Size control of giant unilamellar vesicles prepared from inverted emulsion droplets

The production of giant lipid vesicles with controlled size and structure will be an important technology in the design of quantitative biological assays in cell-mimetic microcompartments. For establishing size control of giant vesicles, we investigated the vesicle formation process, in which inverted emulsion droplets are transformed into giant unilamellar vesicles (GUVs) when they pass through an oil/water interface. The relationship between the size of the template emulsion and the converted GUVs was studied using inverted emulsion droplets with a narrow size distribution, which were prepared by microfluidics. We successfully found an appropriate centrifugal acceleration condition to obtain GUVs that had a desired size and narrow-enough size distribution with an improved yield so that emulsion droplets can become the template for GUVs.     

Photodamage in a Mitochondrial Membrane Model Modulated by the Topology of Cationic and Anionic Meso‐Tetrakis Porphyrin Free Bases

The photodynamic effects of the cationic TMPyP (meso‐tetrakis [N‐methyl‐4‐pyridyl]porphyrin) and the anionic TPPS4 (meso‐tetrakis[4‐sulfonatophenyl]porphyrin) against PC/CL phosphatidylcholine/cardiolipin (85/15%) membranes were probed to address the influence of phorphyrin binding on lipid damage. Electronic absorption spectroscopy and zeta potential measurements demonstrated that only TMPyP binds to PC/CL large unilamellar vesicles (LUVs). The photodamage after irradiation with visible light was analyzed by dosages of lipid peroxides (LOOH) and thiobarbituric reactive substance and by a contrast phase image of the giant unilamellar vesicles (GUVs). Damage to LUVs and GUVs promoted by TMPyP and TPPS4 were qualitatively and quantitatively different. The cationic porphyrin promoted damage more extensive and faster. The increase in LOOH was higher in the presence of D₂O, and was impaired by sodium azide and sorbic acid. The effect of D₂O was higher for TPPS4 as the photosensitizer. The use of DCFH demonstrated that liposomes prevent the photobleaching of TMPyP. The results are consistent with a more stable TMPyP that generates long‐lived singlet oxygen preferentially partitioned in the bilayer. Conversely, TPPS4 generates singlet oxygen in the bulk whose lifetime is increased in D₂O. Therefore, the affinity of the porphyrin to the membrane modulates the rate, type and degree of lipid damage.     

Multilamellar LipoCEST Agents Obtained from Osmotic Shrinkage of Paramagnetically Loaded Giant Unilamellar Vescicles (GUVs)

Moving from nano‐ to micro‐systems may not just be a matter of scale, but it might imply changes in the properties of the systems that can open new routes for the development of efficient MRI contrast agents. This is the case reported in the present paper, where giant liposomes (giant unilamellar vesicles, GUVs) loaded with Ln^III complexes have been studied as chemical exchange saturation transfer (CEST) MRI contrast agents. The comparison between nanosized liposomes (small unilamellar vesicles, SUVs) and GUVs sharing the same formulation led to differences that could not be accounted for only in terms of the increase in size (from 100–150 nm to 1–2 μm). Upon osmotic shrinkage, GUVs yielded a saturation‐transfer effect three order of magnitude higher than SUVs consistent with the increase in vesicles volume. Confocal microscopy showed that the shrinkage of GUVs resulted in multilamellar particles whereas SUVs are known to yield asymmetrical, discoidal shape.     

On‐chip generation of monodisperse giant unilamellar lipid vesicles containing quantum dots

Monodispersed lipid vesicles have been used as a drug delivery vehicle and a biochemical reactor. To generate monodispersed lipid vesicles in the nano‐ to micrometer size range, an extrusion step should be included in conventional hand‐shaking method of lipid vesicle synthesis. In addition, lipid vesicles as a drug carrier still need to be improved to effectively encapsulate concentrated biomolecules such as cells, proteins, and target drugs. To overcome these limitations, this paper reports a new microfluidic platform for continuous synthesis of small‐sized (～10 μm) giant unilamellar vesicles (GUVs) containing quantum dots (QDs) as a nanosized model drug. To generate GUVs, we introduced an additional cross‐flow to break vesicles into small size. 1,2 ‐ dimyristoyl‐sn‐glycero ‐ 3 ‐ phosphocholine (DMPC) in an octanol–chloroform mixture was used in the construction of self‐assembled membrane. Consequently, we have successfully demonstrated the fabrication of monodispersed GUVs with 7?12 μm diameter containing QDs. The proposed synthesis method of cell‐sized GUVs would be highly desirable for applications such as multipurpose drug encapsulation and delivery.     

Steady state and time-resolved spectroscopic studies on zinc(II) phthalocyanine in liposomes.

Zinc(II) phthalocyanine (ZnPc), a potential second-generation phototherapeutic agent for tumours, has been incorporated into small unilamellar vesicles (SUVs) (diameter, 52 nm) and large unilamellar vesicles (LUVs) (diameter, 84 nm) of dipalmitoyl-phosphatidylcholine (DPPC). Absorption spectroscopy, as well as steady state and time-resolved fluorescence emission studies, indicate that ZnPc is monomeric in SUVs at a stoichiometric concentration below 0.25 microM (corresponding to an actual endoliposomal concentration of about 0.5 mM), while in LUVs it is monomeric below 2 microM. The fluorescence lifetime of the monomer is 3-3.5 ns. Upon increasing the ZnPc concentration, aggregated derivatives are formed, which are characterized by shorter fluorescence lifetimes (1.2-1.5 ns; 0.4-0.6 ns). The possible implications of these observations for the phototherapeutic efficiency of ZnPc are briefly discussed.     

Wetting fibers with liposomes

Giant unilamellar vesicles (GUVs) are deposited on glass microfibers. The vesicles adopt the classical "onduloidal" shape of liquid droplets on fibers. They spread by two simultaneous mechanisms: envelopment and emission of a precursor film. This film spreads faster than on a uniform plane surface and eventually stops, signaling the presence of defects on the rod. This fast spreading tenses the vesicles; transient pores open on the GUVs and the internal liquid leaks out. This process leads to a new technique for fiber coating.     

Electroformation in a flow chamber with solution exchange as a means of preparation of flaccid giant vesicles

A recently described technique [Estes and Mayer, Biochim. Biophys. Acta 1712 (2005) 152-160] for the preparation of giant unilamellar vesicles (GUVs) in solutions with high ionic strength is examined. By observing a series of osmotic swellings followed by vesicle bursts upon a micropipette transfer of a single POPC GUV from a sucrose solution into an iso-osmolar glycerol solution, a value for the permeability of POPC membrane for glycerol, P=(2.09+/-0.82) x 10(-8)m/s, has been obtained. Based on this result, an alternative mechanism is proposed for the observed exchange of vesicle interior. With modifications, the method of Estes and Mayer is then applied to preparation of flaccid GUVs.     

Efficient electroformation of supergiant unilamellar vesicles containing cationic lipids on ITO-coated electrodes

Giant unilamellar vesicles (GUVs) represent a versatile in vitro system widely used to study properties of lipid membranes and their interaction with biomacromolecules and colloids. Electroformation with indium tin oxide (ITO) coated coverslips as electrodes is a standard approach to GUV production. In the case of cationic GUVs, however, application of this approach leads to notorious difficulties. We discover that this is related to aging of ITO-coated coverslips during their repeated use, which is reflected in their surface topography on the nanoscale. We find that mild annealing of the ITO-coated surface in air reverts the effects of aging and ensures efficient reproducible electroformation of supergiant (diameter > 100 μm) unilamellar vesicles containing cationic lipids.     

UV-induced bursting of cell-sized multicomponent lipid vesicles in a photosensitive surfactant solution

We study the behavior of multicomponent giant unilamellar vesicles (GUVs) in the presence of AzoTAB, a photosensitive surfactant. GUVs are made of an equimolar ratio of dioleoylphosphatidylcholine (DOPC) and dipalmitoylphosphatidylcholine (DPPC) and various amounts of cholesterol (Chol), where the lipid membrane shows a phase separation into a DPPC-rich liquid-ordered (L(o)) phase and a DOPC-rich liquid-disordered (L(d)) phase. We find that UV illumination at 365 nm for 1 s induces the bursting of a significant fraction of the GUV population. The percentage of UV-induced disrupted vesicles, called bursting rate (Y(burst)), increases with an increase in [AzoTAB] and depends on [Chol] in a non-monotonous manner. Y(burst) decreases when [Chol] increases from 0 to 10 mol % and then increases with a further increase in [Chol], which can be correlated with the phase composition of the membrane. We show that Y(burst) increases with the appearance of solid domains ([Chol] = 0) or with an increase in area fraction of L(o) phase (with increasing [Chol] ≥ 10 mol %). Under our conditions (UV illumination at 365 nm for 1 s), maximal bursting efficiency (Y(burst) = 53%) is obtained for [AzoTAB] = 1 mM and [Chol] = 40 mol %. Finally, by restricting the illumination area, we demonstrate the first selective UV-induced bursting of individual target GUVs. These results show a new method to probe biomembrane mechanical properties using light as well as pave the way for novel strategies of light-induced drug delivery.     

FtsZ Reorganization Facilitates Deformation of Giant Vesicles in Microfluidic Traps**

The geometry of reaction compartments can affect the local outcome of interface-restricted reactions. Giant unilamellar vesicles (GUVs) are commonly used to generate cell-sized, membrane-bound reaction compartments, which are, however, always spherical. Herein, we report the development of a microfluidic chip to trap and reversibly deform GUVs into cigar-like shapes. When trapping and elongating GUVs that contain the primary protein of the bacterial Z ring, FtsZ, we find that membrane-bound FtsZ filaments align preferentially with the short GUV axis. When GUVs are released from this confinement and membrane tension is relaxed, FtsZ reorganizes reversibly from filaments into dynamic rings that stabilize membrane protrusions; a process that allows reversible GUV deformation. We conclude that microfluidic traps are useful for manipulating both geometry and tension of GUVs, and for investigating how both affect the outcome of spatially-sensitive reactions inside them, such as that of protein self-organization.     

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.     

Interaction of the meso-tetrakis (4-N-methylpyridyl) porphyrin with gel and liquid state phospholipid vesicles

The interaction of the cationic meso-tetrakis 4-N-methylpyridyl porphyrin (TMPyP) with large unilamellar vesicles (LUVs) was investigated in the present study. LUVs were formed by mixtures of the zwitterionic 1,2-dipalmitoyl-sn-glycero-phosphatidylcholine (DPPC) and anionic 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG) phospholipids, at different DPPG molar percentages. All investigations were carried out above (50 °C) and below (25 °C) the main phase transition temperature of the LUVs (~41 °C). The binding constant values, K(b), estimated from the time-resolved fluorescence study, showed a significant increase of the porphyrin affinity at higher mol% DPPG. This affinity is markedly increased when the LUVs are in the liquid crystalline state. For both situations, the increase of the K(b) value was also followed by a higher porphyrin fraction bound to the LUVs. The displacement of the vesicle-bound porphyrins toward the aqueous medium, upon titration with the salt potassium chloride (KCl), was also studied. Altogether, our steady-state and frequency-domain fluorescence quenching data results indicate that the TMPyP is preferentially located at the LUVs Stern layer. This is supported by the zeta potential studies, where a partial neutralization of the LUVs surface charge, upon porphyrin titration, was observed. Dynamic light scattering (DLS) results showed that, for some phospholipid systems, this partial neutralization leads to the LUVs flocculation.     

Controlled hydrogel formation in the internal compartment of giant unilamellar vesicles

The introduction of poly(ethylene dioxythiophene) (PEDOT)/poly(styrene sulfonate) (PSS) polyelectrolyte into giant unilamellar phospholipid vesicles (GUVs) and cross-linking with Ca2+ ions to generate a hydrogel within the internal compartment are reported. The aqueous colloidal suspension of PEDOT with excess PSS was microinjected into the internal compartment of liposomes as well as networks of GUVs and lipid nanotubes. The subsequent introduction of calcium ions as cross-linking agent in order to induce hydrogel formation was achieved by three different methods: vesicle fusion, electroporation, and direct microinjection. Gel formation was probed by coinjection of fluorescent nanoparticles and tracking of Brownian motion. Particle mobility was shown to be distinctly reduced in the gel-filled vesicles. Diffusion constants for the particles were calculated from the projected movement of the particles and compared to particles in reference gels and solutions.     

Point‐to‐Plane Nonhomogeneous Electric‐Field‐Induced Simultaneous Formation of Giant Unilamellar Vesicles (GUVs) and Lipid Tubes

It is well‐known that homogeneous electric fields can be used to generate giant unilamellar vesicles (GUVs). Herein we report an interesting phenomenon of formation of GUVs and lipid tubes simultaneously using a nonhomogeneous electric field generated by point‐to‐plane electrodes. The underlying mechanism was analyzed using finite element analysis. The two forces play main roles, that is, the pulling force (F) to drag GUVs into lipid tubes induced by fluid flow, and the critical force (Fc) to prevent GUVs from deforming into lipid tubes induced by electric fields. In the center area underneath the needle electrode, the GUVs were found because F is less than Fc in that region, whereas in the edge area the lipid tubes were obtained because F is larger than Fc. The diffusion coefficient of lipid in the tubes was found to be 4.45 μm² s^?1 using a fluorescence recovery after photobleaching (FRAP) technique. The method demonstrated here is superior to conventional GUV or lipid tube fabrication methods, and has great potential in cell mimic or hollow material fabrication using GUVs and tubes as templates.     

Rapid screening of membrane protein activity: electrophysiological analysis of OmpF reconstituted in proteoliposomes

Solvent-free planar lipid bilayers were formed in an automatic manner by bursting of giant unilamellar vesicles (GUVs) after gentle suction application through micron-sized apertures in a borosilicate glass substrate. Incubation of GUVs with the purified ion channel protein of interest yielded proteoliposomes. These proteoliposomes allow for immediate recording of channel activity after GUV sealing. This approach reduces the time-consuming, laborious and sometimes difficult protein reconstitution processes normally performed after bilayer formation. Bilayer recordings are attractive for investigations of membrane proteins not accessible to patch clamp analysis, like e.g. proteins from organelles. In the presented work, we show the example of the outer membrane protein OmpF from Escherichia coli. We reconstituted OmpF in proteoliposomes and observed the characteristic trimeric conductance levels and the typical gating induced by pH and transmembrane voltage. Moreover, OmpF is the main entrance for beta-lactam antibiotics and we investigated translocation processes of antibiotics and modulation of OmpF by spermine. We suggest that the rapid formation of porin containing lipid bilayers is of potential for the efficient electrophysiological characterization of the OmpF protein, for studying membrane permeation processes and for the rapid screening of antibiotics.     

Membrane fusion of giant unilamellar vesicles of neutral phospholipid membranes induced by La3+

Membrane fusions of vesicles of biomembranes play various important roles in cells, but their mechanisms are unclear and controversial. In the present study, we found that 30 microM to 1 mM La3+ induced membrane fusion of two giant unilamellar vesicles (GUVs) composed of a mixture of dioleoylphosphatidylcholine (DOPC) and dipalmitoleoylphosphatidylethanolamine (DPOPE). We succeeded in observing a process of this membrane fusion in detail. First, two GUVs became strongly associated, with a partition membrane between them composed of two bilayers, one from each GUV. Then, the partition membrane was suddenly broken at one site on its edge. The area of this breakage site gradually spread, until it was completely separated from the GUV to complete the membrane fusion. Here, we propose a new model (i.e., the partition breakage model) for the mechanism of La3+ -induced membrane fusion of GUVs.     

Controlling the internal structure of giant unilamellar vesicles by means of reversible temperature dependent sol-gel transition of internalized poly(N-isopropyl acrylamide)

In this work, we present preparation and basic applications of lipid-bilayer-enclosed picoliter volumes (microcontainers) of solutions of poly(N-isopropylacrylamide) (PNIPAAm). Giant unilamellar vesicles (GUVs) were prepared from phospholipids using a standard swelling procedure and subsequently surface immobilized. Clear, slightly viscous solutions of PNIPAAm of varying concentration in aqueous buffer were directly pressure-microinjected into the GUVs, using a submicrometer-sized, pointed capillary. The GUV was subjected to changing temperature over a 21-40 degrees C range. The typical phase transition of the polymeric material upon heating and cooling across the lower critical solution temperature was followed using optical microscopy and shown to be reversible over multiple sequential heating/cooling cycles without compromising the integrity of the GUV membrane. Fluorescent, carboxylic acid modified 200 nm latex beads, co-injected with the PNIPAAm solution, were temperature-reversibly immobilized during the phase transition, practically freezing the Brownian motion of the entrapped particles in the volume. Furthermore, a co-injected water soluble fluorescent polysaccharide-dye conjugate was shown not to migrate from the aqueous phase into the hydrophobic polymer part upon heating, whereas the fluorescent beads were completely but reversibly immobilized in the hydrophobic domains of dense polymer agglomerates. The system reported here provides a feasible method for the reversible stabilization and solidification of GUV interior volumes, e.g., as a micrometer-sized model system for controlled drug release.     

Lipid diffusion from single molecules of a labeled protein undergoing dynamic association with giant unilamellar vesicles and supported bilayers

It is demonstrated that single-molecule tracking of a fluorescently labeled protein undergoing transient binding to model membranes presents a useful method of obtaining fluid properties. The labeled ACBP protein was tracked during its binding to free-standing giant unilamellar vesicles (GUVs) and supported bilayers prepared from the GUVs in the same environment. The analysis of images that are blurred as a result of fast probe diffusion was discussed. An examination of the lateral diffusion trajectories revealed a homogeneous diffusion on the top segments of the GUVs with D = 6.9 +/- 0.3 microm(2)/s. The supported bilayer experiments revealed two diffusion processes, one with Df = 3.1 +/- 0.4 microm(2)/s and the other with Ds = 0.078 +/- 0.001 microm(2)/s. The 2-fold difference in the lipid bilayer mobility for the free-standing and fast components in the supported bilayers is attributed to the known effect of frictional coupling with the solid support. The slow mobile fraction in the bilayer is suggested to be associated with the migration of pore-like structures, originating from the interaction of the membrane with the glass support.     

Spatiotemporal Propagation of a Minimal Catalytic RNA Network in GUV Protocells by Temperature Cycling and Phase Separation

Compartmentalization is key to many cellular processes and a critical bottleneck of any minimal life approach. In cells, a complex chemistry is responsible for bringing together or separating biomolecules at the right place at the right time. Lipids, nucleic acids and proteins self-organize, thereby creating boundaries, interfaces and specialized microenvironments. Exploiting reversible RNA-based liquid-liquid phase separation (LLPS) inside giant unilamellar vesicles (GUVs), we present an efficient system capable of propagating an RNA-based enzymatic reaction across a population of GUVs upon freezing-thawing (FT) temperature cycles. We report that compartmentalization in the condensed RNA-rich phase can accelerate such an enzymatic reaction. In the decondensed state, RNA substrates become homogeneously dispersed, enabling content exchange between vesicles during freeze-thawing. This work explores how a minimal reversible phase separation system in lipid vesicles could help to implement spatiotemporal control in cyclic processes, as required for minimal cells.     

多肽诱导的巨型脂质体出芽和泄露行为

细胞膜与膜蛋白之间的相互作用与生命中许多过程息息相关.以巨型脂质体(GUV)和多肽分别作为细胞膜和膜蛋白的简化模型,我们设计了四种仅包含亮氨酸(L)和赖氨酸(K)的多肽,即K₁₄、(KL₂KL₂K)₂、(KL₂KL₃)₂和K₆L₈,并对比研究了它们与中性和负电性脂质体的相互作用.电荷密度最高的K₁₄只是涂层在脂质体表面,不破其囊泡结构,但能够引起负电性脂质体发生微相分离,属建设性相互作用.能够形成两亲性α螺旋的(KL₂KL₂K)₂和(KL₂KL₃)₂则引起脂质体发生泄露和破裂,属破坏性作用.但二者引起泄露的速率在中性脂质体和负电性脂质体中的结果恰好相反,说明泄露分两步进行:表面吸附多肽达到一定浓度,继而对膜进行干扰.表面活性剂型多肽K₆L₈的氨基酸组成与(KL₂KL₂K)₂相同,但K₆L₈只是引起负电性脂质体发生泄露,造成中性脂质体发生外出芽.这些简单氨基酸造成的脂质体的复杂构象变化可以统一用静电和疏水相互作用在膜上的位置和强度来进行解释.这些结论对于深入理解膜蛋白的作用机理是有帮助的.