A novel process for producing cubic liquid crystalline nanoparticles (cubosomes) has been developed. The process entails simple mixing of two waterlike solutions with a minimal input of energy. The key to this process is the inclusion of hydrotrope. Most lipids, such as monoolein, used to form cubic liquid crystals are essentially insoluble in water. The hydrotrope dissolves the lipid to create a waterlike solution. Water is added to the hydrotrope solution, resulting in a precipitous decrease in lipid solubility. Provided that the dilution trajectory falls into a cubic phase-water miscibility gap, nanometer-scale cubic liquid crystalline particles form spontaneously, presumably from a homogeneous nucleation mechanism. The process is versatile enough to accommodate any lipid and hydrotrope combination that forms cubic liquid crystalline material upon dilution. Actives and stabilizers can be formulated into either of the two solutions, allowing the production of colloidally stabilized, controlled-release dispersions. The phase diagram of the monooleinethanol-water system is determined to assess appropriate formulation of solutions and to develop dilution trajectories. This process replaces current processes that require long hold times, processing of solidlike materials, and very high-energy inputs to create cubosome nanoparticle dispersions. This process produces smaller, more stable cubosomes than by conventional bulk dispersion techniques.

Bilayer-to-nonbilayer phase transitions in phospholipids occur by means of poorly characterized intermediates. Many have proposed that membrane fusion can also occur by formation of these intermediates. Structures for such intermediates were proposed in a recent theory of these transition mechanisms. Using time-resolved cryo-transmission electron Microscopy (TRC-TEM), we have directly visualized the evolution of inverted phase micro-structure in liposomal aggregates. We have identified one of the proposed intermediates, termed an interlamellar attachment (ILA), which has the structure and dimensions predicted by the theory. We show that ILAs are likely to be the structure corresponding to "lipidic particles" observed by freeze-fracture electron microscopy. ILAs appear to assemble the inverted cubic (III) phase by formation of an ILA lattice, as previously proposed. ILAs are also observed to mediate membrane fusion in the same systems, on the same time scale, and under nearly the same conditions in which membrane fusion was observed by fluorescence methods in earlier studies. These earlier studies indicated a linkage between a membrane fusion mechanism and III phase formation. Our micrographs suggest that the same intermediate structure mediates both of those processes.

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