Rapid report Liquid-liquid immiscibility in lipid monolayers

Some binary lipid mixtures form coexisting liquid phases when spread at the airrwater interface. This work describes the pressure–composition phase diagrams of binary mixtures of four unsaturated phosphatidylcholines with dihydrocholesterol. These four binary mixtures have critical compositions of approximately fifty mole percent, and average critical exponents of 0.25 "0.07. The data can also be approximated by a regular solution thermodynamic model, yielding parameters for the non-ideality of these mixtures. Fluorescence microscopy has been used to study lipid monolayers at the airrwater interface w1–3 . x Monolayers composed of mixtures of cholesterol and phospholipids are of particular interest as biological w x cell membranes contain these lipids 4 . Some of these w mixtures exhibit liquid-liquid immiscibility x 5,6 . It is of interest to know what molecular features w x give rise to liquid-liquid phase separation 7,8 . This paper presents the room-temperature surface pressure–composition phase diagrams for binary mixtures Ž . of dimyristoleoylphosphatidylcholine DMoPC , diŽ palmitoleoylphosphatidylcholine . DPoPC , dioleoylŽ . phosphatidylcholine DOPC , and dieicosenoylphosŽ phatidylcholine . DEPC with dihydrocholesterol Materials and methods. The DMoPC, DPoPC, DOPC, and DEPC were purchased from Avanti Polar Lipids, Inc. The fluorescent lipid Phase diagrams for binary mixtures of DMoPC, DPoPC, DOPC, and DEPC with DChol were mapped using epifluorescence microscopy. A fluorescent probe was incorporated into the monolayer. The probe partitions unequally into the two phases, providing contrast. Under the experimental conditions de­ scribed, low concentrations of the fluorescent probe phospholipids have a negligible effect on the derived Ž0.1–1 mol%. phase diagram. ŽDChol . . The acyl chains are 14, 16, 18, and 20 carbon atoms in length, respectively. tion on the binary phase diagrams with DChol. effect of phospholipid acyl chain length and unsatura­ tion 11. The phospholipids were chosen to probe the -unsaturated at posi­ cis with 9 position These acyl chains are cis-unsaturated at the exception of DEPC which is Ž . probe NTexas Red sulfonyl -1,2-dihexadecanoyl­ sn-glycero-3-phosphoethanolamine, triethylammo­ nium salt ŽTR-DPPE. was purchased from Molecular Probes, Inc. The synthetic cholesterol analog dihy­ drocholesterol ŽDChol. was purchased from Sigma Chemical Company. All chemicals were used without further purification. The experiments were carried out at 20–228C. DChol rather than cholesterol itself is used to minimize air oxidation of the steroid. The monolayers were spread from 1 mM chloro­ form solution containing 0.25 mol% TR-DPPE dye onto a subphase of distilled, deionized water. The monolayers were blanketed with argon to minimize oxidation of the lipids. Compression and expansion of the film were carried out with a movable barrier and the surface pressure was measured with a Wil­ helmy plate. The monolayer was viewed with a Zeiss epifluorescence microscope fitted with a Cohu lowlight-level video camera. After spreading, the mono­ layers were compressed while under observation. Phase boundaries were determined by noting the pressure at which the two phases become homoge­ neous. For the lipid mixtures described the compres­ sion–expansion of the monolayer giving rise to the disappearance–appearance of the two phases is re­ versible and reproducible in the vicinity of the phase boundary, to within less than plus or minus one mNrm. Results and discussion. The derived phase dia­ grams for the binary systems of DMoPC, DPoPC, DOPC, and DEPC with DChol are shown in Fig. 1. These binary phase diagrams reveal several trends when compared to each other and with the phase diagrams of other systems. To make these compar­ isons it is useful to fit the data to models that characterize liquid-liquid immiscibility with critical points. The data can be fit to the following critical expo­ nent equation: b 1 < XyX <sF Žp yp . Ž . c c Here X is the mole fraction of dihydrocholesterol in either phase, Xc is the mole fraction of dihydroFig. 1. The pressure–composition phase diagrams for four phospholipids with dihydrocholesterol. The data are fit to the equation b < XyX <sFŽp yp . where X is the mole fraction of dihydrocholesterol in either phase, X is the mole fraction of dihydrocholesterol c c c Ž . of the critical mixture, b is the critical exponent, and F is an adjustable parameter. a : The DMoPC-DChol phase diagram. Ž . X s0.48"0.01, p s18"1, bs0.23"0.06, and Fs0.2 "0.4. b : The DPoPC-DChol phase diagram. X s0.52"0.01, p s12 c c c c Ž . "1, bs0.25"0.03, and Fs0.2 "0.2. c : The DOPC-DChol phase diagram. Xc s0.48 "0.01, pc s6"1, bs0.27 "0.07, and Ž . Fs0.2 "0.3. d : The DEPC-DChol phase diagram. Xc s0.49"0.01, pc s2.0"0.4, bs0.3"0.1, and Fs0.2"0.6. cholesterol at the critical composition, b is the criti­ cal exponent, and F is an adjustable parameter. The average value of b for these systems was determined to be 0.25 "0.07. For comparison, the classical theo­ retical value of ß for mathematically two-dimensional systems is 0.125 while that of three-dimensional sys­ tems is 0.32 w x 9 . Although these monolayers are only one molecule in thickness the intermolecular forces are not strictly two-dimensional w x 10 . The measured value of b is thus plausible. The values of the critical pressure pc given for these unsaturated phosphatidylcholines contrast with those known for similar saturated phosphatidyl­ cholines. The saturated analog of DMoPC is dimyris­ toylphosphatidylcholine, DMPC. Its mixtures have a critical pressure, pc , of 10.2 mNrm w x 11 , while we see in Fig. 1 that pc for DMoPC is 18 mNrm. The saturated analog of DPoPC is dipalmitoylphospha­ tidylcholine, or DPPC. Its mixtures with DChol have a pc of approximately 2mNrm ŽS. Perkovic, unpub­ ́ . lished data . Other work suggests that these mixtures with cholesterol have a pc of approximately 4 mNrm w x c for DPoPC is 12 7 . However, Fig. 1 shows that p mNrm. Unsaturation at position 9 in phosphatidyl­ cholines therefore raises pc by 8–10 mNrm. The values of the critical composition Xc , given by the fits for these unsaturated phosphatidylcholines also contrast with those known for similar saturated phosphatidylcholines. The saturated phosphatidyl­ cholines DMPC and DPPC have critical compositions w x Xc of approximately 0.3 11 , while all four of the Fig. 2. The critical pressures of phospholipidrdihydrocholesterol mixtures vs. phospholipid acyl chain length. The dashed line is a linear fit. unsaturated phosphatidylcholines used in this study have critical compositions Xc of approximately 0.5. It is also instructive to compare the values of pc for the various unsaturated lipids with each other. In Fig. 2, pc is plotted as a function of phospholipid acyl chain length. Note that there is a linear trend towards greater miscibility with increasing phospho­ lipid acyl chain length. A similar trend of greater miscibility with increasing acyl chain length was found by Slotte w x 7 in mixtures of saturated phos­ phatidylcholines with cholesterol. These phase diagrams can be accounted for with the following simple thermodynamic model. We be­ gin by considering a binary mixture of liquids that w x can be modeled as a regular solution 12 . The chemi­ cal potential of component 1 is written 0 m sm1 qRT ln X1 q Ž A1 qaX 2 . Žpyp 0 . 1 2 q2 RT 0 X2 2 Ž . c 2 Here m 1 is the chemical potential of pure compo­ nent 1 at the pressure psp 0, X1 and X2 are the mole fractions of components 1 and 2, Tc 0 is the critical temperature when the pressure is p 0, A1 is the molar area of component 1, and a is an area contraction parameter. A similar equation holds for component 2 with the subscripts 1 and 2 reversed. Mixtures of phosphatidylcholines and cholesterol are known to contract in area when mixed at constant pressure, with contraction parameters of the order of ́ minus 40 square Angstroms per molecule. From Eq. Ž . 2 it follows that the critical temperature of the mixture when the pressure is p c p c 0 is T Ž .sT q Žp yp .ar2 R. When the pressure p is equal to the 0 critical pressure pc the critical temperature is room temperature, T Ž .sT. Thus T Ž . p p sT q Žp y c c c pc .ar2 R. When the pressure is above the critical pressure, room temperature is above the critical tem­ perature and the system is one phase, T%Tc Žp%pc ... When the pressure is below the critical pressure, room temperature is below the critical temperature and the monolayer shows two immiscible liquid phases T$Tc Žp$pc .. The simple thermodynamic chemical potential in Eq. Ž . gives rise to a symmet­ 2 ric phase diagram for which the equation is pspc q Ž2 RTra. ln Ž X rX2 .r2Ž X1 yX2 .y1 1