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

Jay Preston Kitt and Joel M. Harris

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-phospho¬choline (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.

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