Confocal Raman Microscopy of Hybrid-Supported ... - ACS Publications

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Confocal Raman Microscopy of Hybrid-Supported Phospholipid Bilayers within Individual C18-Functionalized Chromatographic Particles Jay P. Kitt and Joel M. Harris* Department of Chemistry, University of Utah, 315 South 1400 East, Salt Lake City, Utah 84112-0850, United States S Supporting Information *

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.

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INTRODUCTION Measuring interactions between aqueous-phase molecules and phospholipid membranes is critical for predicting and understanding bioavailability in pharmaceutical screening and in toxicology studies.1−7 A stable model membrane for these types of studies has been developed, comprising an upper leaflet of amphiphilic lipid molecules that forms a monolayer with a lower leaflet of hydrophobic n-alkyl chains covalently bound to a solid support. These model membranes, termed hybridsupported phospholipid bilayers (HSBs), were originally generated by adsorption of phospholipids to self-assembled nalkanethiol monolayers on gold and silver substrates8,9 and used for electrochemical,10−12 infrared reflection,10 surface-enhanced Raman,12,13 and surface-plasmon resonance measurements.14,15 More recently, this concept has been adapted to produce hybrid bilayers in C18-modified porous silica for use in chromatographic retention studies,16−18 where the affinities of both small molecules and membrane-active peptides with hybrid bilayers were found to be comparable to their affinities © XXXX American Chemical Society

for phospholipid vesicle membranes. Although chromatographic studies have produced promising results for these studies, the structure of hybrid bilayers within the pore network of chromatographic silica has not been characterized. The techniques employed to investigate planar hybrid bilayers cannot generally be applied in porous silica frameworks either because the internal surface is inaccessible to the probe, as in atomic force microscopy, or because the probing techniques are incompatible with the water-filled pores, as in infrared spectroscopy. Phospholipid bilayers can be characterized using Raman scattering spectroscopy, providing insight into their formation, structure, and functioning.19−22 However, Raman scattering is a weak phenomenon, and detection of a small population of lipid molecules in a planar supported bilayer would be challenging. Received: June 22, 2016 Revised: July 15, 2016

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DOI: 10.1021/acs.langmuir.6b02309 Langmuir XXXX, XXX, XXX−XXX

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dependent structure of the bilayer acyl chains. The temperature dependence of the hybrid-bilayer structure is contrasted with the behavior of DMPC vesicles, where the hybrid-bilayer phase transition is shifted to higher temperature and is much broader than for DMPC vesicle membranes. The structural changes that occur through the hybrid-bilayer phase transition are further investigated by using deuterated DMPC (DMPC-D54) to resolve the Raman bands of the acyl chains of the phospholipid from those of the C18 alkyl chains grafted to the silica surface.

The sensitivity challenge of Raman spectroscopy for studying interfacial chemistry at silica surfaces has been overcome previously, however, by exciting and collecting Raman scattering from within high-surface-area porous silica particles using confocal Raman microscopy.23−26 The confocal probe volume is confined to the interior of an individual chromatographic particle, where Raman scattering from the solid−liquid interface is enhanced by the high surface area of the porous support, overcoming the sensitivity challenge and allowing in situ probing of the interface. The ability to measure Raman scattering from within a porous silica particle is unexpected, as one would anticipate significant loss of excitation and collection efficiency due to losses from multiple scattering into and out of the porous structure. The ∼30 nm pores in xerogel silica,27 however, lie well below the Mie scattering limit of a 647.1 nm excitation source.28 When combined with the nearly matching refractive indices of the silica framework and aqueous solution in the pores, a well-defined sub-micrometer laser focus can be produced and high quality Raman spectra can be collected from the resulting confocal volume at the center of a 10 μm silica particle.23−25 Confocal Raman microscopy has been utilized to probe in situ the chemistry within individual chromatographic silica particles, including studies of functionalization of the silica surface,26 the interfacial solvation environment of C18-stationary phases,24 the mechanism of surfactant-mediated ionpair retention,23 femtoliter-scale solid-phase extraction of PAH compounds,25 and partitioning into octanol confined to the intraparticle pores.29 In this work, we extend within-particle confocal Raman spectroscopy to investigate the structure of hybrid-supported phospholipid bilayers deposited on the interior surfaces of chromatographic C18-silica particles. Raman spectroscopy is well-suited to study lipid bilayer structure.20−22,30 Acyl chain disorder can be monitored by measuring Raman scattering intensities from structural indicators in the acyl chain C−C stretching (1030−1150 cm−1), C−H bending (1400−1500 cm−1), CH2 twisting (1260−1340 cm−1), and C−H stretching (2800−3100 cm−1) regions of the spectrum.19,30 Although it is common to measure changes in indicator bands at specific frequencies elucidate structural changes,22,31,32 this approach allows only investigation of isolated intensity changes or peak shifts, which may not be resolved from neighboring bands. A more effective approach to spectroscopic analysis that allows resolution of correlated spectral changes as a function of experimental conditions (e.g., time, temperature, composition) is self-modeling curve resolution (SMCR).33,34 SMCR is a multivariate statistical method that resolves correlated spectral changes through eigenvector decomposition of the spectral data; it has been used previously to detect temperaturedependent spectral changes from lipid membranes of individual optically trapped vesicles.20 Within-particle confocal Raman and self-modeling curve resolution are employed in this work to investigate the formation and temperature-dependent structure of hybrid supported phospholipid bilayers on the interior surfaces of individual C18 chromatographic silica particles. Raman spectra of a 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) hybrid bilayer deposited onto C18-modified silica measured within chromatographic particles are compared with carbon analysis to determine phospholipid surface coverage. Differential scanning calorimetry (DSC) to is used to measure hybrid-bilayer melting transitions and compared with temperature-controlled Raman microscopy to reveal the temperature-

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EXPERIMENTAL SECTION

Reagents and Materials. Chromatographic silica particles were obtained from YMC America (YMC-Pack ODS-A, YMC America Inc., Allentown, PA). The particles were spherical, C18-derivitized, monofunctional, and end-capped with trimethychlorosilane. The particles have a mean diameter of 10 μm, mean pore diameter of 31.9 nm, specific surface area of 93 m2/g, and pore volume of 0.80 mL/g as reported by the manufacturer. Elemental analysis of carbon content (MHW Laboratories, Phoenix, AZ) and the particle specific surface area were used to determine the C18 surface coverage of 3.7 ± 0.3 μmol/m2. Isopropanol (>99.9%) was obtained from Thermo Fisher Scientific (Waltham, MA). Chloroform (Omnisolv) was obtained from EMD Millipore (Billerica, MA). Deuterium oxide (>99.9%), was obtained from Sigma-Aldrich (St. Louis, MO). Phospholipids used in this study (1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dimyristoyl-d54-sn-glycero-3-phosphocholine (DMPC-D54)) were obtained from Avanti Polar Lipids (Alabaster, AL). Phospholipids were diluted into chloroform and stored at −15 °C until use. Water used for this experiment was filtered using a Barnstead GenPure UV filtration system (ThermoFisher Scientific, Waltham, MA) and had a minimum resistivity of 18.0 MΩ·cm. Microfluidic flow cells for Raman microscopy were constructed by drilling two 2.5 mm holes in a 3 mm thick, 25 mm diameter circular Pyrex glass top plate (VWR, Radnor, PA) and attaching 2.4 mm o.d. Luer adapters (Value Plastics, Inc., Fort Collins, CO) using Devcon 5 min epoxy (ITW Devcon, Danvers, MA). The top plate was attached to a 22 mm × 22 mm No. 1 glass coverslip (Gold Seal, Erie Scientific Co., Portsmouth, NH) using 140 μm thick 3M double-stick tape (TapeCase Ltd., Elk Grove Village, IL) where a 2.5 mm × 11 mm channel was cut between the inlet and outlet ports, allowing flow through a channel between the Luer adapters. All tubing used was 1.6 mm i.d. × 2.4 mm o.d. Viton elastomer (Cole-Parmer, Vernon Hills, IL). Microscopy cells for measuring standards in solution were constructed by gluing a 12 mm length of 10 mm i.d., 13 mm o.d. Pyrex glass tubing to a No. 1 fused silica coverslip using Devcon 5 min epoxy (ITW Devcon, Danvers, MA). A diagram of the cell has been published previously.25 Temperature-controlled experiments were conducted in a brass microscopy sample cell consisting of a cylindrical 3 mm o.d., 2 mm i.d. brass tube which transitions to a 5 cm wide base. The 2 mm i.d. of the opening at the top of the tube was tapered to 0.5 mm at the base to prevent formation of a temperature gradient across the fluid in the measurement portion of the cell. A 22 mm × 22 mm glass coverslip was attached to the base of the cell using 140 μm thick double-stick tape with a 0.25 mm radius hole cut in the center to allow fluid in the sample cell to contact the glass coverslip. The brass microscopy cell was covered by a jacketed copper block and mounted on a silver stage (Technical Video Ltd., Port Townsend, WA). The block is cooled by flowing chilled 50/50 water/ethylene glycol solution through the copper jacket and Peltier stacks on the silver stage using a magnetic drive pump (Micropump Inc., Vancouver, WA). Cell temperature is adjusted using a proportional integral derivative (PID) controller to vary the current supplied to the pair of Peltier stacks on the silver stage. Sample temperature was measured using a thermocouple inserted into the base of the sample cell. A diagram of the temperaturecontrol apparatus and brass cell has been described previously.35 B


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confirmed by solvent wash-off and ex situ analysis.16 Smallmolecule partitioning measurements show similar partition coefficients as those measured in lipid vesicle membranes. While these results suggest the formation of a hybrid bilayer on the C18-functionalized silica surface, little is known about structure of lipid that resides within the pores of reversed-phase chromatographic silica. To investigate this question, we measure Raman scattering from phospholipid molecules adsorbed to the C18 surface within individual chromatographic silica particles. DMPC-filled particles were prepared by equilibration of C18-silica particles with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) in 15% v/v isopropanol/water,16 centrifuged, and resuspended three times in 18 MΩ·cm water and transferred to a microscopy cell, where the particles were allowed to settle onto the coverslip surface. Raman spectra from the interior of individual 10 μm particles were collected by manipulating the focused laser spot and confocal probe volume to the center of the particle. Representative spectra of a C18-silica particle and of a C18 particle that has been equilibrated with DMPC (23 °C) are presented in Figure 1A. Adsorption of phospholipid is confirmed by appearance of Raman scattering from the C−

Sample Preparation and Characterization. For phase-transition studies, hybrid-supported bilayers were prepared on the interior surfaces of porous C18 particles by adsorption of DMPC or DMPCD54 from 15% v/v isopropanol/water. This was accomplished by adding 10 mg of C18-derivatized silica to 2 mL of 5 mg/mL DMPC or DMPC-D54 in 15% isopropanol/water and stirring overnight for ∼15 h. Particles were rinsed three times by centrifugation and resuspension in 18 MΩ·cm water to ensure no phospholipid remained in the exterior solution. For Raman spectroscopy experiments, a 100 μL aliquot of the final 0.5 mg/mL solution of suspended particles was transferred to the measurement cell and allowed to settle to the coverslip surface. The initial temperature of the cell was set at 15 °C and then increased at 1 °C intervals to the final temperature (50 °C for DMPC and 45 °C for DMPC-D54). At each temperature, Raman scattering was collected from within three individual particles with 30 s integration times. To determine C18 and phospholipid surface coverages, bare C18 and hybrid supported bilayer coated particles were sent for elemental analysis (M.H.W. Laboratories, Phoenix, AZ). These particles were prepared as described above with the addition of the following steps. Subsequent to rinsing, particles were dried under a stream of nitrogen, placed in an oven at 120 °C for 15 min, and finally dried under vacuum (120 mTorr) on a warm block (∼80 °C) for 1 h to ensure removal of any remaining water. Temperature-Controlled Confocal Raman Microscopy. A detailed description of the confocal Raman microscope used in this work has been published previously.25,26 Briefly, the beam from a Kr+ laser (Innova 90, Coherent Inc., Santa Clara, CA) operating at 647.1 nm is passed through a band-pass filter, beam expanded, and reflected off a dichroic mirror to slightly overfill the back aperture of a 1.4 NA 100× oil immersion objective mounted on an inverted fluorescence microscope frame. Laser radiation is focused by the objective to an ∼600 nm diameter spot. For porous-particle experiments the focused spot is translated to the center of an individual particle. For phospholipid vesicle experiments, polarizability contrast between the lipid bilayer and water allows optical trapping of a single vesicle at the laser focus.36,37 The scattered light is collected back through the objective and passed through the dichroic mirror. The image is then collimated and directed through a high pass filter, and Raman scattered light is focused on the monochromator slit set to 50 μm, defining the horizontal dimension of a confocal aperture, where the vertical dimension of the confocal aperture is defined by binning three rows of CCD pixels (78 μm)38 to acquire a spectrum within a confocal-probe volume within the sample.38 To excite and collect Raman scattering within a particle, the focused laser beam is brought to the coverslip− solution interface, where a tightly focused reflected spot is observed. The microscope stage is then translated in the x and y dimensions to center a single particle above the reflected spot. The microscope objective is then translated 5 μm upward in the z dimension to center the confocal probe volume within the particle. Data Analysis. All Raman spectra were baseline-corrected by subtracting a fifth-order polynomial fitted to baseline regions of the spectrum using a script executed in Matlab (MathWorks, Natick, MA). Prior to analysis, spectra were offset to eliminate any negative values along the baseline; within-particle Raman spectra were normalized to the phospholipid headgroup CN stretching mode (716 cm−1), which is insensitive to bilayer acyl chain structure.39 Self-modeling curve resolution33,34 of temperature-dependent Raman spectra of phospholipid bilayers has been described previously.20 Details of the analysis algorithm can be found in the Supporting Information.

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RESULTS AND DISCUSSION Raman Scattering from Hybrid Bilayers within C18Silica Particles. Hybrid bilayers have been previously prepared in reversed-phase C18-functionalized silica particles and packed in columns for chromatographic determination of lipid bilayer partition coefficients of small molecules.16−18 Retention of lipid and formation of hybrid bilayers within the particles was established by the elution volume for lipid breakthrough and

Figure 1. (A) Raman spectrum of the alkyl chains within an individual C18-silica particle (black) is compared with the Raman spectrum of a within-particle DMPC hybrid bilayer (red). (B) Raman spectrum of a within-particle hybrid bilayer (red) is compared with the Raman spectrum of an individual optically trapped vesicle (black); spectra are normalized to the headgroup CN-stretching mode at 716 cm−1. Spectra were collected at 23 °C. C


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Langmuir N-stretching mode of the phospholipid headgroup (716 cm−1), along with significantly increased scattering intensity of the CH2-twisting mode (1296 cm−1) from the lipid acyl chains.25,39 DMPC accumulates uniformly throughout the particle, as illustrated by a depth profile of the scattering intensity from the CH2-twisting mode (see Supporting Information). The accumulation of DMPC at the C18-modified surface does not block the pores 32 nm diameter silica pores. This is evident in the uniform distribution of water (as a D2O tracer) throughout the particle, producing a scattering signal compared to bulk solution that is consistent with the fractional pore volume of the particle (see Supporting Information). The DMPC phospholipid that accumulates within the particle exhibits similar features as the Raman spectrum of the phospholipid bilayer of an optically trapped DMPC phospholipid vesicle, as shown in Figure 1B; however, these spectra reveal differences in the bilayer structure, as discussed in the next section. Raman Spectra of Within-Particle Hybrid Bilayers. One might anticipate that a within-particle hybrid bilayer is structurally similar to a vesicle bilayer at the same temperature with the inner leaflet of C18 and outer leaflet of DMPC interacting primarily at the methyl-terminated ends of the lipid tails. If this were the case, one would anticipate that the Raman spectrum of a DMPC hybrid bilayer would exhibit similar structural features as a vesicle bilayer at the same temperature. When the hybrid bilayer spectrum at room temperature (23 °C) is compared with spectrum of an optically trapped DMPC vesicle at the same temperature, however, several differences are observed (Figure 1B). A brief discussion of the structurally informative Raman bands and the differences observed between the vesicle spectrum and hybrid-bilayer spectrum is presented here. Carbon−Carbon Stretching Region (1030−1150 cm−1). Raman bands in the carbon−carbon stretching region are indicative of the ratio of acyl chain gauche and trans conformers. The pair of narrow peaks observed in the hybrid bilayer spectrum at 1061 and 1126 cm−1 correspond to the antisymmetric and symmetric C−C stretching modes of the bilayer acyl chains, respectively, which increase in intensity as the C−C bonds adopt trans conformations in the more ordered phospholipid gel phase.19−21,30,40−43 The broad peak observed at 1086 cm−1 is due to a superposition of the C−C stretching of the acyl chain gauche conformers and the phospholipid headgroup PO2-stretching mode;21,40−42,44 the area of this peak increases in proportion to the number of gauche conformers and also broadens toward lower frequency as the acyl chains adopt a wider range of vibrational frequencies and greater conformational freedom in the liquid-crystalline phase.20,41−43,45,46 Comparing the Raman spectrum of the hybrid bilayer to that of a DMPC vesicle, there are clear differences in the acyl chain structure. The proportion of transto-gauche conformers as indicated by the ratio of the antisymmetric C−C intensities (I1061/I1086 = 1.73 for the hybrid bilayer and 1.62 for a vesicle bilayer) is greater for the hybrid bilayer; additionally, the antisymmetric or out-of-phase C−C-stretching peak is narrower in the Raman spectrum of the hybrid bilayer than the DMPC vesicle. These results indicate that the interactions of the DMPC acyl chains with the underlying surface-bound C18 chains lead to a more ordered acyl chain structure and to less conformational freedom than for a phospholipid vesicle bilayer. Carbon−Hydrogen Twisting Region (1260−1340 cm−1). The methyl group carbon−hydrogen twisting region comprises

several modes including CH2-rocking and CH2-wagging modes. The most prominent feature is the CH2-twisting mode at 1303 cm−1.40,42 It has been shown previously that the CH2-twisting region is indicative of both acyl chain order (gauche-to-trans ratio) and interchain coupling.30,42 As the bilayer melts, the decrease in acyl chain order allows for more freedom of motion about the carbon backbone due to greater gauche-to-trans ratio in the acyl chains. This increase in conformational and rotational freedom results in a range of vibrational energies, leading to broadening and asymmetry of the peak. The typical melting response is a shift in the CH2-twisting mode to higher frequencies and broadening indicating decoupling of the acyl chain vibrations and greater disorder.20,30,40 In the spectrum of the hybrid bilayer at room temperature, the CH2-twisting modes appear less asymmetric, less broad, and at lower frequency than the same modes in the vesicle spectrum. This again implies greater acyl chain order and chain-to-chain coupling in the hybrid bilayer compared with a DMPC vesicle. Carbon−Hydrogen Bending Region (1400−1500 cm−1). The C−H bending region of lipid Raman spectra is indicative of lattice order.20,42,47 The broad band observed in this region is also a superposition of several Raman-active vibrational modes, with the most intense bands due to scattering from the antisymmetric methyl bend (1436 cm−1) and methylene scissoring mode (1455 cm−1).19,30,42,48 The ratio of the intensity of the antisymmetric methyl bend to the intensity of the methylene bend is indicative of chain decoupling and increasing gauche conformers where the resulting increased freedom of motion allows CH2 functional groups to undergo greater scissoring/bending motion.42 In the Raman spectrum of the hybrid bilayer, the ratio of the methyl scissoring peak intensities is greater than observed in the DMPC vesicle bilayer (1.28 as compared to 1.19). This suggests greater interchain coupling in the gel phase as compared with the DMPC vesicle, where the ratio of the two bands indicates the bilayer has begun the transition from the gel to fluid phase. This result further indicates the hybrid bilayer is more structured and contains fewer gauche defects than the DMPC vesicle bilayer. The methyl C−H scissoring mode also indicates the presence of lipid microdomains through correlation field splitting associated with lipid demixing.21,49 The field splitting observed in the spectrum of the hybrid bilayer is very similar to that observed in a gel-phase vesicle bilayer,20,22,35 indicating the hybrid bilayer acyl-chain lattice structure is ordered and has minimal domains, much like that of a gel-phase lipid vesicle bilayer. Carbon−Hydrogen Stretching Region (2800−3100 cm−1). The carbon−hydrogen stretching region of phospholipid Raman spectra is a complex but well-studied region. The broad band of overlapping peaks in this region consists of three main Raman modes: the CH2-symmetric-stretching mode (2847 cm−1), the CH2-antisymmetric-stretching mode (2883 cm−1), and the CH3-terminal-methyl-stretching mode (2930 cm−1) which lie superimposed on a number of broad Fermi resonance bands.19,30,32,41,42,44,50−54 The characteristics of these Raman bands can provide further insight into the acyl chain structure and have been characterized as follows. The ratio of the intensity of the CH2-antisymmetric stretching mode to the CH2-symmetric-stretching mode (I2883/I2847) provides an indicator of the acyl chain lateral packing density.6,22,30,42,53 During a phospholipid thermal phase transition, this ratio undergoes two significant changes: an initial decrease is due to increased rotational freedom in the acyl chains, followed by a larger, more temperature-sensitive decrease, indicative of D


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Langmuir increasing gauche conformers.20 The ratio of the CH3-terminalmethyl-stretching mode to the CH2-symmetric-stretching mode (I2930/I2847) is associated with both structural disorder in the acyl chains (gauche versus trans conformers) as well as a reduction of acyl chain packing density as the bilayer becomes disordered.6,22,30,42,53 The change in this peak ratio as a function of temperature is the most commonly used spectral indicator of the lipid bilayer main melting transition. Frequency shifts of the CH2-antisymmetric- and symmetric-stretching mode also indicate the level of interchain coupling. Here, decreased interchain order results in decreased C−H interactions between chains and increases in the frequency of both modes of C−H-stretching. In the hybrid bilayer, the Raman spectrum in the C−H stretching region indicates greater order at the same temperature compared with the vesicle bilayer. The I2883/I2847 ratio in the hybrid bilayer is 1.11 compared with 1.06 in the vesicle membrane, suggesting a greater interchain packing and fewer gauche defects. The I2930/I2847 ratio is larger for the hybrid bilayer than the vesicle membrane (1.99 vs 1.70), leading to the same conclusion. In addition, the frequencies of both the CH2symmetric stretch and the CH2-antisymmetric stretch in the hybrid bilayer are comparable to a gel-phase membrane,20,22,35 indicating strong dipolar coupling between methylene C−Hstretching modes of neighboring acyl chains. All of the spectroscopic evidence above points to a much more ordered acyl chain structure in the hybrid bilayer compared to a vesicle membrane. Even the C18 chains that are originally disordered and dominated by gauche defects prior to lipid accumulation join the highly ordered structure of the hybrid bilayer and participate in strong dipolar coupling between methylene C− H-stretching modes than acyl chains of phospholipids in a vesicle bilayer. A key to understanding this ordered acyl chain structure of the hybrid bilayer lies in an analysis of the surface density of phospholipid molecules. Quantitative Determination of Phospholipid Surface Coverage in the Hybrid Bilayer. An important specification of any bilayer membrane model is the phospholipid surface density. To determine the DMPC density in the within-particle hybrid bilayer, the Raman spectra of the hybrid bilayer and of the bare C 18 particle (Figure 1A) can be compared quantitatively. The observed 1.6-fold greater scattering intensities of the carbon−carbon and carbon−hydrogen regions of the spectrum reveal a significant increase in the acyl chain content of the interface. To determine the acyl chain surface density from these results, the area under the CH2-twisting mode at 1303 cm−1, which has been shown to increase linearly with number of methylene groups,25 was compared before and after hybrid bilayer formation. By dividing the total Raman scattering of the CH2-twisting peak from the C18 particle by the number of methylene groups in a C18 chain (17), we obtain a calibration factor for the Raman scattering signal per CH2 at the known surface coverage of C18 chains on the silica surface. Taking the difference between CH2 peak areas for the hybrid bilayer and bare C18 particle and dividing by the above calibration factor, we obtain the number of CH2 groups (9.1) added by phospholipid relative to the number C18 chains. Dividing this result by the number of methylene groups in DMPC acyl chains (24), we obtain the surface coverage of DMPC relative to C18 chains (0.37). Multiplying this result by the known surface coverage of C18 chains, 3.7 ± 0.3 μmol/m2 (see Experimental Section), we obtain the DMPC surface coverage of 1.4 ± 0.1 μmol/m 2 . This spectroscopic

determination of DMPC surface coverage was compared with carbon microanalysis of a sample of hybrid bilayer particles (see Supporting Information) and found to be in close agreement, 1.3 ± 0.1 μmol/m2. The surface coverage of DMPC on the hybrid bilayer prepared on the C18 surface corresponds to a larger effective DMPC cross-sectional area of 120 ± 10 Å2 compared with the molecular area of DMPC in a gel-phase bilayer of 47.2 ± 0.1 Å2.55 Relative to a close-packed gel-phase DMPC bilayer in a lipid vesicle, the lipid cross-sectional area of the hybrid bilayer is 2.5-fold greater than the area in a lipid area of a vesicle membrane, so that the acyl chains of the phospholipid monolayer are separated by an additional area equivalent to 1.5 lipid molecules or 3 acyl chains. The mole ratio of C18 chains to lipid in the hybrid bilayer is 2.8 ± 0.1, which is consistent with having the C18 chains provide additional 3-acylchain spacing between lipid molecules required to produce the observed DMPC area per molecules. Given that trans conformers dominate the structure of the hybrid bilayer, for both the lipid and the underlying C18 acyl chains, the results are consistent with an interdigitated bilayer structure, which is more ordered than a DMPC vesicle bilayer, with dipolar coupling between methylene C−H stretching modes comparable to a gel-phase membrane. Bayerl and co-workers56 have hypothesized that phospholipids in C18 hybrid bilayers are interdigitated, based on their broader thermal phase transitions, shifted to higher temperatures compared with vesicles of the same lipid. Their results suggested the ordered acyl chain structure is stabilized by interaction with the immobile C18 chains. To test this hypothesis, they measured deuterium nuclear magnetic resonance (2H NMR) spectra of hybrid bilayers and confirmed hindered axial motions of selectively deuterated acyl chains, thereby providing further evidence of interdigitation.56 Similar results have been reported for hybrid bilayers at a planar substrate, where neutron scattering from a DMPC monolayer adsorbed to a C18-modified oxide layer on a planar silicon substrate was consistent with DMPC acyl chains being interdigitated with the C18 chains.57 These results are in contrast with those of Swanson et al.58 where infrared spectroscopy, X-ray scattering, and ellipsometric experiments suggested a non-interdigitated structure at a planar C18modified interface.58 These results, however, did not include any calorimetric evidence about the melting behavior of these bilayers. Interestingly, Bayerl and co-workers56 reported that in unpublished infrared spectroscopic results the underlying C18 chain structure was constant through the melting transition, despite their calorimetric evidence for interdigitation in a higher temperature and broader phase transition and 2H NMR results.56 These conflicting results motivate investigation the temperature-dependent structure of within-particle hybrid bilayers. Temperature-Dependent Raman Spectroscopy and Differential Scanning Calorimetry of Within-Particle Hybrid Bilayers. Phospholipid bilayer membranes are well-known to exhibit temperature-dependent phase transitions that can affect small molecule and protein interactions59,60 Because the structure of the within-particle hybrid bilayer differs from that of a vesicle bilayer of the same phospholipid at room temperature (Figure 1B), a temperature-dependent study of the hybrid bilayer was conducted by placing DMPC hybrid bilayer particles in a temperature-controlled microscopy cell61 and collecting Raman spectra of the within-particle hybrid E


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Langmuir bilayer as a function of temperature (Figure 2). Principalcomponents analysis (PCA)34 of the temperature-dependent

Figure 2. Raman spectra collected in situ as a function of temperature for the DMPC hybrid bilayer through its melting transition.

Raman spectra of within-particle hybrid bilayers indicated two significant (principal-component) eigenvectors. The spectra of the underlying components and respective composition vectors were resolved by self-modeling curve resolution (SMCR) and are presented in Figures 3A and 3B. The spectra exhibit characteristics expected for lipid bilayer melting, resulting in acyl chain disordering as temperature is increased.20,47,61 In the C−C region of the spectrum, the peaks at 1061 and 1126 cm−1 corresponding to the in-phase and out-of-phase C−C stretching modes of the bilayer acyl chains are present as intense narrow bands in the low-temperature gel-phase spectrum, indicating a majority of symmetric-trans conformers in the acyl chains; these peaks are lost at high temperature in the more disordered fluid-phase spectrum. The superposition of the out-of-phase C−C stretching of the acyl chain gauche conformers with the phospholipid headgroup PO2-stretching mode (the broad peak at 1086 cm−1) shows increased intensity and is broadened toward lower frequency, indicating an increase in the proportion of gauche conformers in the acyl chains at higher temperatures. In the carbon−hydrogen twisting region, the gel-phase spectrum shows a sharp peak at 1296 cm−1 which becomes broad and shifted to higher frequency (1306 cm−1) in the fluid phase. This indicates acyl chain disordering as temperature is increased, resulting from a greater gauche-to-trans conformer ratio and a decrease in interchain coupling in the fluid phase. Similarly, in the C−H bending region, the ratio of the intensity of the antisymmetric methyl bend (1436 cm−1) to the intensity of the methylene bend (1455 cm−1) changes from 1.3 in the gel-phase spectrum to 1.2 in the fluid-phase spectrum, indicating chain decoupling, a change in lattice order, and increasing gauche conformers. The spectra of the gel and fluid phases in the carbon− hydrogen stretching region are also indicative of chain disordering and similar to vesicle spectra over the main phase transition. The ratio of the intensity of the CH2-antisymmetricstretching mode to the CH2-symmetric-stretching mode (I2883/ I2847) changes from 1.2 in the gel-phase spectrum to 1.1 in the fluid-phase spectrum, indicating a decrease in the acyl chain lateral packing density. The ratio of the CH3-terminal-methyl-

Figure 3. (A) Component spectra resolved from the temperaturedependent Raman spectrum of the DMPC hybrid bilayer. The red spectrum corresponds to the lower temperature gel-phase. The black spectrum corresponds to the higher-temperature fluid phase. (B) Corresponding composition vectors showing the change in relative amplitude of each of the spectra in (A) as a function of temperature.

stretching mode to the CH2-symmetric-stretching mode (I2930/ I2847) changes from 0.47 to 0.69, demonstrating an increase in both structural disorder in the acyl chains (gauche vs trans character) and, again, a shift in the acyl chain lateral packing density to less dense in the fluid phase. Frequency shifts of the CH2-antisymmetric- and symmetric-stretching mode from 2848 and 2881 cm−1, respectively, in the gel phase, to 2851 and 2886 cm−1 in the fluid phase indicate a decrease in interchain coupling in the fluid hybrid bilayer, where decreased interchain order results in less dipole interactions between neighboring acyl chains and increases in the frequency of both modes of C− H stretching. Although the spectral changes in transitioning from the gel phase to the fluid phase are similar to those that occur across a DMPC vesicle phase transition, the temperature dependence of the changes is quite different. In a DMPC vesicle, the main phase transition occurs at 24 °C and is narrow, occurring over a 2 °C range (see calorimetry data in Supporting Information). Raman spectra of DMPC vesicles measured through their melting transition follow the calorimetric response.61 The Raman spectra of the hybrid bilayer exhibit temperaturedependent changes across a broad range from 20 to 40 °C (Figure 3B), suggesting the hybrid bilayer phase transition may F


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Langmuir be less cooperative than the transition in a vesicle bilayer.27 Additionally, the phase transition temperature is shifted to higher temperature, 40 °C, consistent with the observations of Bayerl and co-workers.56 These results suggest that the interactions between the upper leaflet of DMPC and the covalently bound lower leaflet of C18 chains inhibit chain melting and broaden the phase transition. To confirm the spectroscopic results and investigate the nature of the within-particle bilayer phase transition, the melting transition of the within-particle DMPC hybrid bilayer was measured using DSC, and the results are shown in Figure 4. The resulting calorimetric transition curve is in good

interdigitated structure as suggested by the surface coverage and strong methylene C−H coupling results, discussed above. It has been shown previously that incorporation of even small mole fractions (0.02) of long chain hydrocarbons (dodecane and hexadecane) can lead to broadening of the DMPC vesicle phase transition,63 and similarly shaped transitions have been observed for 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) when anesthetic drug compounds are solubilized in vesicle membranes.64 This type of broadening has been treated by incorporation of solid−solution theory into a van’t Hoff treatment of calorimetric data, where it was demonstrated that the phase transition can be broadened in the presence of a solute while maintaining significant cooperativity.63 The role of interchain interactions in the hybrid bilayer phase transition can be tested in an experiment where isotope labeling is used to separate the observations of the phospholipid acyl chains and the underlying C18 chains. Temperature-Dependent Raman Spectroscopy of Hybrid Bilayers with Deuterated Phospholipid Acyl Chains. To investigate the broadened, higher-temperature phase transition observed in the within-particle hybrid bilayers, and to gain a better understanding of the structure of the within-particle hybrid bilayer, Raman scattering from the phospholipid acyl chains was resolved from that of the surface C18 chains by using acyl chain deuterated DMPC (1,2-dimyristoyl-d54-sn-glycero-3phosphocholine, DMPC-D54). Raman spectra of the deuterated phospholipid hybrid bilayers within particles were collected as a function of temperature through the hybrid bilayer phase transition (Figure 5), where changes in both the

Figure 4. Calorimetric endotherms of the DMPC (black) and DMPCD54 (red) hybrid bilayer phase transitions. The y-axis scale is on the molar basis of phospholipid.

agreement with the spectroscopy in the broadness and location of the main transition. The calorimetry shows a small pretransition very near the DMPC vesicle phase transition; this pretransition has been observed previously in DSC endotherms of hybrid bilayers in porous silica56 and has been suggested due to phospholipid multilayers adsorbed to the hybrid bilayer, despite multiple washing steps. Unfortunately, we were unable to resolve Raman spectral changes associated with this pretransition. Phospholipid membrane melting is typically characterized by highly cooperative pseudo-first-order transitions.27,62 Cooperativity arises from the balance between intermolecular reorganization during acyl chain disordering and interfacial energy at the phase boundary and typically leads to phase transitions which occur across a narrow temperature range.62 Interestingly, the hybrid bilayer phase transition, measured both spectroscopically and calorimetrically, is broad compared to the DMPC vesicle phase transition. It was previously hypothesized56 that this broadening is due to decreased cooperativity between adjacent phospholipid molecules due to the immobility of the covalently bound C18-acyl chains. A common measure of the cooperativity of a phase transition is the magnitude of the cooperative unit (CU, the number of lipid molecules in a domain which melt “together”).27 The cooperativity of the hybrid bilayer phase transition was compared to the cooperativity of a suspension of DMPC vesicles (see Supporting Information). The cooperativity of the hybrid bilayer phase transition (CU = 104) was found to be a factor of ∼1/2 smaller than the cooperativity of DMPC vesicles (CU = 195). A possible source of the lower cooperativity is the

Figure 5. Raman spectra collected as a function of temperature for the DMPC-D54 hybrid bilayer through its melting transition. Raman band assignments are coded according to their molecular origins: blue/ dashed correspond to DMPC-D54, orange/solid correspond to C18, and green/dash-dot correspond to directly overlapped Raman bands from both components.

deuterated and nondeuterated acyl chain structure are observed. Spectral changes occur across a broad temperature range, but the melting transition occurs at a lower temperature. This is anticipated, however, based on previous calorimetric studies where the deuterated lipid phase transitions of vesicles are shifted to lower temperature.65 Because of the significant overlap in the Raman spectra, the deuterated phospholipid G


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discuss all deuterated modes in this region with respect to the corresponding C−H modes. Carbon−Carbon Stretching Region. The antisymmetric C−C-stretching mode (1061 cm−1) corresponding to the surface-bound C18 acyl chain trans C−C stretching decreases in intensity at high temperatures, indicating loss of order in the C18 acyl chain structure. Similarly, the antisymmetric C−Cstretching mode of the deuterated phospholipid (shifted to 830 cm−1) decreases in intensity with increased temperature, indicating more gauche defects in the phospholipid acyl chains. The out-of-phase C−C modes for the deuterated and nondeuterated chains overlap to form the peak at 1139 cm−1.66 Here, both bands decrease simultaneously to a smaller broad peak. The C−C-stretching mode due to the deuterated lipid has been shown to be more intense for both gel and fluid phases,66 explaining the intensity of the fluid-phase peak in the deuterated hybrid bilayer, which has greater amplitude in the fluid phase than in the nondeuterated case. The gauche, symmetric C−C-stretching mode (1086 cm−1) of the C18-acyl chains also increases in intensity and broadens, indicating disordering of the C18-acyl chains. Carbon−Hydrogen and Carbon−Deuterium Twisting. The CH2-twisting mode from the C18-acyl chains is decreased in intensity and broadened in the fluid-phase spectrum compared with the gel-phase spectrum, consistent with decreased order in the C18 chains as temperature is increased. Again, the CD2-twisting mode has been shown to qualitatively undergo the same changes on melting, as is observed in the deuterated phospholipid bilayer, where the frequency-shifted CD2 twist (916 cm−1)66 decreases in intensity and broadens, indicating decreased ordering in the phospholipid acyl chains at raised temperatures. Carbon−Hydrogen and Carbon−Deuterium Bending. The overlapping CH-bending and scissoring modes at 1436 and 1455 cm−1, respectively, are not overlapping with deuterated modes; thus, the ratio of the peaks is a valid indicator of C18 structure. Here, I1436/I1455 changes from 1.36 in the gel-phase spectrum to 1.30 in the fluid-phase spectrum, consistent with increasing gauche conformers. The CD-bending and scissoring modes in the deuterated acyl chains do not overlap, and additionally, analogous changes in the CD-scissoring mode are not expected due to the lack of an infrared Fermi resonance that leads to this behavior in the CH spectrum (see ref 66). The CD-scissoring mode (981 cm−1) instead decreases in intensity and shifts to lower frequency as a function of increased temperature indicating fluid acyl chains.66 The behavior of the CD-scissoring mode is consistent with this result. Carbon−Deuterium Stretching Region. Unlike the low-tomid wavenumber region where many deuterated and nondeuterated acyl chain Raman bands overlap, the C−H- and C− D-stretching regions of the spectrum are entirely resolved. The C−D-stretching modes occur in the region from 2000 to 2250 cm−1 and the C−H-stretching modes in the region from 2800 to 3100 cm−1.30,66 In the C−D-stretching region, several changes indicative of acyl chain disordering at higher temperature are observed. The most obvious change occurs in the CD2-symmetric-stretching band (2101 cm−1), where the decrease in intensity and shift to higher frequency (2106 cm−1) as temperature is raised are indicative of decreased coupling between the acyl chains and an increase in gauche defects; this is analogous to the C−H symmetric stretch, where decreased coupling results in less damping, higher frequency, and lower polarizability vibrations.30,66 The CD3-symmetric-stretching

phase transition spectral data were also analyzed using SMCR. PCA for the deuterated lipid yielded, similarly to the nondeuterated case, two principal components across the melting transition. The component spectra and concentration vectors from SMCR analysis are presented in Figures 6A and

Figure 6. (A) Component spectra resolved from the temperaturedependent Raman spectra of the DMPC-D54 hybrid bilayer. The red spectrum corresponds to the gel phase. The black spectrum corresponds to the fluid phase. (B) Corresponding composition vectors showing the change in relative amplitude of each of the spectra in (A) as a function of temperature. Note: the Raman band assignments are coded according to their molecular origins: blue/ dashed correspond to DMPC-D54, orange/solid correspond to C18, and green/dash-dot correspond to overlapped Raman bands from both components.

6B. Here, structure changes observed in the Raman spectra, and the composition vectors describing the changes as a function of temperature, clearly demonstrate the phospholipid acyl chains and underlying C18-chains undergo simultaneous disordering as the melting transition progresses. Spectral changes for the gelphase and fluid-phase deuterated hybrid bilayer are discussed as follows. The lower wavenumber region of the deuterated phospholipid hybrid bilayer (750−1520 cm−1) contains the deuterated and nondeuterated C−C stretching modes and the C−H and C−D bending, scissoring, and stretching modes. The spectral components are not shifted uniformly; thus, we will H


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Langmuir mode at 2073 cm−1 decreases as the bilayer acyl chains decouple, similar to the nondeuterated case as does the antisymmetric C−D stretching mode at 2195 cm−1, while the CD2-symmetric stretch increases slightly as shown by Gaber.66 These results all confirm decoupling and disordering of the CD tails of the phospholipid at higher temperature. Carbon−Hydrogen Stretching Region. The C−H-stretching region of the Raman spectrum is particularly sensitive to acyl chain packing and interchain vibrational coupling, where many modes lie superimposed on broad Fermi resonances. These characteristics make this region valuable for characterizing the asymmetric bilayer in this work. The lowest wavenumber peak at 2847 cm−1 which is attributed to the CH2-symmetric-stretching mode is interestingly less intense in the low-temperature spectrum than expected for an all-trans gel-phase bilayer. Previous work has shown that increased intensity at low temperatures of the narrow CH2-symmetricstretching mode is due to interchain interactions resulting in vibrational coupling of neighboring CH2-symmetric-stretching modes.30,51,67 The absence of the intense CH2-symmetric stretching mode, despite all-trans configuration of the corresponding acyl chains, indicates that while the structure is likely is a close-packed layer, the methylene C−H vibrations of the C18-acyl chains are not coupled due to their dilution with C−D methylene groups of the phospholipid acyl chains. This is consistent with a fully interdigitated gel phase, where the deuterated acyl chains, which do not vibrationally couple with protonated methylene chains, pack between the surface-bound acyl chains, preventing vibrational coupling event at low temperatures. In the high-temperature spectrum, the CH2-symmetric stretch is slightly increased in intensity and shifted to 2853 cm−1 typical of melting of phospholipid acyl chains and indicating adoption of triclinic crystal structure typical of liquidcrystalline phases. The 2853 cm−1 mode is attributed to splitting of the CH2-symmetric-stretching mode and also a strong Fermi resonance between the terminal methyl group and the symmetric methylene stretching mode.51−53 The increase, then, is likely due to decreased acyl-chain mobility in the fluid phase, where the bilayer acyl chains can adopt configurations where terminal methyl groups on the protonated chains can couple with methylene symmetric stretching modes. This indicates increased gauche conformers and increased methyl-tomethylene C−H coupling. The prominent peak at 2881 cm−1 in the low-temperature spectrum has been attributed to the headgroup methylene stretching mode in the deuterated phospholipid66 which lies superimposed on the CH2-antisymmetric-stretching mode at 2883 cm−1,50,54 both of which have been shown to decrease with increased gauche defects as is seen in the high temperature spectrum. The broad peak which grows in at 2900 cm−1 is due to the sum of the decreased broadened 2883 cm−1 band and the Fermi resonance due to the asymmetric CH-bending mode and the symmetric CH3-stretching mode.50,54 The increase in this band is then again indicative of decreased order in the bilayer acyl chains, where an increase in gauche defects again results in greater likelihood of vibrational interaction between terminal methyl groups and protonated acyl chains. The temperature-dependent concentration profiles determined using SMCR (Figure 6B) show two distinct components which, on examination of the corresponding spectra (Figure 6A), represent the gel phase and fluid phase, which exchange as a function of temperature. Similarly to the protonated lipid

bilayer, the phase transition temperature predicted using spectroscopy is shifted from that of DMPC-D54 vesicles and is quite broad. Therefore, to confirm the spectroscopic result, the melting transition of the within-particle DMPC-D54 hybrid bilayer was measured using DSC (Figure 4) following the protocol previously described for protonated lipid. The resulting calorimetric transition curve is again in good agreement with the spectroscopic result in broadness and location of the main transition. Interestingly, the calorimetry again shows a low-temperature transition, which we are unable to resolve in Raman spectral changes. This pretransition is near the DMPC-D54 vesicle melting transition65 and may be attributable to DMPC-D54 bilayers formed at defects in the C18 layer on the silica. The results of Raman spectral analysis and calorimetry of the deuterated phospholipid hybrid bilayer are informative of the bilayer structure. The bands indicative of acyl chain trans character in all regions of the spectrum show that both the deuterated and nondeuterated acyl chains are in all-trans conformations at low temperatures and exhibit a much greater fraction of gauche conformers at high temperature. The C−Hstretching region indicates a lack of vibrational coupling despite the all-trans acyl chain character and other indicators of close packing in the gel phase. The spectroscopically resolved “composition” vectors and calorimetric endotherms are in good agreement, showing that the structural changes associated with melting the deuterated acyl chains and the surface C18 chains occur together, as the temperature is raised. These results suggest a fully interdigitated gel-phase bilayer where the deuterated and nondeuterated acyl chain structures are mixed, which explains the increase in melting temperature and broadening of the hybrid bilayer phase transitions. The results show a single melting transition for both the lipid and the C18 chains, which means that they melt together, providing further evidence of mixing of substrate and lipid acyl chains through interdigitation. This result contrasts with the infrared and ellipsometric measurements reported for a planar, oxidized silicon−C18 interface, which were consistent with a structurally decoupled, noninterdigitated DPPC layer at the C18 interface.58 We hypothesize that the differences between these two results could be due to a greater density of grafted n-alkyl chains at the planar silica interface; the planar surface films were prepared by self-assembly of a polymeric silane reagent.58 Polymeric silanes have been shown to produce greater acyl chain packing density than corresponding monomeric silanes,68 where a higher density n-alkyl silane monolayer could prevent penetration and interdigitation of phospholipid acyl chains. In the present work, the chromatographic silica particles were derivatized with a monofunctional silane reagent, which produces a lower C18 surface coverage on the silica support.68 The resulting sparsity of C18 chains on the surface (∼2.4 C18 chains/nm2) provides space where the phospholipid acyl chains can form an interdigitated structure between the surface-bound C18 chains.

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CONCLUSIONS In this work, the formation and temperature-dependent structure of DMPC hybrid bilayer membranes formed at the C18 surfaces within chromatographic silica particles were investigated using confocal Raman microscopy to probe the interior surfaces of individual porous particles. The DMPC surface coverage was quantified by measuring the increase in Raman scattering intensities from the CH2-twisting modes from the accumulation of a DMPC monolayer on the C18 chains I


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grafted to the silica surface. The lipid surface coverage, verified by carbon analysis, corresponds to a DMPC cross-sectional area of 120 ± 10 Å2 and is 2.5 times greater than the DMPC area in the bilayer of a lipid vesicle. The increased area corresponds to ∼3 n-alkyl chains and is consistent with the mole ratio of C18 chains to lipid in the hybrid bilayer. This stoichiometry and the highly ordered (all-trans) conformations of both lipid and (previously disordered) C18 acyl chains suggest an interdigitated structure for the hybrid bilayer. Further support for this structure derives from the strong vibrational coupling between methylene C−H-stretching modes of both the C18 and lipid acyl chains. This coupling disappears when a hybrid bilayer is prepared from a lipid having deuterated acyl chains, which is consistent with the C−D-labeled lipid acyl chains residing between C18 chains and disrupting C−H vibrational coupling. By monitoring acyl chain conformations versus temperature, it was possible to observe the hybrid bilayer melting transition, which is broader and shifted to higher temperature than a DMPC vesicle, in agreement with differential scanning calorimetry (DSC) results. This result is also consistent with an interdigitated structure, where immobility of the C18 chains decreases the transition cooperativity and increases the melting temperature of hybrid bilayers. Finally, through the use of deuterated DMPC to resolve the Raman scattering of lipid acyl chains from that of the C18 chains, it was learned that the lipid monolayer and underlying C18 chains melt in unison in a single transition, providing more evidence for their interdigitation. Despite their interdigitated structure, within particle hybrid bilayers do show a main phase transition with similar calorimetric and spectroscopic characteristics as vesicle membranes. Additionally, previous chromatographic retention studies of small molecules in hybrid bilayers reported comparable partitioning into hybrid bilayers and vesicle membranes, indicating that hybrid bilayers can serve as a model for measuring small-molecule lipid membrane partition coefficients. Future spectroscopic studies of small-molecule partitioning into hybrid bilayers, measured in situ within individual chromatographic particles, could provide structural insight into the utility of within-particle hybrid bilayers for small molecule partitioning studies. Caution should be exercised, however, in using within-particle hybrid bilayers as models to measure protein−bilayer interactions. The interdigitated structure of hybrid bilayers would likely influence the insertion of hydrophobic residues into the membrane, and the covalently bound lower leaflet would not allow membrane-spanning proteins to adopt a natural conformation within this membrane model.

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AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected] (J.M.H.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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REFERENCES

This research was supported in part with funds from the U.S. Department of Energy under Grant DE-FG03-93ER14333. This article is dedicated to the memory of Professor Eli Grushka, whose pioneering research in phospholipid-modified reversed-phase columns was an inspiration for this work.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.6b02309. Additional information is provided on the algorithm for self-modeling curve resolution, measurements of the distribution of lipid through the particles, and calorimetry comparing the melting transitions and their cooperativities for DMPC vesicle bilayers and DMPC hybrid bilayers (PDF) J


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