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.     

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.     

Aqueous Self‐Assembly of Y‐Shaped Amphiphilic Block Copolymers into Giant Vesicles

The preparation and aqueous self‐assembly of newly Y‐shaped amphiphilic block polyurethane (PUG) copolymers are reported here. These amphiphilic copolymers, designed to have two hydrophilic poly(ethylene oxide) (PEO) tails and one hydrophobic alkyl tail via a two‐step coupling reaction, can self‐assemble into giant unilamellar vesicles (GUVs) (diameter ≥ 1000 nm) with a direct dissolution method in aqueous solution, depending on their Y‐shaped structures and initial concentrations. More interesting, the copolymers can self‐assemble into various distinct nano‐/microstructures, such as spherical micelles, small vesicles, and GUVs, with the increase of their concentrations. The traditional preparation methods of GUVs generally need conventional amphiphilic molecules and additional complicated conditions, such as alternating electrical field, buffer solution, or organic solvent. Therefore, the self‐assembly of Y‐shaped PUGs with a direct dissolution method in aqueous solution demonstrated in this study supplies a new clue to fabricate GUVs based on the geometric design of amphiphilic polymers.

     

Visualization and Quantification of Transmembrane Ion Transport into Giant Unilamellar Vesicles

Transmembrane ion transporters (ionophores) are widely investigated as supramolecular agents with potential for biological activity. Tests are usually performed in synthetic membranes that are assembled into large unilamellar vesicles (LUVs). However transport must be followed through bulk properties of the vesicle suspension, because LUVs are too small for individual study. An alternative approach is described whereby ion transport can be revealed and quantified through direct observation. The method employs giant unilamellar vesicles (GUVs), which are 20–60 μm in diameter and readily imaged by light microscopy. This allows characterization of individual GUVs containing transporter molecules, followed by studies of transport through fluorescence emission from encapsulated indicators. The method provides new levels of certainty and relevance, given that the GUVs are similar in size to living cells. It has been demonstrated using a highly active anion carrier, and should aid the development of compounds for treating channelopathies such as cystic fibrosis.     

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.     

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.     

Freezing or wrapping: the role of particle size in the mechanism of nanoparticle-biomembrane interaction

Understanding the interactions between nanoparticles (NPs) and biological matter is a high-priority research area because of the importance of elucidating the physical mechanisms underlying the interactions leading to NP potential toxicity as well as NP viability as therapeutic vectors in nanomedicine. Here, we use two model membrane systems, giant unilamellar vesicles (GUVs) and supported monolayers, to demonstrate the competition between adhesion and elastic energy at the nanobio interface, leading to different mechanisms of NP-membrane interaction relating to NP size. Small NPs (18 nm) cause a "freeze effect" of otherwise fluid phospholipids, significantly decreasing the phospholipid lateral mobility. The release of tension through stress-induced fracture mechanics results in a single microsize hole in the GUVs after interaction. Large particles (>78 nm) promote membrane wrapping, which leads to increased lipid lateral mobility and the eventual collapse of the vesicles. Electrochemical impedance spectroscopy on the supported monolayer model confirms that differently sized NPs interact differently with the phospholipids in close proximity to the electrode during the lipid desorption process. The time scale of these processes is in accordance with the proposed NP/GUV interaction mechanism.     

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.     

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.     

Shape changes and vesicle fission of giant unilamellar vesicles of liquid-ordered phase membrane induced by lysophosphatidylcholine

Liquid-ordered phase (lo phase) of lipid membranes has properties that are intermediate between those of liquid-crystalline phase and those of gel phase and has attracted much attention in both biological and biophysical aspects. Rafts in the lo phase in biomembranes play important roles in cell function of mammalian cells such as signal transduction. In this report, we have prepared giant unilamellar vesicles (GUVs) of lipid membranes in the lo phase and investigated their physical properties using phase-contrast microscopy and fluorescence microscopy. GUVs of dipalmitoyl-phosphatidylcholine (DPPC)/cholesterol membranes and also GUVs of sphingomyelin (SM)/cholesterol membranes in the lo phase in water were formed at 20-37 degrees C successfully, when these membranes contained >/=30 mol % cholesterol. The diameters of GUVs of DPPC/cholesterol and SM/cholesterol membranes did not change from 50 to 28 degrees C, supporting that the membranes of these GUVs were in the lo phase. To elucidate the interaction of a substance with a long hydrocarbon chain with the lo phase membrane, we investigated the interaction of low concentrations (less than critical micelle concentration) of lysophosphatidylcholine (lyso-PC) with DPPC/cholesterol GUVs and SM/cholesterol GUVs in the lo phase. We found that lyso-PC induced several shape changes and vesicle fission of these GUVs above their threshold concentrations in water. The analysis of these shape changes indicates that lyso-PC can be partitioned into the external monolayer in the lo phase of the GUV from the aqueous solution. Threshold concentrations of lyso-PC in water to induce the shape changes and vesicle fission increased greatly with a decrease in chain length of lyso-PC. Thermodynamic analysis of this result indicates that shape changes and vesicle fission occur at threshold concentrations of lyso-PC in the membrane. These new findings on GUVs of the lo phase membranes indicate that substances with a long hydrocarbon chain such as lyso-PC can enter into the lo phase membrane and also the raft in the cell membrane. We have also proposed a mechanism for the lyso-PC-induced vesicle fission of GUVs.     

Crystalline protein domains and lipid bilayer vesicle shape transformations

Cellular membranes can take on a variety of shapes to assist biological processes including endocytosis. Membrane-associated protein domains provide a possible mechanism for determining membrane curvature. We study the effect of tethered streptavidin protein crystals on the curvature of giant unilamellar vesicles (GUVs) using confocal, fluorescence, and differential interference contrast microscopy. Above a critical protein concentration, streptavidin domains align and percolate as they form, deforming GUVs into prolate spheroidal shapes in a size-dependent fashion. We propose a mechanism for this shape transformation based on domain growth and jamming. Osmotic deflation of streptavidin-coated GUVs reveals that the relatively rigid streptavidin protein domains resist membrane bending. Moreover, in contrast to highly curved protein domains that facilitate membrane budding, the relatively flat streptavidin domains prevent membrane budding under high osmotic stress. Thus, crystalline streptavidin domains are shown to have a stabilizing effect on lipid membranes. Our study gives insight into the mechanism for protein-mediated stabilization of cellular membranes.     

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.     

Fabrication of Thickness‐Controllable Micropatterned Polyelectrolyte‐Film/Nanoparticle Surfaces by Using the Plasma Oxidation Method

We have demonstrated a novel way to form thickness‐controllable polyelectrolyte‐film/nanoparticle patterns by using a plasma etching technique to form, first, a patterned self‐assembled monolayer surface, followed by layer‐by‐layer assembly of polyelectrolyte‐films/nanoparticles. Octadecyltrimethoxysilane (ODS) and (3‐aminopropyl)triethoxysilane (APTES) self‐assembled monolayers (SAMs) were used for polyelectrolyte‐film and nanoparticle patterning, respectively. The resolution of the proposed patterning method can easily reach approximately 2.5 μm. The height of the groove structure was tunable from approximately 2.5 to 150 nm. The suspended lipid membrane across the grooves was fabricated by incubating the patterned polyelectrolyte groove arrays in solutions of 1,2‐dioleoyl‐sn‐glycero‐3‐phosphocholine (DOPC) giant unilamellar vesicles (GUVs). The method demonstrated here reveals a new path to create patterned 2D or 3D structures.     

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.     

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.     

Supported lipid bilayer formation by the giant vesicle fusion induced by vesicle–surface electrostatic attractive interaction

The effect of the electrostatic attractive force between giant unilamellar vesicles (GUVs) and the SiO₂ surface on the formation of a Ca²⁺-free supported lipid bilayer (SLB) was investigated by atomic force microscopy and fluorescence microscopy. When negatively charged GUVs were incubated for 1 h without Ca²⁺, the surface coverage of lipid bilayer was <1% on the SiO₂ surface. In contrast, a high coverage was obtained without addition of Ca²⁺ on the positively charged surface modified by aminopropyldimethylethoxysilane, and the coverage of SLBs decreased with increasing KCl concentrations. The thickness of the water layer under SLB was reduced by modification of APS.     

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.     

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.     

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.