Binary Lipid Mixtures Research Articles

Structural characterization of liposomes made of diether archaeal lipids and dipalmitoyl-L-α-phosphatidylcholine

The physicochemical properties of binary lipid mixtures of diether C 25,25 lipids and dipalmitoyl-L-α-phosphatidylcholine (DPPC) were studied using photon correlation, fluorescence and electron paramagnetic resonance spectroscopy, and transmission electron microscopy. These two types of lipids can be mixed at all molar ratios to form unilamellar and multilamellar liposomes. Fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatrien in mixed liposomes indicates that the abrupt changes in order parameter in the hydrophobic part of bilayer membranes made of DPPC lipids disappears with increasing mol% C 25,25 lipids. Electron paramagnetic resonance spectroscopy shows that at temperatures below 50 °C, the interfacial regions of membrane bilayer of mixed liposomes is more fluid than for pure DPPC liposomes, while at higher temperatures, the impact of the long isoprenoid chains on the membrane stability becomes more pronounced. Photon correlation spectroscopy and transmission electron microscopy show that mixed liposomes do not fuse or aggregate, even after 41 days at 4 °C.

Fluid-Fluid Phase Separation in Cholesterol-Containing Membranes: Rafts and Non-Rafts

Fluid-fluid phase separation is the functionally important mode of in-plane domain formation in lipid-bilayer membranes. The most common, if not sole, well-established case of fluid phase separation is that between liquid-ordered (Lo) and liquid-disordered (Lα) phases in cholesterol-containing membranes. The liquid-ordered phase is characterised by short-range orientational order, but long-range translational disorder. It is well documented for gel-fluid (Lα-Lo) phase coexistence in binary lipid mixtures with cholesterol but, surprisingly, direct evidence for the functionally significant fluid-fluid (Lα-Lo) phase coexistence is rather sparse. Interestingly, evidence for Lα-Lo coexistence is more abundant in ternary mixtures of a high-chain-melting lipid and a low-chain-melting lipid with cholesterol than in binary mixtures. Here, I shall review the current status of the field: it seems that no aspect is without some controversy. Published phase diagrams for the canonical “raft” lipid mixtures, N-palmitoyl sphingomyelin/palmitoyl-oleoyl phosphatidylcholine/cholesterol will be compared, and also new spin-label EPR results on this system will be presented.Marsh, D., 2009. Cholesterol-induced fluid membrane domains: a compendium of lipid-raft ternary phase diagrams, Biochim. Biophys. Acta 1788, 2114-2123.Marsh, D., 2010. Liquid-ordered phases induced by cholesterol: a compendium of binary phase diagrams, Biochim. Biophys. Acta 1798, 688-699.

The Competitive Binding of Ca 2+ vs. Mg 2+ to PIP2-Containing Lipid Monolayers and the Comparison of Pi(4,5)P2 and its Isomers

Interactions between divalent cations and PtdIns(4,5)2-containing lipid model membrane are investigated. Surface pressure measurements of lipid monolayers formed by binary lipid mixtures containing 25mol% PIP2 were used to quantify divalent cation binding. Direct titration and competitive binding assays show that divalent cations including Mg2+, Ca2+, Sr2+, Ba2+, Co2+, Ni2+, Cu2+, Zn2+ and some multivalent polyamines bind PIP2 with Kd ranges from 200nM-50uM. Some of these cations, which are considered physiologically important, were examined more closely and evidence for divalent cation-induced lateral segregation of PIP2 is presented. Such studies are based on fluorescence and atomic force microscopic studies of Langmuir-Schaeffer lipid monolayers, and are coupled with numerical studies. Ca2+ and Mg2+ have similar binding affinity to PIP2-containing monolayers, but Ca2+ induces PIP2 lateral segregation more efficiently than compared to Mg2+. Ca2+ vs. Mg2+ competitive binding assays are also carried out on PtdIns(3,4)P2- and PtdIns(3,5)P2-containing monolayers. Significant differences in the response of these three PIP2 isomers to divalent cations are found. Besides monolayer studies, PIP2-cation interactions are also examined on a bilayer system. Fluorescence correlation spectroscopy (FCS) and laser scanning confocal microscopy (LSCM) are used following divalent cation titration on asymmetric labeled PIP2-containing giant unilamellar vesicles (GUVs). The diffusion coefficient and the fluorescence intensity variance of fluorescent PIP2 change with increasing divalent cation concentration. Förster resonance energy transfer (FRET) studies using bi-color labeled PIP2 are also carried out in a large unilamellar vesicle (LUV) system. These results together reveal that the lateral inhomogeneity of PIP2 changes with divalent cation concentration on bilayer model membranes. These results may provide insight into divalent cation-induced PIP2 microdomain formation in the cell membrane.

Cardiolipin, a Key Component to Mimic the E. coli Bacterial Membrane in Model System Membranes

The phase transition temperatures of several lipidic systems were determined using two different techniques: dynamic light scattering (DLS) and steady-state fluorescence anisotropy, using two fluorescent probes that report different membrane regions (TMA DPH and DPH). Atomic force microscopy (AFM) was used as a complementary technique to characterize different lipid model systems under study. The systems were chosen due to the increased interest in bacterial membrane studies due to the problem of antibiotic drug resistance. The simpler models studied comprised of mixtures of POPE and POPG lipids, which form a commonly used model system for E. coli membranes. Given the important role of cardiolipin (CL) in natural membranes, a ternary model system, POPE/POPG/CL, was then considered. The results obtained in these mimetic systems were compared to those obtained for the natural systems E. coli polar and total lipid extract. DLS and fluorescence anisotropy are not commonly used to study lipid phase transitions, but it was shown that they can give useful information about the thermotropic behaviors of model systems for bacterial membranes. These two techniques provided very similar results, validating their use as methods to measure phase transitions in lipid model systems. The temperature transitions obtained from these two very different techniques and the AFM results clearly show that cardiolipin is a fundamental component to mimic bacteria membranes. The results suggest that the less commonly used ternary system is a considerably better mimic for natural E. coli membranes than binary lipid mixture.

Enthalpy Drives Phase Separation in Model Membranes: Evidence from Molecular Dynamics Simulations of Non-Lipid Amphiphiles Vitamin-E, triton-X, and Benzyl Alcohol

Phase separation of lipid bilayers into liquid-ordered and liquid-disordered regions facilitates compartmentalization of membrane proteins, an important prerequisite for cellular biochemical signaling. In this study, we investigated the phase behavior of binary and ternary lipid mixtures under the influence of three non-lipid amphiphiles, Vitamin-E, Triton-X 100, and benzyl alcohol. Molecular dynamics (MD) simulations of these additives in fluid-phase lipid bilayers were performed to investigate their effect on membrane thickness and fluidity as readouts of enthalpic and entropic changes, respectively. Simulation results indicate that Vitamin-E increases bilayer thickness and lowers the fluidity, while Triton-X and benzyl alcohol each decrease bilayer thickness and increase fluidity. Experimentally, in both binary and ternary lipid mixtures, Vitamin-E induced ideal mixing of the lipids whereas Triton-X and benzyl alcohol either resulted in sustained phase separation or induced large-scale phase separation. Because changes in bilayer thickness determined by MD correlate better than fluidity with phase separation observed in model membranes we conclude that hydrophobic mismatch of different lipid types (enthalpic effect) dominate the effect of fluidity (entropic effect) as the key driving force for the phase separation behavior observed in lipid bilayers. Moreover, these non-lipid amphiphiles provide new tools to tune raft formation/disruption and to study raft-related cell membrane functions through modulation of non-raft membrane thickness.

Revealing the selective interactions of fibronectin with lipid bilayers

The interaction of extracellular matrix proteins with lipid bilayers is fundamental to cell adhesion and related processes such as tissue formation. In this paper we use atomic force microscopy to examine the interaction between fibronectin and supported lipid bilayers. For binary lipid mixtures that display phase separation, atomic force microscopy shows that fibronectin preferentially and almost exclusively interacts with the gel phase domains, sitting on top of these raised regions. Single molecule force spectroscopy confirms the location of fibronectin on the bilayer and reveals that the mechanical properties of fibronectin are affected by aggregation of the protein on the lipid. We demonstrate that it is possible to measure the thickness of the protein aggregates by identifying a protein layer breakthrough event. Of particular interest are force–distance curves that exhibit a combination of three features, a protein layer breakthrough event, a lipid bilayer breakthrough event, and a sawtooth pattern attributed to fibronectin unfolding. The combination of lipid- and protein-related events on a single force–distance profile, characterizes in detail the lipid/protein interaction. Preferential interactions of fibronectin with lipids suggest that non-specific binding could be important in extracellular matrix protein-cell interactions and that the lateral organization of the cell membrane may play an even greater role in cell adhesion than previously thought.

Synthesis of organic–inorganic hybrid bicelles–lipid bilayer nanodiscs encompassed by siloxane surfaces

Well-defined hybrid nanodiscs were produced by employing bicelle formation of a binary lipid mixture in water. The resulting nanoparticles have a lipid bilayer coated with ceramic layers, which are formed by the sol-gel reaction among the alkoxysilyl headgroups of the hybrid lipid. The hybrid bicelles displayed significant morphological stability against dry environments and surfactant addition, in stark contrast to conventional phospholipid bicelles.

Nanoscale chemical imaging of segregated lipid domains using tip-enhanced Raman spectroscopy

Lipid domains in supported lipid layers serve as a popular model to gain insight into the processes associated with the compartmentalization of biological membranes into so-called lipid rafts. In this paper, we present reproducible tip-enhanced Raman spectra originating from a very small number of molecules in a lipid monolayer on a gold surface, probed by the apex of a nanometer-sized silver tip. For the first time, we show large (128 × 128 pixels), high-resolution (< 50 nm) tip-enhanced Raman images of binary lipid mixtures with full spectral information at each pixel.

An X‐ray diffraction study of model membrane raft structures

Protein sorting and assembly in membrane biogenesis and function involves the creation of ordered domains of lipids known as membrane rafts. The rafts are comprised of all the major classes of lipids, including glycerophospholipids, sphingolipids and sterol. Cholesterol is known to interact with sphingomyelin to form a liquid-ordered bilayer phase. Domains formed by sphingomyelin and cholesterol, however, represent relatively small proportions of the lipids found in membrane rafts and the properties of other raft lipids are not well characterized. We examined the structure of lipid bilayers comprised of aqueous dispersions of ternary mixtures of phosphatidylcholines and sphingomyelins from tissue extracts and cholesterol using synchrotron X-ray powder diffraction methods. Analysis of the Bragg reflections using peak-fitting methods enables the distinction of three coexisting bilayer structures: (a) a quasicrystalline structure comprised of equimolar proportions of phosphatidylcholine and sphingomyelin, (b) a liquid-ordered bilayer of phospholipid and cholesterol, and (c) fluid phospholipid bilayers. The structures have been assigned on the basis of lamellar repeat spacings, relative scattering intensities and bilayer thickness of binary and ternary lipid mixtures of varying composition subjected to thermal scans between 20 and 50 °C. The results suggest that the order created by the quasicrystalline phase may provide an appropriate scaffold for the organization and assembly of raft proteins on both sides of the membrane. Co-existing liquid-ordered structures comprised of phospholipid and cholesterol provides an additional membrane environment for assembly of different raft proteins.

Cardiolipin, a key component to mimic the E. coli bacterial membrane in model systems revealed by dynamic light scattering and steady-state fluorescence anisotropy

The phase transition temperatures of several lipidic systems were determined using two different techniques: dynamic light scattering (DLS) and steady-state fluorescence anisotropy, using two fluorescent probes that report different membrane regions (TMA-DPH and DPH). Atomic force microscopy (AFM) was used as a complementary technique to characterize different lipid model systems under study. The systems were chosen due to the increased interest in bacterial membrane studies due to the problem of antibiotic drug resistance. The simpler models studied comprised of mixtures of POPE and POPG lipids, which form a commonly used model system for Escherichia coli membranes. Given the important role of cardiolipin (CL) in natural membranes, a ternary model system, POPE/POPG/CL, was then considered. The results obtained in these mimetic systems were compared with those obtained for the natural systems E. coli polar and total lipid extract. DLS and fluorescence anisotropy are not commonly used to study lipid phase transitions, but it was shown that they can give useful information about the thermotropic behaviors of model systems for bacterial membranes. These two techniques provided very similar results, validating their use as methods to measure phase transitions in lipid model systems. The temperature transitions obtained from these two very different techniques and the AFM results clearly show that cardiolipin is a fundamental component to mimic bacteria membranes. The results suggest that the less commonly used ternary system is a considerably better mimic for natural E. coli membranes than binary lipid mixture.

Pattern Formation and Molecular Transport of Histidine-Tagged GFPs Using Supported Lipid Bilayers

We fabricated a heterogeneous supported lipid bilayer (SLB) by employing binary lipid mixtures comprising a saturated acyl chain DSPC and an unsaturated acyl chain nickel-chelating lipid. By using the specific adsorption properties of histidine-tagged proteins (His-tagged GFPs) in relation to nickel-chelating lipids, we demonstrated protein pattern formation on the SLB corresponding to the phase separation pattern of the SLB. In addition, by using a lipid mixture consisting of an unsaturated acyl chain DOPC and a nickel-chelating lipid, and His-tagged GFPs, we succeeded in transporting the proteins along a hydrophilic micropattern on a SiO(2) substrate. The protein transport is induced by the self-spreading behavior of a fluid SLB with a kinetic spreading coefficient beta = 10.4 microm(2) s(-1). This method provides a guide for strategically carrying various biomolecules to specific positions by using a soft biointerface on a solid surface. In addition, the results demonstrate the importance of using techniques that allow the controlled manipulation of biomolecules based on the static or dynamic properties of the SLB platform.

Solid-Supported Bilayer Lipid Membranes from Lipid Mixtures: Structure and Composition

Biological membranes are of overarching importance for all aspects of cell structure and function in living organisms. Planar tethered bilayer lipid membranes (tBLMs) are synthetic membrane models stabilized by the proximity of a solid substrate that enhances its long-time stability by orders of magnitude.[1,2] A nanometer-thin hydration layer between the bilayer and the substrate ensures that the biomimetic lipid membrane remains fluid with in-plane lipid dynamics similar to that in vesicles.[3] In this work we establish tBLMs composed of binary and ternary lipid mixtures as more complex, and hence more realistic, membrane models.

Lipid multilayered particles: the role of chitosan on structure and morphology

Multilayered nanovectors made up from a controlled binary lipid mixture (POPC and DMPS) and trimethyl chitosan (TMC) have been prepared and characterized by light- and small angle neutron scattering. The morphology and the multilayer structure of the particle outer shell has been described in detail. By varying the amount of TMC in the starting solution it is possible to tune the overall surface particle charge as well as its multilamellarity. In this way the drug loading/release properties of the particles can be controlled. Therefore the use of controlled POPC/DMPS mixtures can be a valid alternative to commercial lecithin to obtain nanovectors with specific release properties.

Properties of phosphatidylcholine in the presence of its monofluorinated analogue

In aqueous solution, the monofluorinated phospholipid 1-palmitoyl-2-[16-fluoropalmitoyl]sn-glycero-3-phosphocholine (F-DPPC) interdigitates without the use of inducing agents. To understand the thermal and physical properties of this unique lipid, F-DPPC was combined with the non-fluorinated 1,2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC), and 1,2-diarachidoyl- sn-glycero-3-phosphocholine (DAPC). Differential scanning calorimetry (DSC) was used to determine the miscibility and thermotropic phase behavior of these binary lipid mixtures. In addition, the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH) and a DPH-labeled analogue of DPPC, 2-(3-(diphenylhexatrienyl) propanoyl)-1-hexadecanoyl- sn-glycero-3-phosphocholine (β-DPH HPC, aka DPH-PC or DPHpPC), were used to detect interdigitation. In F-DPPC, the fluorescence intensity of both probes decreased a similar amount and to a degree that is consistent with an interdigitated system. We also determined that there are two separate effects of increasing the ratio of F-DPPC in the DPPC/F-DPPC system. With low amounts of F-DPPC, there is little evidence that the system is heavily interdigitated. Instead, we hypothesize that the introduction of F-DPPC provides nucleation sites that alter the kinetics, reversibility, and temperature of the main transition ( T m). At higher mol% of F-DPPC, we propose that interdigitated F-DPPC-rich domains form to create a phase-segregated system. While DPPC/F-DPPC was highly miscible, the DAPC/F-DPPC system was significantly less miscible. Additionally, we observed that DAPC/F-DPPC samples have reduced solubility in water, which affected the acquisition of fluorescence data. However, our DSC results indicate the existence of DAPC-rich and F-DPPC-rich components. Furthermore, this data support that the mixing was disruptive to lipid packing and that the presence of DAPC hinders the interdigitation of F-DPPC.

Micron-scale phase segregation in lipid monolayers induced by myelin basic protein in the presence of a cholesterol analog

It was previously shown that myelin basic protein (MBP) can induce phase segregation in whole myelin monolayers and myelin lipid films, which leads to the accumulation of proteins into a separate phase, segregated from a cholesterol-enriched lipid phase. In this work we investigated some factors regulating the phase segregation induced by MBP using fluorescent microscopy of monolayers formed with binary and ternary lipid mixtures of dihydrocholesterol (a less-oxidable cholesterol analog) and phospholipids. The influence of the addition of salts to the subphase and of varying the lipid composition was analyzed. Our results show that MBP can induce a dihydrocholesterol-dependent segregation of phases that can be further regulated by the electrolyte concentration in the subphase and the composition (type and proportion) of non-sterol lipids. In this way, changes of the lipid composition of the film or the ionic strength in the aqueous media modify the local surface density of MBP and the properties (phase state and composition) of the protein environment.

Lipid reorganization induced by membrane-active peptides probed using differential scanning calorimetry

The overlapping biological behaviors between some cell penetrating peptides (CPPs) and antimicrobial peptides (AMPs) suggest both common and different membrane interaction mechanisms. We thus explore the capacity of selected CPPs and AMPs to reorganize the planar distribution of binary lipid mixtures by means of differential scanning calorimetry (DSC). Additionally, membrane integrity assays and circular dichroism (CD) experiments were performed. Two CPPs (Penetratin and RL16) and AMPs belonging to the dermaseptin superfamily (Drs B2 and C-terminal truncated analog [1–23]-Drs B2 and two plasticins DRP-PBN2 and DRP-PD36KF) were selected. Herein we probed the impact of headgroup charges and acyl chain composition (length and unsaturation) on the peptide/lipid interaction by using binary lipid mixtures. All peptides were shown to be α-helical in all the lipid mixtures investigated, except for the two CPPs and [1–23]-Drs B2 in the presence of zwitterionic lipid mixtures where they were rather unstructured. Depending on the lipid composition and peptide sequence, simple binding to the lipid surface that occur without affecting the lipid distribution is observed in particular in the case of AMPs. Recruitments and segregation of lipids were observed, essentially for CPPs, without a clear relationship between peptide conformation and their effect in the lipid lateral organization. Nonetheless, in most cases after initial electrostatic recognition between the peptide charged amino acids and the lipid headgroups, the lipids with the lowest phase transition temperature were selectively recruited by cationic peptides while those with the highest phase transition were segregated. Membrane activities of CPPs and AMPs could be thus related to their preferential interactions with membrane defects that correspond to areas with marked fluidity. Moreover, due to the distinct membrane composition of prokaryotes and eukaryotes, lateral heterogeneity may be differently affected by cationic peptides leading to either uptake or/and antimicrobial activities.

Actin-induced perturbation of PS lipid–cholesterol interaction: A possible mechanism of cytoskeleton-based regulation of membrane organization

To obtain insight into the potential role of the cytoskeleton on lipid mixing behavior in plasma membranes, the current study explores the influence of physisorbed actin filaments (F-actin) on lipid-lipid phase separations in planar model membrane systems containing raft-mimicking lipid mixtures of well-defined compositions using a complementary experimental approach of epifluorescence microscopy, fluorescence anisotropy, wide-field single molecule fluorescence microscopy, and interfacial rheometry. In particular, we have explored the impact of F-actin on cholesterol (CHOL)-phospholipid interactions, which are considered important for the formation of CHOL-enriched lipid raft domains. By using epifluorescence microscopy, we show that physisorbed filamentous actin (F-actin) alters the domain size of lipid-lipid phase separations in the presence of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine (POPS) and cholesterol (CHOL). In contrast, no actin-induced modification in lipid-lipid phase separations is observed in the absence of POPS or when POPS is replaced by another anionic lipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG). Wide-field single molecule fluorescence microscopy on binary lipid mixtures indicate that PS and PG lipids show similar electrostatic interactions with physisorbed actin filaments. Complementary fluorescence anisotropy experiments on binary PS lipid-containing lipid mixtures are provided to illustrate the actin-induced segregation of anionic lipids. The similarity of electrostatic interactions between actin and both anionic lipids suggests that the observed differences in actin-mediated perturbations of lipid phase separations are caused by distinct PS lipid-CHOL versus PG lipid-CHOL interactions. We hypothesize that the actin cytoskeleton and some peripheral membrane proteins may alter lipid-lipid phase separations in plasma membranes in a similar way by interacting with PS lipids.

Path Dependence of Three-Phase or Two-Phase End Points in Fluid Binary Lipid Mixtures

The phase behavior of anionic/zwitterionic mixtures can be controlled by tuning the charge state of the anionic lipid. In the case of dioleoylphosphatidic acid (DOPA)/dioleoylphosphatidylcholine (DOPC) mixtures, demixing occurs either when DOPA is protonated or when DOPA(2-):Ca(2+) complexes form. Herein it will be shown that the final end point, a three-phase or two-phase system, depends on the order in which the charge state is manipulated. The facile accessibility of different end points is a clear demonstration of the inherent flexibility of biological systems.

Membrane Protein Frustration: Protein Incorporation into Hydrophobic Mismatched Binary Lipid Mixtures

Bacteriophage M13 major coat protein was reconstituted in different nonmatching binary lipid mixtures composed of 14:1PC and 22:1PC lipid bilayers. Challenged by this lose-lose situation of hydrophobic mismatch, the protein-lipid interactions are monitored by CD and site-directed spin-label electron spin resonance spectroscopy of spin-labeled site-specific single cysteine mutants located in the C-terminal protein domain embedded in the hydrophobic core of the membrane (I39C) and at the lipid-water interface (T46C). The CD spectra indicate an overall α-helical conformation irrespective of the composition of the binary lipid mixture. Spin-labeled protein mutant I39C senses the phase transition in 22:1PC, in contrast to spin-labeled protein mutant T46C, which is not affected by the transition. The results of both CD and electron spin resonance spectroscopy clearly indicate that the protein preferentially partitions into the shorter 14:1PC both above and below the gel-to-liquid crystalline phase transition temperature of 22:1PC. This preference is related to the protein tilt angle and energy penalty the protein has to pay in the thicker 22:1PC. Given the fact that in Escherichia coli, which is the host for M13 bacteriophage, it is easier to find shorter 14 carbon acyl chains than longer 22 carbon acyl chains, the choice the M13 coat protein makes seems to be evolutionary justified.

Formation of Block Liposomes is a General Phenomenon of Charged Membranes

We have recently reported on the discovery of block liposomes (BLs), a new class of chain-melted (liquid) vesicles, formed in mixtures of curvature-stabilizing hexadecavalent cationic lipid MVLBG2, DOPC and water (Zidovska et al., Langmuir, 2008). BLs consist of distinctly shaped, yet connected spheres, pears, tubes, or rods. Unlike typical liposome systems, where spherical vesicles, tubular vesicles, and cylindrical micelles are separated on the macroscopic scale, within a BL, shapes are separated on the nanometer scale. We carried out structural studies of BLs with differential-contrast-microscopy (DIC) and cryo-transmission-electron-microscopy (cryo-TEM) and identified membrane charge and spontaneous membrane curvature as key parameters controlling the BL-formation. BL-formation was believed to be a special capacity of MVLBG2, a newly synthesized highly charged (16+) lipid (Ewert et al., JACS, 2006) with giant dendrimer-like headgroup leading to a conical molecular shape resulting into high spontaneous membrane curvature, when incorporated into lipid bilayer. In this work we report formation of BLs for other cationic lipids, demonstrating that BL-formation is a general phenomenon of all charged membranes. We carried out systematic study of binary lipid mixtures comprised of DOPC and a cationic lipid, varying the headgroup size and charge of cationic lipid from 1+, 3+ to 5+. We find that all cationic lipids form BLs on the micrometer and nanometer scale. We have also found that pure DOPC forms BLs in presence of monovalent salt, which is known to cause zwitterionic DOPC to become negatively charged. This latter finding confirms that BL-formation is a general capacity of charged membranes independent of the charge nature. Block liposomes may find a range of applications in chemical and nucleic acid delivery and as building blocks in the design of templates for hierarchical structures. Funding by DOE DE-FG-02-06ER46314, NIH GM-59288, NSF DMR-0503347.