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J. Phys. Chem. B 2007, 111, 11428-11436
Detecting Phase Transitions in Phosphatidylcholine Vesicles by Raman Microscopy and Self-Modeling Curve Resolution Christopher B. Fox,† Rory H. Uibel,‡ and Joel M. Harris*,†,§ Department of Bioengineering, UniVersity of Utah, 50 South Central Campus DriVe, Salt Lake City, Utah 84112-9202, Process Instruments Inc., 825 North 300 West Ste 225, Salt Lake City, Utah 84103, and Department of Chemistry, UniVersity of Utah, 315 South 1400 East, Salt Lake City, Utah 84112-0850 ReceiVed: May 10, 2007; In Final Form: July 11, 2007
The study of phospholipid phase transitions is important for understanding drug- and protein-membrane interactions as well as other phenomena such as trans-membrane diffusion and vesicle fusion. A temperaturecontrolled stage on a confocal Raman microscope has allowed phase transitions in optically trapped phospholipid vesicles to be monitored. Raman spectra were acquired and analyzed using self-modeling curve resolution, a multivariate statistical analysis technique. This method revealed the subtle spectral changes indicative of sub- and pretransitions and main transitions in vesicles composed of 1,2-dipalmitoyl-sn-glycero-3phosphocholine (DPPC). The Raman scattering results were compared to differential scanning calorimetry (DSC) experiments and found to be in good agreement. This method of observing lipid phase transition profiles requires little sample preparation and a minimal amount of lipid (e0.1 nmol) for an experiment. The conformational changes of the phospholipid molecules occurring during phase transitions are elucidated from the Raman spectroscopy results. The evolution of chain decoupling, rotational disorder, and gauche defects in the lipid acyl chains as a function of temperature is described.
Introduction The importance of phospholipids as biological building blocks is becoming increasingly apparent.1,2 Phospholipid membranes serve as encapsulators of cells and organelles and are integral participants in the control of molecular transport into and out of cells and in cell communication. Lipid malfunction is implicated in several diseases. Research on lipid structure and dynamics is crucial to understanding the cell membrane and membrane-bound protein behavior and is a critical factor in drug development. Lamellar lipid assemblies undergo characteristic thermal phase transitions.1,3,4 These transitions describe changes in lipid structural order. In phosphatidylcholines, there are four stable phases described as lamellar crystalline orthorhombic (Lc or subgel phase), lamellar orthorhombic (Lβ′ or gel phase), hexagonal periodical (Pβ′ or ripple phase), and disordered liquidcrystalline (LR or fluid phase). The corresponding phase transitions are the subtransition (Ts) for subgel to gel phases, the pretransition (Tp) for gel to ripple phases, and the main transition (Tm) for ripple to fluid phases.5,6 In addition, metastable phases with corresponding transition temperatures have been found in some phosphatidylcholines.7,8 All of these transitions occur at specific temperatures for each lipid depending on headgroup, chain length, chain saturation, hydration, and thermal history. The presence of membrane-active drug molecules will often alter the transition temperature and profile.9-16 Although extensive research has been carried out to study the structural changes occurring during these thermal transitions, * Corresponding author. E-mail address:
[email protected]. † Department of Bioengineering, University of Utah. ‡ Process Instruments Inc. § Department of Chemistry, University of Utah.
the subtransitions and pretransition are still not well understood, and several models have been proffered which differ according to the method of detection.5,6,17-20 Many techniques have previously been used to monitor lipid phase transitions, including differential scanning calorimetry (DSC),4,21-24 Raman and infrared spectroscopy,6,25,26 X-ray diffraction (XRD),13 NMR,13,27 fluorescence spectroscopy,9 and computer simulations.5,17,28 Several studies have examined the influence of drugs and other membraneperturbantsonphospholipidphasetransitionbehavior.9-16,27 Despite the variety of methods employed to study lipid phase transitions and drug-membrane interactions, there are major drawbacks with many of these methods: for high sensitivity methods such as fluorescence and electron spin resonance (ESR), there is a need for labeling; the influence of the fluorescence or spin-resonance probe on the structure and behavior of the system must be considered.29,30 For less sensitive methods such as DSC, NMR, XRD, or traditional IR and Raman spectroscopy, large sample concentrations are required, typically in the 10-100 mM range.11,22,31 Raman spectroscopy is well-suited to study phospholipid structure; the method involves no labeling and easily accommodates aqueous buffers with a minimum of sample preparation. When integrated with a confocal microscope, this technique is much more sensitive for detecting small particles such as lipid vesicles. With a focused excitation laser beam in a confocalRaman microscope, it is possible to optically trap individual vesicles or small vesicle aggregates so that Raman scattering is collected from a small (1.5 fL) volume dominated by the trapped vesicle such that contributions from the surrounding solution are minimized.32 The confocal Raman microscope allows lipid vesicles to be optically trapped indefinitely in the laser focus33 and their spectra to be acquired while the temperature or other environmental conditions are varied.34,35 A temperature-
10.1021/jp0735886 CCC: $37.00 © 2007 American Chemical Society Published on Web 09/12/2007
Phase Transitions in Phosphatidylcholine Vesicles controlled stage and a custom-made metal sample cell allow sample temperatures to be varied from ∼5 to 55 °C using very low sample volumes (
