International Journal of Pharmaceutics 310 (2006) 203–212 Pharmaceutical Nanotechnology Cryo-TEM investigation of phase behaviour and aggregate structure in dilute dispersions of monoolein and oleic acid Denise Alves Ferreira a , Maria Vit´ oria L.B. Bentley a , G¨ oran Karlsson b , Katarina Edwards b,∗ a Faculdade de Ciˆ encias Farmacˆ euticas de Ribeir ˜ ao Preto, Universidade de S˜ ao Paulo, Av do Caf´ e s/n, 14040-903, Ribeir ˜ ao Preto, SP, Brazil b Department of Physical and Analytical Chemistry, Uppsala University, Box 579, S-751 23 Uppsala, Sweden Received 27 June 2005; received in revised form 15 November 2005; accepted 19 November 2005 Available online 24 January 2006 Abstract Cryo-transmission electron microscopy (cryo-TEM) was used to image the microstructure in dilute sonicated dispersions of monoolein and oleic acid. The aim of the study was to explore how different experimental parameters, such as sample composition, total lipid concentration, pH, and ageing affect the phase behaviour and aggregate structure. Our investigations show that a rich variety of lamellar and non-lamellar structures, including liposomes and particles of cubic and inverted hexagonal phase, may form depending on the experimental conditions. The results are analyzed and discussed in relation to existing phase diagrams and earlier investigations concerning phase- and structural behaviour in monoolein/oleic acid/water systems. © 2005 Elsevier B.V. All rights reserved. Keywords: Monoolein; Oleic acid; Aggregate structure; Liposomes; Dispersed liquid crystalline phases; Cryo-TEM; Vaccines 1. Introduction The area of lipid and surfactant self-assembly has over the years received well-deserved attention from a multidisci- plinary science community encompassing chemists, biologists and physicists. In the pharmaceutical field, interest in the use of micelles, microemulsions and dispersed liquid crystalline phases formed by biocompatible surfactants is rapidly growing. As a consequence, the demand has increased for systems where par- ticle size, curvature, charge, and other physico-chemical proper- ties are well defined and possible to control (Mele et al., 2004). Monoolein (MO), a monoglyceride traditionally used as emulsifier and food additive, has during the last decades received significant attention for applications in the pharmaceutical area (Larsson, 2000; Ganem-Quintanar et al., 2000). This polar lipid is essentially insoluble in water but self-associates and may, depending on water content, form a reversed micellar (L 2 ), a lamellar (L  ), or a bicontinuous cubic (C) phase of either the ∗ Corresponding author. Tel.: +46 18 471 36 68; fax: +46 18 471 36 54. E-mail address: Katarina.Edwards@fki.uu.se (K. Edwards). diamond (space group Pn3m) or gyroid (space group Ia3d) type (Lindblom et al., 1979; Hyde et al., 1984; Qiu and Caffrey, 2000). Several publications have reported on the ability of the MO/water system to solubilize both hydrophilic and lipophilic substances and pointed out the potential of this system for con- trolled drug delivery purposes (Shah et al., 2001). A number of studies have focused on modifications in the MO/water phase behaviour induced by addition of a third component (Lawrence, 1994; Larsson, 2000; Ganem-Quintanar et al., 2000), and the observed changes in phase behaviour have been correlated to properties, such as polarity, geometric shape and concentration, of the additive (Caboi et al., 1997; Pitzalis et al., 2000; Caboi et al., 2001). Apart from constituting promising candidates for drug deliv- ery, MO-based dispersions have lately emerged as attractive systems also for the delivery of vaccines. The interest is here primarily sparked by the need to improve the immunogenic- ity of vaccines containing weak or non-traditional antigens, such as recombinant proteins, peptides, and DNA (O’Hagan and Rappuoli, 2004). In particular, MO/oleic acid (OA) dispersions have demonstrated good potential as mucosal vaccine adjuvants 0378-5173/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.ijpharm.2005.11.028