Confocal Raman Microscopy of Hybrid-Supported Phospholipid Bilayers within Individual C₁₈-Functionalized Chromatographic Particles

Abstract

Measuring lipid-membrane partitioning of small molecules is critical to predicting bioavailability and investigating molecule-membrane interactions. A stable model membrane for such studies has been developed through assembly of a phospholipid monolayer on n-alkane-modified surfaces. These hybrid bilayers have recently been generated within n-alkyl-chain (C18)-modified porous silica and used in chromatographic retention studies of small molecules. Despite their successful application, determining the structure of hybrid bilayers within chromatographic silica is challenging because they reside at buried interfaces within the porous structure. In this work, we employ confocal Raman microscopy to investigate the formation and temperature-dependent structure of hybrid-phospholipid bilayers in C18-modified, porous-silica chromatographic particles. Porous silica provides sufficient surface area within a confocal probe volume centered in an individual particle to readily measure, with Raman microscopy, the formation of an ordered hybrid bilayer of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) with the surface C18 chains. The DMPC surface density was quantified from the relative Raman scattering intensities of C18 and phospholipid acyl chains and found to be ∼40% of a DMPC vesicle membrane. By monitoring Raman spectra acquired versus temperature, the bilayer main phase transition was observed to be broadened and shifted to higher temperature compared to a DMPC vesicle, in agreement with differential scanning calorimetry (DSC) results. Raman scattering of deuterated phospholipid was resolved from protonated C18 chain scattering, showing that the lipid acyl and C18 chains melt simultaneously in a single phase transition. The surface density of lipid in the hybrid bilayer, the ordering of both C18 and lipid acyl chains upon bilayer formation, and decoupling of C18 methylene C-H vibrations by deuterated lipid acyl chains all suggest an interdigitated acyl chain structure. The simultaneous melting of both layers is also consistent with an interdigitated structure, where immobility of surface-grafted C18 chains decreases the cooperativity and increases the melting temperature compared to a vesicle bilayer.

Deciphering the Role of Bilayer of a Niosome towards Controlling the Entrapment and Release of Dyes

“…Since, niosome membranes are dominated by C−H groups, C−H stretching region (2800 ‐ 3000 cm −1 ) is the most well‐studied region in a Raman spectrum of a niosome. As shown in Figure , three main peaks were detected in this region which were used to explain the changes in bilayer assembly: symmetric stretch (2840‐2860 cm −1 ) and antisymmetric stretch (2880‐2900 cm −1 ) of the methylene group, along with the CH 3 terminal methyl symmetric stretch (2920‐2930 cm −1 ) . The intensity ratio between the terminal CH 3 ‐symmetric stretching mode to the CH 2 ‐symmetric stretching mode (I 2930 /I 2850 ) provides the information regarding disorder of the surfactant structure along its chain length and packing density of the alkyl chains.…”

Confocal Raman Microscopy Investigation of Self-Assembly of Hybrid Phospholipid Bilayers within Individual Porous Silica Chromatographic Particles

“…Raman spectra are analyzed using a multivariate statistical technique, self-modeling curve resolution (SMCR), , which allows resolution of the time-dependent Raman scattering data into component Raman spectra and corresponding composition vectors that describe the time-dependent changes in intensity of the component spectra. This methodology has been previously used to detect temperature-dependent structural changes in phospholipid membranes of individual optically trapped lipid vesicles and in fully assembled hybrid bilayers in silica particles . In the present work, this analysis provides insight into how the structures of both the lipid and C 18 chains of hybrid lipid bilayers evolve during their deposition and organization into ordered structures on reversed-phase chromatographic silica surfaces.…”

“…Confocal Raman microscopy is well suited to this task because it can probe the internal interfacial composition and structure within individual chromatographic particles; the results showed that the acyl chains of the phospholipid indeed interact with the C 18 alkyl chains bound to the silica surface, as suggested by Tsirkin and Grushka . The hydrocarbon chains were found to be highly ordered, and this ordering arises from chain interdigitation, as indicated by the surface-density of lipid in the hybrid bilayer, the nearly all-trans conformations of both C 18 and lipid alkyl chains upon bilayer formation, and the decoupling of C 18 methylene CH vibrations by deuterated (CD labeled) lipid acyl chains residing between C 18 chains that disrupt CH dipolar coupling . Further insight into the hybrid-bilayer structure was found in temperature-dependent Raman spectra, where a single melting transition was observed that is broader and shifted to higher temperature compared to a lipid vesicle.…”

“…To determine the organization of lipid layers deposited onto the interior surfaces of reversed-phase chromatographic silica, it is helpful to have an in situ spectroscopic measurement of lipid bilayer structure. Raman spectroscopy has previously been employed in situ to probe the composition and structure of reversed-phase chromatographic interfaces, − and this approach was recently used in a confocal-microscopy experiment to probe the structure of hybrid lipid bilayers formed within individual chromatographic silica particles . Confocal Raman microscopy is well suited to this task because it can probe the internal interfacial composition and structure within individual chromatographic particles; the results showed that the acyl chains of the phospholipid indeed interact with the C 18 alkyl chains bound to the silica surface, as suggested by Tsirkin and Grushka .…”

Replacing a Century Old Technique – Modern Spectroscopy Can Supplant Gram Staining

“…The primary role of the lipid-bilayers is to isolate the homeostatic interior cytoplasm from the outside environment 26 . This spontaneously organized bacterial organ has relatively strong Raman shifts in the 2800–3000 cm −1 region that are attributed to symmetric and antisymmetric methylene stretching vibrations 27, 28 .…”