Optically Trapping Confocal Raman Microscopy of Individual Lipid Vesicles: Kinetics of Phospholipase A 2 -Catalyzed Hydrolysis of Phospholipids in the Membrane Bilayer
ABSTRACT Phospholipase A2 (PLA2)-catalyzed hydrolysis at the sn-2 position of 1,2-dimyristoyl-sn-glycero-3-phosphocholine in optically trapped liposomes is monitored in situ using confocal Raman microscopy. Individual optically trapped liposomes (0.6 microm in diameter) are exposed to PLA2 isolated from cobra (Naja naja naja) venom at varying enzyme concentrations. The relative Raman scattering intensities of C-C stretching vibrations from the trans and gauche conformers of the acyl chains are correlated directly with the extent of hydrolysis, allowing the progress of the reaction to be monitored in situ on a single vesicle. In dilute vesicle dispersions, the technique allows the much higher local concentration of lipid molecules in a single vesicle to be detected free of interferences from the surrounding solution. Observing the local composition of an optically trapped vesicle also allows one to determine whether the products of enzyme-catalyzed hydrolysis remain associated with the vesicle or dissolve into solution. The observed reaction kinetics exhibited a time lag prior to the rapid hydrolysis. The lag time varied inversely with the enzyme concentration, which is consistent with the products of enzyme-catalyzed lipid hydrolysis reaching a critical concentration that allows the enzyme to react at a much faster rate. The turnover rate of membrane-bound enzyme determined by Raman microscopy during the rapid, burst-phase kinetics was 1200 s(-1). Based on previous measurements of the equilibrium for PLA2 binding to lipid membranes, the average number of enzyme molecules responsible for catalyzing the hydrolysis of lipid on a single optically trapped vesicle is quite small, only two PLA2 molecules at the lowest enzyme concentration studied.
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ABSTRACT: We present the first experimental study of self-forming synthetic lipids, trademarked as QuSomesTM, using Raman spectroscopy in the spectral range of 500 to 3100 cm-1. Raman spectra of these new artificial lipids composed of 1,2- dimyristoyl-rac-glycerol-3-dodecaethylene glycol (GDM-12) and 1,2-dioleoyl-rac-glycerol-3-dodecaethylene glycol (GDO-12) have been obtained in pure form and in aqueous suspensions with Phosphate Buffered Saline (PBS) by using an inverted confocal laser-tweezers-Raman-microscopy system. This spectrometer works with an 80 mW diode-pumped solid-state laser, operating at a wavelength of 785 nm in the TEM00 mode. The laser is used both for optical trapping and Raman excitation. The two amphiphiles considered in this study, differ in their hydrophobic chain length and contain similar units of hydrophilic polyethylene glycol (PEG) head groups. Such synthetic PEG coated lipids exist in liquid form at room temperature and spontaneously form liposomes (nano type vesicles) upon hydration. In this work, we have focused on the band assignments for the spectra of single QuSomesTM nano particles in pure form and in aqueous media acquired by means of Raman spectroscopy. In particular, we have found that the most prominent peaks in the studied spectral region are dominated by vibrational modes arising from C-C and C-H bonds. Furthermore, we have noticed that some of the distinct peaks observed below 1800 cm-1 in pure sample are preserved in aqueous environment. These retained intense bands are located at 1449, 1128, 1079, and 1065 cm-1. This effect might be due to the strong chain-chain interactions, because the chains have to orient themselves and become tightly packed in the vesicles wall rather than adopt random orientations in bulk. This technique has proven to be an excellent tool to establish the fingerprint region revealing the molecular structure and conformation of QuSomesTM particles. The Raman spectroscopic data of these novel lipids and its vesicles formed in suspensions confirm high stability and are therefore considered as potential candidate for varieties of future applications including lipid based novel substances and drug delivery systems.Proceedings of SPIE - The International Society for Optical Engineering 01/2008; DOI:10.1117/12.804822 · 0.20 Impact Factor
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ABSTRACT: In this article, a temperature-controlled Raman spectro-microscopic technique has been utilized to detect and analyze the phase behaviors of two newly developed synthetic PEGylated lipids trademarked as QuSomes, which spontaneously form liposomes upon hydration in contrast to conventional lipids. The amphiphiles considered in this study differ in their hydrophobic hydrocarbon chain length and contain different units of polyethylene glycol (PEG) hydrophilic headgroups. Raman spectra of these new artificial lipids have been recorded in the spectral range of 500-3100 cm(-1) by using a Raman microscope system in conjunction with a temperature-controlled sample holder. The gel to liquid phase transitions of the sample lipids composed of pure 1,2-dimyristoyl-rac-glycerol-3-dodecaethylene glycol (GDM-12) and 1,2-distearoyl-rac-glycerol-3-triicosaethylene glycol (GDS-23) have been revealed by plotting peak intensity ratios in the C-H stretching region as a function of temperature. From this study, we have found that the main phase transitions occur at a temperature of approximately 5.2 and 21.2 degrees C for pure GDM-12 and GDS-23, respectively. Furthermore, the lipid GDS-23 also shows a postphase transition temperature at 33.6 degrees C. To verify our results, differential scanning calorimetry (DSC) experiments have been conducted and the results are found to be in an excellent agreement with Raman scattering data. This important information may find application in various studies including the development of lipid-based novel substances and drug delivery systems.Biopolymers 11/2008; 89(11):1012-20. DOI:10.1002/bip.21051 · 2.29 Impact Factor
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ABSTRACT: We review the combinations of optical micro-manipulation with other techniques and their classical and emerging applications to non-contact optical separation and sorting of micro- and nanoparticle suspensions, compositional and structural analysis of specimens, and quantification of force interactions at the microscopic scale. The review aims at inspiring researchers, especially those working outside the optical micro-manipulation field, to find new and interesting applications of these methods.Electrophoresis 02/2009; 29(24):4813-51. DOI:10.1002/elps.200800484 · 3.16 Impact Factor