Formation and Characterization of Ordered Bicontinuous Microemulsions Anna Kogan, † Deborah E. Shalev, ‡ Uri Raviv, § Abraham Aserin, † and Nissim Garti* ,† Casali Institute of Applied Chemistry, The Institute of Chemistry, and Wolfson Centre for Applied Structural Biology, The Hebrew UniVersity of Jerusalem, 91904 Jerusalem, Israel ReceiVed: February 21, 2009; ReVised Manuscript ReceiVed: June 10, 2009 Ordered bicontinuous microstructures formed in a fully water-dilutable, pseudoternary unique nonionic microemulsion were obtained and characterized. The concentrate contained a mixture of triacetin/D-R-tocopherol acetate/ethanol/Tween 60. Upon dilution, the concentrate was transformed from a reversed micellar system to oil-in-water microemulsion droplets. The transformation occurred through an intermediate phase of ordered bicontinuous structures. The factors that governed the construction of this unique phase, and its physical and structural properties, were characterized in detail. The techniques used included small angle X-ray scattering (SAXS), self-diffusion and quantum filtered NMR, differential scanning calorimetry, rheology measurements, electrical conductivity, and dynamic light scattering. This mesophase displays microemulsion properties along with some characteristics of lyotropic liquid crystals (but is not a mixture of the two). Similar to microemulsions, the structures were transparent and spontaneously formed and exhibited thermodynamic stability. Yet, unlike microemulsions, they showed short-range order at room temperature. Additionally, the microstructures exhibited non-Newtonian flow behavior, characteristic of lamellar structures. The bicontinuous ordered microemulsions were obtained upon heating (to 25 °C) from the lamellar phase existing at low temperatures (5 °C). The main feature governing the bicontinuous mesophase formation was the amphiphilic nature of oil blends composed of D-R-tocopherol acetate and triacetin. The oils functioned as cosurfactants, altering the packing parameter of the surfactant and leading to the construction of bicontinuous structures with short-range order. These unique structures might have drug or nutraceutical delivery advantages. Introduction Microemulsions are optically isotropic and thermodynami- cally stable nanostructured mixtures of water, oil, and am- phiphile(s). 1 They frequently require cosolvents or cosurfactants to achieve very low interfacial tension and to facilitate proper packing parameters. Water-in-oil (W/O), bicontinuous, and oil- in-water (O/W) are the most common structures. 2 Bicontinuous microemulsion structures contain oil and water domains that are chaotically intertwined but are stabilized by sheetlike surfactant regions in the boundary zones between domains. These sheetlike regions are formed due to the tendency of the surfactant to localize between water-rich and oil-rich regions. Nonetheless, the long-range order remains stochastic. 3 The absence of geometric order, verified by the lack of secondary scattering maxima in the small-angle X-ray scattering (SAXS) patterns, is what distinguished microemulsions from ordered isotropic liquid crystals 3 until this work was done. Different models for bicontinuous microemulsion structures have been suggested: Scriven suggested that bicontinuous structures may be present in mesomorphic, liquid-crystal-like phases regarded as dispersions of spheres, cylinders, or lamel- lae. 4 Talmon and Prager used a Voronoi theoretical model, where the bicontinuous structure was generated from many microscopic cells which are filled randomly with oil and water, while surfactant molecules are confined to the internal interfaces between oil and water. 5 Saito and Shinoda described a bicon- tinuous microstructure in terms of a thermally coiled lamellar liquid crystal. 6,7 Later, Kaler’s group also described bicontinuous structures based on the model of a disordered lamellar phase. 8 It is generally agreed that bicontinuous microemulsions do not have the same long-range order as liquid crystals but rather exhibit a disordered or melted structure. 9 Despite the existence of long-range order within the liquid crystalline phases, their application is limited due to their high viscosity. Consequently, low viscosity dispersions of liquid crystal phases in the aqueous phase are being explored (e.g., cubosome, hexosome, and liposome dispersions). 10-12 Dilution with water lowers the viscosity but also decreases the solubi- lization capacity and the stability. 10-12 There is as yet no experimental evidence for formulation of a bicontinuous me- sophase that will combine the advantages of both microemul- sions and liquid crystalline phases. Therefore, in the current work we aimed at selecting the proper ingredients that will allow us to obtain such structures and to explore their physical properties. In our studies, a complex mixture of pharmaceutically permitted components was used to form fully dilutable nonionic microemulsions. The oil phase consisted of triacetin (TA), D-R- tocopherol acetate (TocAc), and ethanol (EtOH) and as a surfactant, Tween 60 (T60) was used. The aqueous phase was comprised only of water. Under in ViVo conditions a micro- emulsion is projected to infinite dilution by body fluids. The concentrating effect is observed when microemulsions are applied topically (due to the evaporation of water). Thus, we determined the behavior of the system upon dilution with water, unlike many studies that neglect to investigate the effect of dilution on the structure and on the stability of microemulsions. 13-15 * Author to whom correspondence should be addressed at the Casali Institute of Applied Chemistry, E. Safra Campus, Givat Ram, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel. Telephone: 972-2-658- 6574/5. Fax: 972-2-652-0262. E-mail: garti@vms.huji.ac.il. † Casali Institute of Applied Chemistry and The Institute of Chemistry. ‡ Wolfson Centre for Applied Structural Biology. § The Institute of Chemistry. J. Phys. Chem. B 2009, 113, 10669–10678 10669 10.1021/jp901617g CCC: $40.75  2009 American Chemical Society Published on Web 07/13/2009