Confocal Raman Microscopy for in Situ Measurement of Phospholipid–Water Partitioning into Model Phospholipid Bilayers within Individual Chromatographic Particles

Abstract

The phospholipid-water partition coefficient is a commonly measured parameter that correlates with drug efficacy, small-molecule toxicity, and accumulation of molecules in biological systems in the environment. Despite the utility of this parameter, methods for measuring phospholipid-water partition coefficients are limited. This is due to the difficulty of making quantitative measurements in vesicle membranes or supported phospholipid bilayers, both of which are small-volume phases that challenge the sensitivity of many analytical techniques. In this work, we employ in situ confocal Raman microscopy to probe the partitioning of a model membrane-active compound, 2-(4-isobutylphenyl) propionic acid or ibuprofen, into both hybrid- and supported-phospholipid bilayers deposited on the pore walls of individual chromatographic particles. The large surface-area-to-volume ratio of chromatographic silica allows interrogation of a significant lipid bilayer area within a very small volume. The local phospholipid concentration within a confocal probe volume inside the particle can be as high as 0.5 M, which overcomes the sensitivity limitations of making measurements in the limited membrane areas of single vesicles or planar supported bilayers. Quantitative determination of ibuprofen partitioning is achieved by using the phospholipid acyl-chains of the within-particle bilayer as an internal standard. This approach is tested for measurements of pH-dependent partitioning of ibuprofen into both hybrid-lipid and supported-lipid bilayers within silica particles, and the results are compared with octanol-water partitioning and with partitioning into individual optically trapped phospholipid vesicle membranes. Additionally, the impact of ibuprofen partitioning on bilayer structure is evaluated for both within-particle model membranes and compared with the structural impacts of partitioning into vesicle lipid bilayers.

Using single-vesicle technologies to unravel the heterogeneity of extracellular vesicles

“…Therefore, RTM, with a typical processing capacity of 0.2 particles per min, is not considered a high-throughput methodology 71 . RTM can, however, be used to obtain interesting, unique information not only for EVs 57,60,61,[70][71][72][73][74][75][76][77] but also for many other bioparticles such as liposomes, lipid layers on synthetic nanoparticles and others [78][79][80][81][82][83] .…”

Hybrid-Lipid Bilayers Induce n-Alkyl-Chain Order in Reversed-Phase Chromatographic Surfaces, Impacting their Shape Selectivity for Aromatic Hydrocarbon Partitioning

“…Quantification of each solute was achieved by measuring the intensities of the within-particle Raman scattering from the symmetric ring stretching mode of fluorene or biphenyl (1612 or 1616 cm –1 ) relative to scattering from the CH 2 -twisting modes (1296–1301 cm –1 ) of the C 18 ligands and phospholipid acyl chains, used as an internal standard. − Converting these signals to concentrations was achieved by measuring scattering intensities from standard solutions of fluorene and biphenyl in n -heptane (Figures S-4 and S-5). The PAH surface coverages were determined from their CC scattering intensities relative to intensity of the CH 2 twisting mode of the n -alkyl chains, where the CH 2 surface coverage of each of stationary phase was determined from carbon analysis and specific surface area (Supporting Information).…”

“…We use confocal Raman microscopy to characterize the degree of chain order of these hybrid bilayers from the ratio of scattering intensities from the asymmetric C–C trans-conformer stretching mode to the symmetric C–C stretching of the gauche conformer. The partitioning of two model PAH molecules, fluorene (planar) versus biphenyl (nonplanar), was determined from their Raman scattering intensities using scattering from the CH 2 –twisting mode of the hybrid bilayer as an internal standard. − Interfacial PAH concentrations and corresponding partition coefficients were measured for each of the four hybrid bilayers. The retention selectivities for the planar versus nonplanar PAH compounds determined from the ratios of partition coefficients were found to vary significantly and systematically with the degree of order of the acyl/alkyl chains in the hybrid lipid bilayers.…”

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

“…Confocal Raman microscopy has also been used to investigate the partitioning of small molecules into hybrid-lipid bilayers in C 18 chromatographic silica particles compared to their partitioning into lipid vesicle membranes . While there are structural differences between hybrid (interdigitated) versus vesicle (noninterdigitated) lipid bilayers, a comparison of the pH-dependent partitioning of a model solute (2-(4-isobutylphenyl) propionic acid) showed an equivalent response at low pH for the neutral form of the solute; at high pH, the deprotonated (anionic) form of the solute showed somewhat greater partitioning into the hybrid-bilayer.…”

“…Confocal Raman microscopy has also been used to investigate the partitioning of small molecules into hybridlipid bilayers in C 18 chromatographic silica particles compared to their partitioning into lipid vesicle membranes. 26 While there are structural differences between hybrid (interdigitated) versus vesicle (noninterdigitated) lipid bilayers, a comparison of the pH-dependent partitioning of a model solute (2-(4isobutylphenyl) propionic acid) showed an equivalent response at low pH for the neutral form of the solute; at high pH, the deprotonated (anionic) form of the solute showed somewhat greater partitioning into the hybrid-bilayer. This difference likely arises from greater headgroup spacing of the hybrid bilayer that arises from interdigitation, 25 which increases solution-phase contact with hydrophobic regions of the interface and contributes to hybrid bilayer association of the surface-active anionic solute by its lowering of the interfacial tension.…”