We investigate a method for the controlled assembly of unilamellar vesicles consisting of bilayers assembled one leaflet at a time. We use water-in-oil emulsions stabilized by the material for the inner leaflet and produce vesicles by passing the water droplets through a second oil−water interface, where they become coated with the outer leaflet. We have used this technique to form vesicles from lipids, mixed lipid and surfactant systems, and diblock copolymers. The stability of lipid-stabilized emulsions limits the range of sizes that can be produced and the vesicle yield; nevertheless, there are several advantages with this emulsion-based technique:  It is possible to make unilamellar vesicles with sizes ranging from 100 nm to 1 μm. Moreover, the process allows for efficient encapsulation and ensures that the contents of the vesicles remain isolated from the continuous aqueous phase. To illustrate possible applications of this technique, we demonstrate the use of vesicles as microreactors where we polymerize actin through the addition of magnesium and show that the polymerization kinetics are unaffected by the encapsulation.

Vesicles are bilayers of lipid molecules enclosing a fixed volume of aqueous solution. Ubiquitous in cells, they can be produced in vitro to study the physical properties of biological membranes and for use in drug delivery and cosmetics. Biological membranes are, in fact, a fluid mosaic of lipids and other molecules; the richness of their chemical and mechanical properties in vivo is often dictated by an asymmetric distribution of these molecules. Techniques for vesicle preparation have been based on the spontaneous assembly of lipid bilayers, precluding the formation of such asymmetric structures. Partial asymmetry has been achieved only with chemical methods greatly restricting the study of the physical and chemical properties of asymmetric vesicles and their use in potential applications for drug delivery. Here we describe the systematic engineering of unilamellar vesicles assembled with two independently prepared monolayers; this process produces asymmetries as high as 95%. We demonstrate the versatility of our method by investigating the stability of the asymmetry. We also use it to engineer hybrid structures comprised of an inner leaflet of diblock copolymer and an independent lipid outer leaflet. V esicles are produced in the laboratory by a variety of methods including sonication (1), extrusion (2), swelling (3), electroformation (4), and reverse evaporation (5); all methods rely on self-assembly and lead to a symmetric distribution of lipids on the inner and outer leaflets of the bilayer. Realistic models of biological membranes must incorporate lipid asymmetry (6, 7); moreover, asymmetric vesicles consisting of completely different types of molecules on the inner and outer leaflets would greatly increase the flexibility of vesicle drug delivery systems. Partial asymmetry can be achieved by altering the distribution of specific phospholipids using pH gradients, osmotic pressure, or molecules that promote lipid redistribution (8). However, the chemical constraints of these methods severely limit the applicability of such systems.In this article, we describe a method for systematically engineering vesicles with asymmetric bilayers where each leaflet is assembled independently. A schematic of the process is shown in Fig. 1. We begin with an inverted emulsion of water droplets dispersed in dodecane and stabilized by the lipids intended for the inner leaflet. This phase is placed over an intermediate phase of the same oil containing the lipids for the outer leaflet. The intermediate phase is placed over the final aqueous phase, and a monolayer of the second lipid forms at the interface. The water droplets in the emulsion are heavier than the oil and thus sediment, pulling the second monolayer from the interface to complete the bilayer, resulting in the formation of asymmetric vesicles in the final aqueous phase. A similar strategy for making vesicles was first demonstrated using a benzene:water:eggphosphatidylcholine (PC) emulsion (9). The uniqueness of our method lies in the introduction of this distinct i...

We demonstrate ordered orientation of the hydration water at the surface of phospholipid bilayers by use of coherent anti-Stokes Raman scattering (CARS) microscopy, a highly sensitive vibrational imaging method recently developed. We investigated negatively charged POPS (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine) and neutral POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) multilamellar onions dispersed in deuterated dodecane. The imaging contrast based on the CARS signal from the H 2O stretching vibration shows a clear dependence on the excitation field polarization. Our results provide direct experimental evidence that water molecules close to the phospholipid bilayer surface are ordered with the symmetry axis along the direction normal to the bilayer. Moreover, the amount of ordered water molecules depends on the lipid polar group. The spectral profile for the interlamellar water shows that the water molecules bound to the bilayer surface are less hydrogen-bonded and exhibit a higher vibrational frequency than bulk water.M embranes formed by bilayers of phospholipids play a pervasive and central role in many cellular functions. Whenever two such membranes come in contact, short-range interactions between the membranes are critical for a multitude of processes. These interactions are mainly hydration forces resulting from partial orientation of interlamellar water by the bilayer (1) and steric repulsions resulting from contact between lipid molecules in the opposing bilayers (2, 3). Hydration forces (4) have been identified as the dominant nonspecific short-range interactions that account for the repulsive pressure between two lipid bilayers when their separation is in the range of 1.0 to 3.0 nm (1, 5). Several models have been developed that relate the hydration force with the ordering of water molecules on a membrane surface (6-8). Molecular dynamics (MD) simulation studies (9, 10) have suggested that water is partially ordered within 1.0 nm from the membrane surface. However, experimental results have supplied only partial information regarding the exact nature of liquid water organization in the vicinity of bilayers. Some evidence for water bound to a membrane surface was provided by [ 2 H]NMR studies, in which the quadrupolar splitting frequency of deuterated water was found to decrease with increasing hydration level (11,12). In addition, Fourier transform infrared (FTIR) studies (13) supported by a recent MD simulation (14) have shown that water molecules are hydrogen-bonded to the oxygen atoms of the PO 2 Ϫ group in the phospholipid polar head moiety and the ester CAO group of the sn-2 acyl chain. In principle, the net orientation of the interfacial water molecules at a membrane surface can be probed by polarization measurements. In fact, such an attempt has been made in a sum frequency generation study of water͞lipid monolayer interface (15). However, ordering of water was not observed for the particular sample used. To the best of our knowledge, no direct experimental evidence for ord...