Phase Behavior of Planar Supported Lipid Membranes Composed of Cholesterol and 1,2-Distearoyl-sn-Glycerol-3-Phosphocholine Examined by Sum-Frequency Vibrational Spectroscopy

Abstract

The influence of cholesterol (CHO) on the phase behavior of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) planar supported lipid bilayers (PSLBs) was investigated by sum-frequency vibrational spectroscopy (SFVS). The intrinsic symmetry constraints of SFVS were exploited to measure the asymmetric distribution of phase segregated phospholipid domains in the proximal and distal layers of DSPC + CHO binary mixtures as a function of CHO content and temperature. The SFVS results suggest that cholesterol significantly affects the phase segregation and domain distribution in PSLBs of DSPC in a concentration dependent manner, similar to that found in bulk suspensions. The SFVS spectroscopic measurements of phase segregation and structure change in the binary mixture indicate that membrane asymmetry must be present in order for the changes in SFVS signal to be observed. These results therefore provide important evidence for the delocalization and segregation of different phase domain structures in PSLBs due to the interaction of cholesterol and phospholipids.

Figure 1

Molecular structures of CHO, CHO-d₇, DSPC, DSPC-d₇₀ and DSPC-d₈₃.

Figure 2

(a) SFVS spectra of DSPC PSLBs containing various concentrations of CHO-d₇ recorded at 23 °C. (b) SFVS spectra of 60 mol% CHO-d₇ in a DSPC bilayer (black) and 60 mol% CHO-d₇ in a DSPC-d₈₃ bilayer (red) recorded at 23 °C. Spectrum (c) is the difference of the DSPC+60 mol% CHO-d₇ spectrum minus the spectrum of DSPC-d₈₃ + 60 mol% CHO-d₇ shown in (b). Also shown in blue are the individual peaks used in the fitting of the difference spectrum (spectra are offset for clarity). All SFVS spectra were collected with s-polarized sum-frequency, s-polarized visible, and p-polarized IR (ssp).

Figure 3

SFVS spectra of pure CHO (black) and CHO-d₇ (red) monolayers at the silica/air interface. The SFVS spectra were collected with s-polarized sum-frequency, s-polarized visible, and p-polarized IR (ssp).

Figure 4

SFVS CH₃ v_s intensity as a function of temperature for increasing concentrations of CHO-d₇ in a DSPC bilayer recorded with the ssp polarization combination. Black curves represent samples heated at a rate of 0.24 °C/min. Red curves represent samples which were heated at a rate of 0.24 °C/min after the bilayer was heated to 70 °C at a rate of 0.24 °C /min and rapidly cooled to 30 °C at 1 °C/min. Blue curves represent samples cooled with at a rate of 0.24 °C/min after the bilayer was heated to 70 °C at a rate of 0.24 °C/min.

Figure 5

Composite phase diagram of a DSPC+CHO binary mixture extrapolated from the data of Vist et al., and Ipsen et al.[5,11] Definitions: solid-ordered (so), liquid-disordered (ld), and liquid-ordered (lo) phases. The blue arrow indicates the direction of increasing CHO concentration at 23 °C examined by SFVS. The red arrows indicate the temperature paths traversed at the fixed CHO concentration of 5, 10, 15, 25, 40 and 60 mol% for the SFVS experiments.

Figure 6

Illustrations of CHO+DSPC structures: (a) symmetric distribution of CHO in a so (blue) and ld phases (red), (b) symmetric distribution of CHO in the so+lo phase coexistence region (blue and green respectively) and the lo+ld phase coexistence region (green and red respectively), and (c) an asymmetric distribution of CHO in the lo phase at low temperature and a symmetric distribution at high temperature.

Figure 7

(a) SFVS spectrum of a CHO-d₇ monolayer on a sapphire substrate and (b) a spectrum of 60 mol% CHO-d₇ in a DSPC-d₇₀ bilayer on sapphire, both shown in gray. Also shown are the spectral fits to the data (solid black lines) and the individual peaks used to obtain the fits which have been offset for clarity. An illustration of the CHO arrangement in a monolayer and bilayer are shown as inserts to (a) and (b) respectively.