Spontaneous vesicles, disks, threadlike and spherical micelles found in the solubilization of DMPC liposomes by the detergent DTAC Raquel F. Correia a , Maria Isabel Viseu a,⇑ , Telmo J.V. Prazeres b , José M.G. Martinho b a Centro de Química Estrutural, Instituto Superior Técnico, Universidade Técnica de Lisboa, 1049-001 Lisboa, Portugal b Centro de Química-Física Molecular and Institute of Nanosciences and Nanotechnologies, Instituto Superior Técnico, Universidade Técnica de Lisboa, 1049-001 Lisboa, Portugal article info Article history: Received 25 January 2012 Accepted 10 April 2012 Available online 25 April 2012 Keywords: 1,2-Dimyristoyl-sn-glycero-3- phosphocholine Dodecyltrimethylammonium chloride Chlorophyll-a Liposomes Discoidal micelles Cylindrical micelles Neutral-sphere-equivalent hydrodynamic diameters Spontaneous colloidal nanostructures abstract The spontaneous colloidal nanostructures formed in water by the zwitterionic phospholipid DMPC (1,2- dimyristoyl-sn-glycero-3-phosphocholine) with the cationic detergent DTAC (n-dodecyltrimethylammo- nium chloride) were investigated at a fixed DMPC concentration and variable detergent:lipid total molar ratios (D:L). Apparent (neutral-sphere-equivalent) hydrodynamic diameters (U e ) of liposomes and micelles were obtained by dynamic light scattering (DLS). Fluorescence lifetime imaging microscopy (FLIM), using chlorophyll-a as a probe, showed the morphology of giant vesicles and threadlike micelles. Micro-differential scanning calorimetry (micro-DSC) detected the presence of bilayers, in vesicles and discoidal micelles (disks). Pure DMPC liposomes are multilamellar and polydisperse (U e  100– 10,000 nm). As D:L increased, smaller vesicles were found, due to the bigger spontaneous curvature of the bilayer: at D:L = 1, ULVs (unilamellar vesicles; U e  100 nm) appeared and, at D:L = 2–10, ULVs coex- isted with disks (U e  30 nm). Bilayers totally disappeared at D:L P 15, giving rise to spheroidal (U e  2– 16 nm) and threadlike (U e  100–10,000 nm) micelles. A quasi-equilibrium structural diagram for the DMPC–DTAC–water system shows equivalent diameters of the scattering nanoparticles as a function of D:L. The results obtained herein for the system DMPC–DTAC show the role of electrostatic interactions in the formation of the mixed structures. Ó 2012 Elsevier Inc. All rights reserved. 1. Introduction Phospholipid liposomes (or vesicles) are commonly used as models of cell membranes and, in the pharmaceutical and cosmetic industries, as nano-containers for drug delivery [1–3]. Micelle-forming surfactants (detergents) have been widely used as solubilizing agents for isolation, purification, and reconstitution of cell membrane components, especially membrane proteins. Liposome solubilization has been explained using a three-stages model [4,5]. In stage I, the detergent molecules are incorporated into the vesicle bilayers, up to a critical concentration. Above this concentration, the system enters into stage II, where detergent-sat- urated mixed vesicles coexist with the first mixed micelles. Finally, in stage III, vesicle solubilization is complete and only mixed mi- celles exist in the system. In reality, lipid solubilization by a detergent may be more com- plex, because the morphological changes of the process depend on both the total and relative amounts of lipid and detergent in the mixture, and on the preparation method [6]. On the other hand, a variety of mixed colloidal aggregates, such as ruptured vesicles, curved bilayer fragments, and discoidal micelles, have also been found as more or less stable intermediates by cryo-transmission electron microscopy (cryo-TEM) [7–15] and micro-DSC [16]. To fully understand the whole solubilization process, a detailed char- acterization of these colloidal nanostructures is needed. In a previous paper, the structural changes occurring in the DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) liposome solubilization by the cationic detergent DTAC (n-dodecyltrimethyl- ammonium chloride), at a fixed phospholipid concentration and variable detergent:lipid (D:L) total molar ratios, were studied mainly by micro-differential scanning calorimetry (micro-DSC) [16]. Thermodynamic parameters and membrane fluidities of bi- layer-containing structures were determined for pure and mixed liposomes, curved bilayer fragments, and discoidal micelles (disks). Spheroidal and cylindrical mixed micelles were detected by dy- namic light scattering (DLS). However, the size and morphology of these nanostructures were not systematically studied. The main purpose of the present work is the determination of mean hydrodynamic diameters of the spontaneous colloidal nano- structures formed in the DMPC–DTAC aqueous system, as a func- tion of the total D:L molar ratio. Spherical micelles and vesicles differ in sizes by orders of magnitude, showing a very distinct scat- tering behavior: indeed, micelles (<5–10 nm in diameter) make 0021-9797/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jcis.2012.04.027 ⇑ Corresponding author. Address: Centro de Química Estrutural, Instituto Supe- rior Técnico, Universidade Técnica de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal. Fax: +351 21 8464455. E-mail address: isabelviseu@ist.utl.pt (M.I. Viseu). Journal of Colloid and Interface Science 379 (2012) 56–63 Contents lists available at SciVerse ScienceDirect Journal of Colloid and Interface Science www.elsevier.com/locate/jcis