二棕榈酰磷脂酰胆碱(DPPC)/胆固醇(Chol)的巨型囊泡, 以TR-DPPE为荧光染色剂, 在荧光显微镜下直接观察膜的侧向相分离与微区相凸起出芽的耦合. 发现囊泡膜内的相分离具有诱导期, 相分离速度很快, 形成的微相区在整个囊泡球面上均衡分布. 各微相区的出芽不是同时进行, 为逐个随机发生. 每次出芽的时间小于0.5 s. 分相与出芽的耦合使球面上的不同微区之间不会相互融合成更大的微区.     

Elimination of autofluorescence in fluorescence correlation spectroscopy using the AzaDiOxaTriAngulenium (ADOTA) fluorophore in combination with time-correlated single-photon counting (TCSPC)

Fluorescence correlation spectroscopy (FCS) is a frequently applied technique that allows for the precise and sensitive analysis of molecular diffusion and interactions. However, the potential of FCS for in vitro or ex vivo studies has not been fully realized due in part to artifacts originating from autofluorescence (fluorescence of inherent components and fixative-induced fluorescence). Here, we propose the azadioxatriangulenium (ADOTA) dye as a solution to this problem. The lifetime of the ADOTA probe, about 19.4 ns, is much longer than most components of autofluorescence. Thus, it can be easily separated by time-correlated single-photon counting methods. Here, we demonstrate the suppression of autofluorescence in FCS using ADOTA-labeled hyaluronan macromolecules (HAs) with Rhodamine 123 added to simulate diffusing fluorescent background components. The emission spectrum and decay rate of Rhodamine 123 overlap with the usual sources of autofluorescence, and its diffusion behavior is well known. We show that the contributions from Rhodamine 123 can be eliminated by time gating or by fluorescence lifetime correlation spectroscopy (FLCS). While the pairing of ADOTA and time gating is an effective strategy for the removal of autofluorescence from fluorescence imaging, the loss of photons leads to erroneous concentration values with FCS. On the other hand, FLCS eliminates autofluorescence without such errors. We then show that both time gating and FLCS may be used successfully with ADOTA-labeled HA to detect the presence of hyaluronidase, the overexpression of which has been observed in many types of cancer.     

On the origin of the polar order of T4 lysozyme on planar model surfaces

Site directed spin labeling is used to investigate the origin of the macroscopic alignment of T4 lysozyme vectorially tethered to planar biomimetic surfaces. T4 lysozyme was adsorbed to a quartz-supported dioleoylphosphatidylcholine (DOPC) bilayer by selective binding of the histidine-tagged protein to functionalized headgroups (1,2-dioleoyl-sn-glycero-3-[[N(5-amino-1-carboxypentyl)iminodiacetic acid]succinyl], DOGS NTA) of the bilayer. This results in a polar oriented ensemble of proteins on the surface, which gives rise to angular-dependent electron paramagnetic resonance (EPR) spectra. In order to reveal the mechanism of the protein alignment, the influence of protein coverage on the order of the molecules was addressed. Along the lines described previously for a full monolayer (Jacobsen, et al. Biophys. J. 2005, 88, 4351), the polar orientation of the molecules was inferred from an analysis of the EPR line shape using the stochastic Liouville equation (SLE) approach developed by Freed and co-workers. The simulations reveal that the orientation of the protein is strongly determined by lateral protein-protein interactions. In comparison to the lipid bilayer, a fusion protein of T4 lysozyme (T4L) with Annexin XII was investigated, where the two-dimensional crystallization of Annexin XII on a dioleoylphosphatidylserine (DOPS) bilayer provides a surface layer of regularly anchored T4L molecules. For this system, it is found that the interaction between T4L and Annexin plays a more important role for understanding the structure in the adsorbed state.     

Interfacial behavior of chroman-6 and chroman-6 palmitoyl ester and their interaction with phospholipids

The surface activity of chroman-6 (CR-6) and chroman-6 palmitoyl ester (PCR-6) and the interactions with lipid membranes, using 1,2-dipamitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) monolayers, were determined. 8-Anilino-1-naphthalenesulfonic acid titration indicates that none of these molecules was able to form aggregates in aqueous media. The presence of a palmitoyl chain in PCR-6 increases strongly the surface activity of the parent compound (CR-6), rendering a molecule to form stable monomolecular layers. The interaction of both compounds with DPPC and DOPC, measured at constant area or in a compression isotherm model, follows the same trend. Miscibility studies performed with DPPC/CR-6 or PCR-6 indicate that the energies involved are small. The presence of CR-6 and PCR-6 has a soft influence on the compressibility of the Langmuir mixed films. Differential scanning calorimetry studies indicate that CR-6 and PCR-6 modify the temperature and cooperativity of the transition from gel to liquid crystal process in DPPC vesicles.