Nanoscale Phase Separation in DSPC-Cholesterol Systems Angela C. Brown † and Steven P. Wrenn* Department of Chemical and Biological Engineering, Drexel University, 3141 Chestnut Street, Philadelphia, Pennsylvania 19104, United States * S Supporting Information ABSTRACT: The lipid arrangement of eukaryotic cell membranes has been shown to be heterogeneous, with domains enriched in cholesterol and saturated phospholipids, coexisting with a continuous phase that is enriched in unsaturated phospholipids. While the existence of these domains is well-established, there is still a lack of consensus regarding domain size and the factors influencing it. In this work, we investigate model membranes consisting of 1,2- dioleoyl-sn-glycero-3-phosphocholine (DOPC)-1,2-distearo- yl-sn-glycero-3-phosphocholine (DSPC)-cholesterol (Chol) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC, 18:1-16:0)-DSPC-Chol with a steady-state fluorescence assay and report the influence of phospholipid chain saturation and chain length on domain size. The spectral shifts of 1-myristoyl-2-[12-[(5-dimethylamino-1-naphthalenesulfonyl)amino]- dodecanoyl]-sn-glycero-3-phosphocholine (DAN-PC) and a Fö rster resonance energy transfer (FRET) assay were used, along with an analytical model, to estimate domain sizes. A region of nanoscale domain existence was observed in both ternary systems; however, the domains formed in the system containing the asymmetric lipid (POPC, 18:1-16:0) were larger than those formed in the diunsaturated lipid (DOPC, 18:1-18:1). This is a new finding, as domains were not previously known to exist in similar POPC-based systems. 1. INTRODUCTION Since the introduction of the raft hypothesis of eukaryotic cell membrane organization was first proposed, 1 extensive research has been performed in both natural and model cell membranes to characterize these ordered, cholesterol-rich regions. It is now believed that the liquid-ordered (l o ) phase, which is observed in binary and ternary lipid mixtures containing cholesterol, is the equivalent of the raft phase in natural membranes. 2-4 This l o phase is enriched in cholesterol, sphingolipids, and saturated phospholipids, much like the raft portion of cell membranes. 3 While much has been learned about lipid rafts, many questions remain unanswered. One such question is what governs raft size. Answering this question in not a straightforward task, given that direct visualization of rafts in natural cell membranes is difficult as their nanometer-scale size is below the resolution of most common direct visualization techniques. In addition, both raft and nonraft phases are liquid phases, and the mechanical and physical properties of the two phases are very similar, making it extremely difficult to differentiate between the two. 4,5 Another complicating factor is the transitory nature of rafts in natural cell membranes. 6 Additionally, the wide variety of membrane components in natural cell membranes precludes systematic studies of the factors influencing domain size. For this reason, many researchers have turned to model membranes where the lipid components can be precisely controlled. We, and others, have found that Fö rster resonance energy transfer (FRET) is well-suited to detect nanometer-scale domains in model membranes, 7-10 and we have presented a method to extract domain size estimates from steady-state FRET data. 7 The use of FRET in model membranes allows for systematic studies of the factors influencing domain size to be ascertained. It has been well-established that lipid mixtures of a high- transition temperature (T m ) lipid, a low-T m lipid, and cholesterol (Chol) form immiscible domains. 11 However, not all such systems form domains, 7,11,12 and it is clear that the factors governing domain formation and the resulting domain size are complex. In this work, we investigate domain formation in two ternary systems containing cholesterol and 1,2- distearoyl-sn-glycero-3-phosphocholine (DSPC, 18:0), as well as either 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC, 18:1), or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC, 18:1-16:0), to determine the effect of phospholipid chain properties (particularly chain length and saturation) on domain formation and size in model membranes. 2. MATERIALS AND METHODS 2.1. Materials. DSPC, DOPC, POPC, and 1-myristoyl-2-[12-[(5- dimethylamino-1-naphthalenesulfonyl)amino]dodecanoyl]-sn-glycero- 3-phosphocholine (DAN-PC) were purchased from Avanti Polar Lipids (Alabaster, AL). Cholesterol, dehydroergosterol (DHE), sodium chloride (NaCl), calcium chloride (CaCl 2 ), sodium azide (NaN 3 ), and 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid Received: September 19, 2012 Revised: June 30, 2013 Article pubs.acs.org/Langmuir © XXXX American Chemical Society A dx.doi.org/10.1021/la401249m | Langmuir XXXX, XXX, XXX-XXX