Characterization and control of lipid layer fluidity in hybrid bilayer membranes

The main gel-to-liquid-crystal (LC) phase transition temperature, T(m), of the distal lipid layer in hybrid bilayer membranes (HBMs) under water was investigated using vibrational sum frequency spectroscopy (VSFS). VSFS has unique sensitivity to order/disorder transitions in the lipid acyl chains and can determine T(m) for the lipid monolayers in HBMs. We recently reported the observation that T(m) is raised and the transition width is broadened for the overlying phospholipid monolayer in HBM systems formed on densely packed crystalline self-assembled monolayers (SAMs) as compared to that of vesicles in solution. In this report, we establish that T(m) for the lipid layer of HBMs can be controlled by proper choice of the SAM underlayer. The SAM underlayer of the HBM was systematically altered by using an alkane thiol, a saturated thiolipid, a mixed SAM of a saturated lipid-pyridine disulfide, and finally a mixed SAM of an unsaturated lipid-pyridine disulfide. T(m) was measured for two different chain length saturated phosphatidylcholine lipid overlayers on these SAMs. The values obtained show that Tm of the lipid layer of HBMs is sensitive to the composition and/or packing density of molecules in the underlying SAM.     

Cholesterol/phospholipid interactions in hybrid bilayer membranes

The interactions between cholesterol and saturated phospholipids in hybrid bilayer membranes (HBMs) were investigated using the interface-sensitive technique of vibrational sum frequency spectroscopy (VSFS). The unique sensitivity of VSFS to order/disorder transitions of the lipid acyl chains was used to determine the main gel to liquid crystal phase transition temperature, Tm, for HBMs of binary cholesterol/phospholipid mixtures on octadecanethiolate self-assembled monolayers. The phase transition temperature and the breadth of the transition were shown to increase with cholesterol content, and the phase boundaries observed in the cholesterol/phospholipid HBMs were comparable to the published phase diagrams of binary cholesterol/phospholipid vesicles. A thermodynamic assessment of the cooperative units of the HBM phase transitions revealed the presence of <10 nm diameter domains that were independent of the cholesterol composition.     

Adsorption of amyloid beta (1-40) peptide to phosphatidylethanolamine monolayers.

The aggregation of soluble, nontoxic amyloid beta (Abeta) peptide to beta-sheet containing fibrils is assumed to be a major step in the development of Alzheimer's disease. Interactions of Abeta with neuronal membranes could play a key role in the pathogenesis of the disease. Herein, we study the adsorption of synthetic Abeta peptide to DPPE and DMPE monolayers (dipalmitoyl- and dimyristoylphosphatidylethanolamine). Both lipids exhibit a condensed monolayer state at 20 degrees C and form a similar lattice. However, at low packing densities (at large area per molecule), the length of the acyl chains determines the phase behavior, therefore DPPE is fully condensed whereas DMPE exhibits a liquid-expanded state with a phase transition at approximately 5-6 mNm(-1). Adsorption of Abeta to DPPE and DMPE monolayers at low surface pressure leads to an increase of the surface pressure to approximately 17 mNm(-1). The same was observed during adsorption of the peptide to a pure air-water interface. Grazing incidence X-ray diffraction (GIXD) experiments show no influence of Abeta on the lipid structure. The adsorption kinetics of Abeta to a DMPE monolayer followed by IRRAS (infrared reflection absorption spectroscopy) reveals the phase transition of DMPE molecules from liquid-expanded to condensed states at the same surface pressure as for DMPE on pure water. These facts indicate no specific interactions of the peptide with either lipid. In addition, no adsorption or penetration of the peptide into the lipid monolayers was observed at surface pressures above 30 mNm(-1). IRRAS allows the measurement of the conformation and orientation of the peptide adsorbed to the air-water interface and to a lipid monolayer. In both cases, with lipids at surface pressures below 20 mNm(-1) and at the air-water interface, adsorbed Abeta has a beta-sheet conformation and these beta-sheets are oriented parallel to the interface.     

Surface phase behavior and microstructure of lipid/PEG-emulsifier monolayer-coated microbubbles

Langmuir trough methods and fluorescence microscopy were combined to investigate the phase behavior and microstructure of monolayer shells coating micron-scale bubbles (microbubbles) typically used in biomedical applications. The monolayer shell consisted of a homologous series of saturated acyl chain phospholipids and an emulsifier containing a single hydrophobic stearate chain and polyethylene glycol (PEG) head group. PEG-emulsifier was fully miscible with expanded phase lipids and phase separated from condensed phase lipids. Phase coexistence was observed in the form of dark condensed phase lipid domains surrounded by a sea of bright, emulsifier-rich expanded phase. A rich assortment of condensed phase area fractions and domain morphologies, including networks and other novel structures, were observed in each batch of microbubbles. Network domains were reproduced in Langmuir monolayers under conditions of heating–cooling followed by compression–expansion, as well as in microbubble shells that underwent surface flow with slight compression. Domain size decreased with increased cooling rate through the phase transition temperature, and domain branching increased with lipid acyl chain length at high cooling rates. Squeeze-out of the emulsifier at a surface pressure near 35 mN/m was indicated by a plateau in Langmuir isotherms and directly visualized with fluorescence microscopy, although collapse of the solid lipid domains occurred at much higher surface pressures. Compression of the monolayer past the PEG-emulsifier squeeze-out surface pressure resulted in a dark shell composed entirely of lipid. Under certain conditions, the PEG-emulsifier was reincorporated upon subsequent expansion. Factors that affect shell formation and evolution, as well as implications for the rational design of microbubbles in medical applications, are discussed.     

Thermodynamics of phase transitions in langmuir monolayers observed by vibrational sum frequency spectroscopy

Vibrational sum-frequency spectroscopy (VSFS) was used to study gauche defects in octadecylamine (ODA) monolayers at the air/water interface. The VSFS spectra provide unique insights into phase transitions that occur as a result of changes in the structure of the monolayer's hydrophobic region. These changes can be attributed to the increased presence of gauche conformers in the ODA alkyl chains during the monolayer's transition from the solid to liquid phase. Temperature-dependent spectra from monolayers at several different pressures were used to assign the phase transition temperature based on the observed changes in microscopic structure. Through application of a two-dimensional form of the Clapeyron equation, the first in situ measurements of the entropy and enthalpy changes associated with gauche conformers in a monolayer were made.     

Structure of mixed micelles formed in PEG-lipid/lipid dispersions

Polyethylene glycol (PEG)-conjugated lipids are commonly employed for steric stabilization of liposomes. When added in high concentrations PEG-lipids induce formation of mixed micelles, and depending on the lipid composition of the sample, these may adapt either a discoidal or a long threadlike shape. The factors governing the type of micellar aggregate formed have so far not been investigated in detail. In this study we have systematically varied the lipid composition in lipid/PEG-lipid mixtures and characterized the aggregate structure by means of cryo-transmission electron microscopy (cryo-TEM). The effects caused by adding sterols, phosphatidylethanolamines, and phospholipids with saturated acyl chains to egg phosphatidylcholine/1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)-2000 (EPC/DSPE-PEG2000) mixtures with a fixed amount (25 mol %) of DSPE-PEG2000 was studied. Further, the aggregate structure in 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine/1,2-dimyristoyl-sn-glycero-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)-2000] (DMPC/DMPE-PEG2000) samples above and below the gel to liquid crystalline phase transition temperature (TC) was investigated. Our results revealed that lipid components, as well as environmental conditions, that reduce the lipid spontaneous curvature and increase the monolayer bending modulus tend to promote formation of discoidal micelles. At temperatures below the gel-to-liquid crystalline phase transition temperature reduced lipid/PEG-lipid miscibility, furthermore, likely contribute to the observed formation of discoidal rather than threadlike micelles.     

Langmuir-Blodgett films of fluorinated glycolipids and polymerizable lipids and their phase separating behavior

This paper describes the phase separating behavior of Langmuir monolayers from mixtures of different lipids that (i) either carry already a glycopeptide recognition site or can be easily modified to carry one and (ii) polymerizable lipids. To ensure demixing during compression, we used fluorinated lipids for the biological headgroups and hydrocarbon based lipids as polymerizable lipids. As a representative for a lipid monomer, which can be polymerized in the hydrophilic headgroup, a methacrylic monomer was used. As a monomer, which can be polymerized in the hydrophobic tail, a lipid with a diacetylene unit was used (pentacosadiynoic acid, PDA). The fluorinated lipids were on the one hand a perfluorinated lipid with three chains and on the other hand a partially fluorinated lipid with a T(N)-antigen headgroup. The macroscopic phase separation was observed by Brewster angle microscopy, whereas the phase separation on the nanoscale level was observed by atomic force microscopy. It turned out that all lipid mixtures showed (at least) a partial miscibility of the hydrocarbon compounds in the fluorinated compounds. This is positive for pattern formation, as it allows the formation of small demixed 2D patterned structures during crystallization from the homogeneous phase. For miscibility especially a liquid analogue phase proved to be advantageous. As lipid 3 with three fluorinated lipid chains (very stable monolayer) is miscible with the polymerizable lipids 1 and 2, it was mostly used for further investigations. For all three lipid mixtures, a phase separation on both the micrometer and the nanometer level was observed. The size of the crystalline domains could be controlled not only by varying the surface pressure but also by varying the molar composition of the mixtures. Furthermore, we showed that the binary mixture can be stabilized via UV polymerization. After polymerization and subsequent expansion of the barriers, the locked-in polymerized structures are stable even at low surface pressures (10 mN/m), where the unpolymerized mixture did not show any segregation.     

Raman spectroscopic study of boundary lipid in 1,2-dipalmitoylphosphatidylcholine/apolipoprotein A—I recombinants

Raman spectroscopy has been used to obtain thermal phase transition profiles for recombinant particles composed of 1,2-dipalmitoylphosphatidylcholine (DPPC) and apolipoprotein A—I. Comparison of these profiles with unilamellar vesicles of DPPC indicates that lateral packing of DPPC acyl chains is tighter in recombinant DPPC/apolipoporotein A—I particles than in uncomplexed lipid of unilamellar vesicles. Consequently, the magnitude of the entropy change associated with acyl chain melting in the recombinants at the main lipid phase transition is almost twice that of unilamellar DPPC. In addition, a second phase transition has been observed for the DPPC/apolipoprotein A—I complex and has been assigned to the acyl chain melting of DPPC molecules which are bound to the apolipoprotein annulus on the periphery of the discoidal complexes. A combination of results from Raman spectroscopy, electron micrograph measurements and chemical analysis leads to the conclusion that these protein-bound lipids, the “boundary layer”, account for about 20% of the total lipid in the recombinant material. Calculations indicate that there are about 55 protein-bound lipid molecules per apolipoprotein A—I molecule in the DPPC/apolipoprotein A—I discoidal complexes prepared for this study.     

Free pyrene probes in gel and fluid membranes: perspective through atomistic simulations

We consider the properties of free pyrene probes inside gel- and fluidlike phospholipid membranes and unravel their influence on membrane properties. For this purpose, we employ atomic-scale molecular dynamics simulations at several temperatures for varying pyrene concentrations. Molecular dynamics simulations show that free pyrene molecules prefer to be located in the hydrophobic acyl chain region close to the glycerol group of lipid molecules. Their orientation is shown to depend on the phase of the membrane. In the fluid phase, pyrenes favor orientations where they are standing upright in parallel to the membrane normal, while, in the gel phase, the orientation is affected by the tilt of lipid acyl chains. Pyrenes are found to locally perturb membrane structure, while the nature of perturbations in the gel and fluid phases is completely different. In the gel phase, pyrenes break the local packing of lipids and decrease the ordering of lipid acyl chains around them, while, in the fluid phase, pyrenes increase the ordering of nearby acyl chains, thus having an opposite effect. Interestingly, this proposes a similarity to effects induced by cholesterol on structural membrane properties above and below the gel-fluid transition temperature. Further studies express a view that the orientational ordering of pyrene is not a particularly good measure of the acyl chain ordering of lipids. While pyrene ordering provides the correct qualitative behavior of acyl chain ordering in the fluid phase, its capability to predict the correct temperature dependence is limited.     

Sum-frequency spectroscopic study of Langmuir monolayers of lipids having oppositely charged headgroups

Sum-frequency vibrational spectroscopy, with the help of surface pressure-area (π-A) isotherm, was used to study lipid Langmuir monolayers composed of molecules with positively and negatively charged headgroups as well as a 1:1 neutral mixture of the two. The spectral profiles of the CH(x) stretch vibrations are similar for all monolayers in the liquid-condensed (LC) phase. They suggest a monolayer structure of closely packed alkyl chains that are nearly all-trans and well oriented along the surface normal. In the liquid-expanded (LE) phase, the spectra of all monolayers appear characteristic of loosely packed chains with significant gauche defects. The OH stretch spectra of interfacial water for both positively and negatively charged monolayers are significantly enhanced in comparison with a neutral water interface, but the phase measurement of SFVS indicates that OH in the two cases points toward the bulk and the interface, respectively. The enhancement results mainly from surface-field-induced polar ordering of interfacial water molecules. For a charge-neutral monolayer composed of an equal number of positively and negatively charged lipid molecules, no such enhancement is observed. This mixed monolayer exhibits a wide range of LC/LE coexistence region extended to very low surface pressure and its CH(x) spectral profile in the coexistence region resembles that of the LC phase. This result suggests that in the LC/LE coexistence region, the mixed monolayer consists of coexisting LC and LE patches in which oppositely charged lipid molecules are homogeneously mixed and dispersed.     

Preparation and characterization of asymmetric planar supported bilayers composed of poly(bis-sorbylphosphatidylcholine) on n-octadecyltrichlorosilane SAMs

Planar supported lipid bilayers (PSLBs) have been widely studied as biomembrane models and biosensor scaffolds. For technological applications, a major limitation of PSLBs composed of fluid lipids is that the bilayer structure is readily disrupted when exposed to chemical, mechanical, and thermal stresses. A number of asymmetric supported bilayer structures, such as the hybrid bilayer membrane (HBM) and the tethered bilayer lipid membrane (tBLM), have been created as an alternative to symmetric PSLBs. In both HBMs and tBLMs, the inner monolayer is covalently attached to the substrate while the outer monolayer is typically composed of a fluid lipid. Here we address if cross-linking polymerization of the lipids in the outer monolayer of an asymmetric supported bilayer can achieve the high degree of stability observed previously for symmetric PSLBs in which both monolayers are cross-linked [E.E. Ross, L.J. Rozanski, T. Spratt, S.C. Liu, D.F. O'Brien, S.S. Saavedra, Langmuir 19 (2003) 1752]. To explore this issue, HBMs composed of an outer monolayer of a cross-linkable lipid, bis-sorbylphosphatidylcholine (bis-SorbPC), and an inner SAM were prepared and characterized. Several experimental conditions were varied: vesicle fusion time, polymerization method, and polymerization time and temperature. Under most conditions, bis-SorbPC cross-linking stabilized the HBM such that its bilayer structure was largely preserved after drying; however these films invariably contained sub-micron scale defects that exposed the hydrophobic core of the HBM. The defects appear to be caused by desorption of low molecular weight oligomers when the film is removed from water, rinsed, and dried. In contrast, poly(bis-SorbPC) PSLBs prepared under similar conditions by Ross et al. were nearly defect free. This comparison shows that formation of a cross-linked network in the outer leaflet of an asymmetric supported bilayer is insufficient to prevent lipid desorption; inter-leaflet covalent linking appears to be necessary to create supported poly(lipid) assemblies that are impervious to repeated drying and rehydration. The difference in stability is attributed to inter-leaflet cross-linking between monolayers which can form in symmetric bis-SorbPC PSLBs.     

Influence of the physical state of phospholipid monolayers on protein binding

Langmuir monolayers were used to characterize the influence of the physical state of phospholipid monolayers on the binding of protein Retinis Pigmentosa 2 (RP2). The binding parameters of RP2 (maximum insertion pressure (MIP), synergy and ΔΠ(0)) in monolayers were thus analyzed in the presence of phospholipids bearing increasing fatty acyl chain lengths at temperatures where their liquid-expanded (LE), liquid-condensed (LC), or solid-condensed (SC) states can be individually observed. The data show that a larger value of synergy is observed in the LC/SC states than in the LE state, independent of the fatty acyl chain length of phospholipids. Moreover, both the MIP and the ΔΠ(0) increase with the fatty acyl chain length when phospholipids are in the LC/SC state, whereas those binding parameters remain almost unchanged when phospholipids are in the LE state. This effect of the phospholipid physical state on the binding of RP2 was further demonstrated by measurements performed in the presence of a phospholipid monolayer showing a phase transition from the LE to the LC state at room temperature. The data collected are showing that very similar values of MIP but very different values of synergy and ΔΠ(0) are obtained in the LE (below the phase transition) and LC (above the phase transition) states. In addition, the binding parameters of RP2 in the LE (below the phase transition) as well as in the LC (above the phase transition) states were found to be indistinguishable from those where single LC and LE states are respectively observed. The preference of RP2 for binding phospholipids in the LC state was then confirmed by the observation of a large modification of the shape of the LC domains in the phase transition. Therefore, protein binding parameters can be strongly influenced by the physical state of phospholipid monolayers. Moreover, measurements performed with the α/β domain of RP2 strongly suggest that the β helix of RP2 plays a major role in the preferential binding of this protein to phospholipids in the LC state.     

Effects of lipid chain unsaturation and headgroup type on molecular interactions between paclitaxel and phospholipid within model biomembrane

Molecular interactions between paclitaxel, an anticancer drug, and phospholipids of various chain unsaturations and headgroup types were investigated in the present study by Langmuir film balance and differential scanning calorimetry. Both the lipid monolayer at the air-water interface and the lipid bilayer vesicles (liposomes) were employed as model cell membranes. It was found that, regardless of the difference in molecular structure of the lipid chains and headgroup, the drug can form nonideal, miscible systems with the lipids at the air-water interface over a wide range of paclitaxel mole fractions. The interaction between paclitaxel and phospholipid within the monolayer was dependent on the molecular area of the lipids at the interface and can be explained by intermolecular forces or geometric accommodation. Paclitaxel is more likely to form thermodynamically stable systems with 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC) and 1,2-dielaidoyl-sn-glycero-3-phosphocholine (DEPC) than with 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). Investigation of the drug penetration into the lipid monolayer showed that DPPC and DEPC have higher incorporation abilities for the drug than DPPE and DSPC. A similar trend was also evidenced by DSC investigation with liposomes. While little change of DSC profiles was observed for the DPPE/paclitaxel and DSPC/paclitaxel liposomes, paclitaxel caused noticeable changes in the thermographs of DPPC and DEPC liposomes. Paclitaxel was found to cause broadening of the main phase transition without significant change in the peak melting temperature of the DPPC bilayers, which demonstrates that paclitaxel was localized in the outer hydrophobic cooperative zone of the bilayer, i.e., in the region of the C1-C8 carbon atoms of the acyl chain or binding at the polar headgroup site of the lipids. However, it may penetrate into the deeper hydrophobic zone of the DEPC bilayers. These findings provide useful information for liposomal formulation of anticancer drugs as well as for understanding drug-cell membrane interactions.     

Determination of the line tension of giant vesicles from pore-closing dynamics

Giant vesicles generated from synthetic and natural lipids such as phosphatidylcholines are useful models for understanding mechanical properties of cell membranes. Line tension is the one-dimensional force enabling the closing of transient pores on cell membranes. Transient pores were repeatedly and reproducibly formed on the membrane edge of giant vesicles generated from synthetic and natural phosphatidylcholines employing a nitrogen-pumped coumarin dye laser (440 nm). Line tension was determined at room temperature from closing of these pores that occurred over several seconds when the radius of the vesicle could be considered to be constant. The value of line tension depends on the nature of the lipid for single lipid systems, which, at room temperature, yielded a vesicle bilayer region in the gel, fluid, or mixed gel and fluid phases. The line tension for vesicles generated from phosphatidylcholines with saturated acyl chains of lengths of 12-18 carbon atoms ranges from 1 to 12 pN, exhibiting an increase with chain length. Vesicles generated from the natural Egg-PC, which is a mixture of lipids, are devoid of phase transition and exhibited the largest value of line tension (32 pN). This value is much larger than that estimated from the line tensions of vesicles obtained from lipids with homologous acyl chains. This study, to our knowledge, is the first to employ laser ablation to generate transient pores and determine line tension from the rate of pore closure and demonstrate a relationship between line tension and acyl chain length.     

Construction of higher-ordered monolayer membranes derived from archaeal membrane lipid-inspired cyclic lipids with longer alkyl chains

A series of artificial cyclic lipids that mimic archaeal membrane ones has been synthesized. The structural features of these molecules include a longer cyclic framework, in which the alkyl chain length ranges from 24 to 32 in carbon number, which is longer than our first analogous molecule with 20-carbon long alkyl chains [K. Miyawaki, T. Takagi, M. Shibakami, Synlett 8 (2002) 1326]. Microscopic observation reveals that these molecules have a self-assembling ability: hydration of the lipids yields multilamellar vesicles in aqueous solution and monolayer sheets on solid supports. High-sensitivity differential scanning calorimetry (24- and 28-carbon alkyl chain lipids) indicates that (i) the alkyl chain length affects their phase behavior and (ii) the enthalpies of endothermic peaks accompanied by phase transition were considerably lower than those of their monomeric phospholipid analogs. Fluorescence polarization measurements suggest that the membranes made from the 24-carbon alkyl chain lipid have a higher polarization factor than membranes composed of DMPC and DMPC plus cholesterol. These findings imply that the cyclic lipids containing 24- and 28-carbon alkyl chain construct well-organized monolayer membranes and, in particular, that the molecular order of the 24-carbon alkyl chain lipid is higher than that of bilayer membranes in the liquid-ordered phase.     

Simulation studies of pore and domain formation in a phospholipid monolayer

Despite extensive study the phase behavior of phospholipid monolayers at an air-water interface is still not fully understood. In particular recent vibrational sum-frequency generation (VSFG) spectra of DPPC monolayers as a function of area density show a sharp transition in the order of the lipid chains at 1.10 nm2/molecule. This is in a region where the lateral pressure as a function of area is effectively constant. We have investigated the nature of this transition by studying the phase behavior of DPPC monolayers as a function of area density using molecular-dynamics simulations. The changes in order within the monolayer as a function of area density correlate well with the experimental signal. At 0.58 nm2/molecule we observe the onset of lateral separation of highly ordered and disordered lipids, indicating the coexistence of a gel-like liquid condensed and a fluidlike liquid expanded phase. At 0.97 nm2/molecule the monolayer ruptures, marking the onset of the liquid-gas (G) coexistence region. This is much earlier than suggested by fluorescence microscopy results and implies that at the point of rupture, the initial pores have an equilibrium size smaller than approximately 500 nm in diameter. The rupture of the monolayer leads to a sharp increase in the overall lipid order that explains the sharp transition observed in the VSFG measurements. VSFG measurements thus may represent a sensitive means to determine the onset of the liquid-gas (G) coexistence region for such systems.     

Infrared reflection absorption spectroscopy coupled with Brewster angle microscopy for studying interactions of amphiphilic triblock copolymers with phospholipid monolayers

Novel water-soluble amphiphilic triblock copolymers poly(glycerol monomethacrylate)-b-poly(propylene oxide)-b-poly(glycerol monomethacrylate) (PGMA-b-PPO-b-PGMA) were synthesized because of their expected enhanced ability to interact with biological membranes compared to the well-known poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) (PEO-b-PPO-b-PEO) block copolymers. Their bulkier hydrophilic PGMA blocks might induce a disturbance in the packing of liquid-crystalline lipid bilayers in addition to the effect caused by the hydrophobic PPO block alone. To gain a better insight into the polymer-membrane interactions at the molecular level, the adsorption kinetics and concomitant interactions of (PGMA14)(2-)PPO(34) with model membranes of dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) were monitored using infrared reflection absorption spectroscopy (IRRAS) coupled with Brewster angle microscopy (BAM) and surface pressure (pi) measurements. The maximum penetration surface pressure of ca. 39 mN/m suggests that (PGMA14)(2-)PPO(34) is able to insert into lipid monolayers even above the so-called monolayer-bilayer equivalent pressure of 30-35 mN/m. Copolymer adsorption to a liquid-expanded DPPC-d62 monolayer proceeds in a two-step mechanism: (i) initially only the more hydrophobic PPO middle block penetrates the lipid monolayer; (ii) following the liquid-expanded-liquid-condensed (LE-LC) phase transition, the bulky PGMA hydrophilic blocks are dragged into the headgroup region as the PPO block inserts further into the fatty acid region. The adsorption kinetics is considerably faster for DMPC-d54 monolayers due to their higher fluidity. Copolymer adsorption to an LC-DPPC-d62 monolayer leads to a change in the monolayer packing by forcing the lipid alkyl chains into a more vertical orientation, their tilt angle with respect to the surface normal being reduced from initially 30 degrees +/- 3 degrees to 18 degrees +/- 3 degrees. BAM images rule out macroscopic phase separation and show that coalescence of DPPC-d62 LC domains takes place at relatively low surface pressures of pi > or = 23 mN/m, suggesting that (PGMA14)(2-)PPO (34) partitions into both LE as well as LC domains.     

High pressure effect on phase transition behavior of lipid bilayers

Phase behavior of lipid bilayers at high pressure is critical to biological processes. Using coarse grained molecular dynamic simulations, we report critical characteristics of dipalmitoylphosphatidylcholine bilayers with applied high pressure, and also show their phase transition by cooling bilayer patches. Our results indicate that the phase transition temperature of dipalmitoylphosphatidylcholine bilayers obviously shifts with pressure increasing in the rate of 37 °C kbar(-1), which are in agreement with experimental data. Moreover, the main phase transition is revealed to be strongly dependent on lipid area. A critical lipid area of ~0.57 nm(2) is found on the main phase transition boundary. Similar structures of acyl chains lead to the same sensitivity of phase transition temperature of different lipids to the pressure. Based on the lateral density and pressure profiles, we also discuss the different effects on bilayer structure induced by high temperature and high pressure, e.g., increasing temperature induces higher degree of interdigitation of lipid tails and thinner bilayers, and increasing pressure maintains the degree of interdigitation and bilayer thickness.     

Structure and thermotropic phase behavior of fluorinated phospholipid bilayers: a combined attenuated total reflection FTIR spectroscopy and imaging ellipsometry study

Lipid bilayers consisting of lipids with terminally perfluoroalkylated chains have remarkable properties. They exhibit increased stability and phase-separated nanoscale patterns in mixtures with nonfluorinated lipids. In order to understand the bilayer properties that are responsible for this behavior, we have analyzed the structure of solid-supported bilayers composed of 1,2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC) and of a DPPC analogue with 6 terminal perfluorinated methylene units (F6-DPPC). Polarized attenuated total reflection Fourier-transform infrared spectroscopy indicates that for F6-DPPC, the tilt of the lipid acyl chains to the bilayer normal is increased to 39 degrees as compared to 21 degrees for native DPPC, for both lipids in the gel phase. This substantial increase of the tilt angle is responsible for a decrease of the bilayer thickness from 5.4 nm for DPPC to 4.5 nm for F6-DPPC, as revealed by temperature-controlled imaging ellipsometry on microstructured lipid bilayers and solution atomic force microscopy. During the main phase transition from the gel to the fluid phase, both the relative bilayer thickness change and the relative area change are substantially smaller for F6-DPPC than for DPPC. In light of these structural and thermotropic data, we propose a model in which the higher acyl-chain tilt angle in F6-DPPC is the result of a conformational rearrangement to minimize unfavorable fluorocarbon-hydrocarbon interactions in the center of the bilayer due to chain staggering.     

Correlation of the physicochemical properties of symmetric 1,3-dialkoylamidopropane-based cationic lipids containing single primary and tertiary amine polar head groups with in vitro transfection activity

The physicochemical properties of a novel series of symmetric 1,3-dialkylamidopropane-based cationic amphiphiles [M. Sheikh, J. Feig, B. Gee, S. Li, M. Savva, In vitro lipofection with novel series of symmetric 1,3-dialkoylamidopropane-based cationic surfactants containing single primary and tertiary amine polar head groups, Chem. Phys. Lipids 124 (2003) 49-61] were studied by several techniques, in an effort to correlate cationic lipid structure with transfection efficacy. It was found that only the unsubstituted amine and tertiary amine dioleoyl derivatives 1,3lmp5 and 1,3lmt5, respectively, mediated in vitro transfection activity in the absence of helper lipids. This activity pattern was consistent with ethidium bromide fluorescence quenching studies, which indicated that only these two derivatives bound to and efficiently condense plasmid DNA at physiological pH. Dynamic light scattering indicated that lipoplexes made by these two cationic lipids were relatively small particles below 1 microm, in sharp contrast to lipoplexes bigger than 3 microm composed of saturated cationic derivatives. Transmission electron microscopy studies clearly indicated that cationic lipid dispersions made by saturated derivatives form multilamellar tubules at physiological pH. Calorimetric studies showed that cationic amphiphiles with saturated acyl chains longer than 12 carbons exhibit solid-to-liquid crystalline phase transitions above 37 degrees C. In agreement with the microscopy and calorimetry studies, Langmuir film balance experiments indicated that saturated derivatives with hydrophobic chains longer that 12 carbons are not well hydrated and exist at a chain-ordered state at ambient temperature. Calculation of compressibility moduli from monolayer compression isotherms at 23 degrees C suggested that monolayers made by cationic lipids bearing saturated acyl chains are less compressible relative to those of the dioleoyl derivatives 1,3lmp5 and 1,3lmt5. In conclusion, high hydration, increased fluidity and high elasticity of cationic lipid assemblies in isolation, all correlate with high in vitro transfection activity.