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...

The method of cumulants is a standard technique used to analyze dynamic light-scattering data measured for polydisperse samples. These data, from an intensity-intensity autocorrelation function of the scattered light, can be described in terms of a distribution of decay rates. The method of cumulants provides information about the cumulants and the moments of this distribution. However, the method does not permit independent determination of the long-time baseline of the intensity correlation function and can lead to inconsistent results when different numbers of data points are included in the fit. The method is reformulated in terms of the moments about the mean to permit more robust and satisfactory fits. The different versions of the method are compared by analysis of the data for polydisperse-vesicle samples.