Time evolution of the thermotropic behavior of spontaneous liposomes and disks of the DMPC–DTAC aqueous system Maria Isabel Viseu * , Raquel F. Correia, Anabela C. Fernandes Centro de Química Estrutural, Instituto Superior Técnico, Universidade Técnica de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal article info Article history: Received 15 April 2010 Accepted 29 June 2010 Available online 1 July 2010 Keywords: 1,2-Dimyristoyl-sn-glycero-3- phosphocholine Dodecyltrimethylammonium chloride Lipid–detergent interactions Spontaneous colloidal nanostructures Discoidal micelles Threadlike micelles Spontaneous vesicles Differential scanning calorimetry Dynamic light scattering abstract In this work, solubilization of the phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) by the cationic detergent n-dodecyltrimethylammonium chloride (DTAC) was studied in aqueous solution, at a fixed DMPC concentration and variable detergent:lipid (D:L) molar ratios. The colloidal nanostruc- tures present in different stages of the solubilization process were characterized using micro-differential scanning calorimetry (DSC) and dynamic light scattering (DLS) techniques. For total (analytical) D:L molar ratios below 1, DTAC monomers incorporate into the DMPC liposome bilayers, forming smaller and more fluid vesicles than pure DMPC liposomes. At D:L  1–2, vesicles begin to rupture, coexisting with intact vesicles and bilayer fragments. At D:L  2–12.5, discoidal and spherical micelles are formed and coexist with vesicles; a slow structural rearrangement of the system, monitored in successive DSC heating/cooling cycles, was observed, and is reported for the first time. Finally, for D:L above 15–20, the bilayers are completely dissolved, and the main aggregates in solution become spherical micelles, which slowly evolve to cylindrical (threadlike) micelles. Based on the dependence of the temperature and enthalpy of transitions on the total D:L molar ratio, at constant DMPC concentration, a schematic model, showing the different colloidal nanostructures present in the solubilization process, is proposed. Ó 2010 Elsevier Inc. All rights reserved. 1. Introduction Solubilization of liposomes by synthetic detergents is an impor- tant tool to model biomembrane systems. In addition, detergents are used to purify, characterize, and crystallize membrane proteins [1] and to reconstitute integral membrane proteins with lipids, in biomimetic systems [2,3]. The dissolution and reconstitution of membrane proteins is based on the detergent ability to breakdown the vesicular structure and form detergent–lipid mixed micelles [4–6]. Furthermore, in the dissolution process of membrane pro- teins, their functional properties are often best preserved when a detergent is present [7]. Another important application of deter- gents in membrane research is the prevention of non-specific bind- ing, in immunoassays [8] and in cell lysis [9]. Pure phospholipids, when dispersed in an aqueous medium, form multilamellar liposomes (or vesicles, MLVs), the size of which depends on the lipid concentration and preparation method. The addition of even small amounts of detergent to a lipid membrane (or vesicle) affects its structure, fluidity, and thermody- namic properties [10]. Above a critical detergent:lipid (D:L) molar ratio, detergents dissolve sparingly soluble lipids by the break- down of lamellar structures and the formation of detergent–lipid micelles [6]. Vesicle solubilization by increasing amounts of detergent was classically described by the three-stages hypothesis [4,11]. In stage 1, the detergent molecules incorporate into the liposome bilayer membrane, without disrupting it. Increasing the detergent amount in the suspension, a critical (effective) D:L molar ratio, R sat , is reached at which the bilayer no longer supports further detergent and starts to disintegrate. Then, the conversion of saturated mixed bilayers into mixed micelles begins, and both structures coexist in equilibrium (stage 2). Increasing even further the amount of deter- gent, another critical D:L ratio, R sol is attained, at which the bilayer structure can no longer be maintained and all lipid molecules are incorporated into micelles. This corresponds to the onset of stage 3 (complete liposome dissolution). With further detergent addition, the effective D:L ratio increases within the micelles, 0021-9797/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.jcis.2010.06.061 Abbreviations: CMC, critical micelle concentration; Cryo-TEM, electron transmis- sion microscopy, with sample cryogenation; DTAC, n-dodecyltrimethylammonium chloride; CTAC, n-hexadecyltrimethylammonium chloride; DMPC, 1,2-dimyristoyl- sn-glycero-3-phosphocholine; DDAB, di(n-dodecyl)dimethylammonium bromide; DSC, differential scanning calorimetry; DLS, dynamic light scattering; D, detergent (usually, DTAC); L, lipid (usually, DMPC); D:L, total (analytical) detergent/lipid molar ratio; R eff , critical (effective) detergent/lipid molar ratio; R sat , effective detergent/lipid molar ratio for saturation; R sol , effective detergent/lipid molar ratio for solubilization; MLVs, multilamellar vesicles (liposomes); ULVs, unilamellar vesicles (liposomes). * Corresponding author. Address: Centro de Química Estrutural, Complexo I, Instituto Superior 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 351 (2010) 156–165 Contents lists available at ScienceDirect Journal of Colloid and Interface Science www.elsevier.com/locate/jcis - 4 -