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. Author manuscript; available in PMC: 2010 Aug 1.
Published in final edited form as: J Colloid Interface Sci. 2009 Apr 8;336(1):90–99. doi: 10.1016/j.jcis.2009.03.068

Ordering Transitions in Micrometer-Thick Films of Nematic Liquid Crystals Driven by Self-Assembly of Ganglioside GM1

I-Hsin Lin 1, Maria-Victoria Meli 1, Nicholas L Abbott 1,*
PMCID: PMC2778293  NIHMSID: NIHMS108770  PMID: 19428021

Abstract

We report an investigation of the self-assembly of the monosialoganglioside (GM1) at interfaces formed between aqueous solutions of 10 µM GM1 (at 25°C) and micrometer-thick films of the nematic liquid crystal (LC) 4’-pentyl-4-cyanobiphenyl (5CB). We observe the process of spontaneous transfer of GM1 onto the interfaces to be accompanied by continuous ordering transitions within the micrometer-thick films of the LC. At saturation coverage, the GM1 orders the LC in an orientation that is perpendicular to the interface, an orientation that is similar to that caused by phospholipids such as dilaurylphosphatidylcholine (DLPC). This result suggests an interaction between the LC and GM1 that is dominated by the hydrophobic tails of the GM1. Relative to DLPC, however, we observe the dynamics of the LC ordering transition driven by GM1 to be slow (2 hours for DLPC versus 100 hours for GM1). To provide insight into the origins of the slow dynamics of the GM1-induced ordering transition in the LC, we performed two additional measurements. First, we quantified the time-dependent adsorption of GM1 at the LC interface by using fluorescently-labeled GM1. Second, we used the Langmuir-Schaefer method to transfer preorganized monolayers of GM1 from an air-water interface to the aqueous-LC interface. Results obtained from these two experiments are consistent with a physical picture in which the final stages of spontaneous adsorption/ordering of GM1 at the aqueous-LC interface dictate the dynamics of the LC ordering transition. This rate limiting process underlying the ordering transition was substantially accelerated by heating the system above the phase transition temperature of GM1 (26 °C), suggesting that the phase state of the GM1 micellar aggregates in bulk solution strongly influences the kinetics of the final stages of ordering/adsorption of GM1 at the LC interface. Overall, these results and others presented in this manuscript reveal that it is possible to decorated interfaces of a nematic LC with GM1, and that the assembly of GM1 at these interfaces impacts the dynamic and equilibrium ordering of the LC.

Keywords: Ganglioside, GM1, Self-Assembly, Liquid Crystals, Biomolecular Interfaces, Langmuir-Schaefer, Ordering Transitions, Anchoring of Liquid Crystals, Biosensors

INTRODUCTION

A series of recent studies have reported on ordering transitions induced in micrometer-thick films of liquid crystals (LC) that are caused by the self-assembly of amphiphiles at aqueous interfaces of LC films.1–13 Of the various different amphiphiles explored in these studies (which includes surfactants, phospholipids, macromolecular amphiphiles), the ordering of nematic LCs by the interfacial self-assembly of the phospholipid l-α-dilauroylphosphatidylcholine (l-DLPC) has been particularly well characterized.3, 5, 6, 9–13 Specifically, equilibration of aqueous dispersions of unilamellar vesicles formed from l-DLPC has been shown to lead to formation of monolayers of DLPC at the interfaces of the LC. The formation of the monolayer of l-DLPC has in turn been demonstrated to cause the LC to adopt a perpendicular (homeotropic) ordering at the aqueous interface. Additional experiments have also revealed that (i) the phase behavior of monolayers of DLPC formed at the interfaces of the nematic LC is substantially different from that observed in the absence of the nematic ordering of the LC, and (ii) that binding of proteins (such as phospholipases) to these lipid-laden LC interfaces leads to easily visualized ordering transitions in the LCs.5, 6 Whereas an increasingly complete understanding of the assembly of phospholipids at aqueous interfaces of LCs is emerging, in this paper, we move to report on the assembly of glycolipids at aqueous-LC interfaces. In particular, as a prototypical example of the wide range of glycolipids found in biological systems, we focus on the self-assembly of the monosialoganglioside GalBeta1-3GalNAcBeta1-4(NeuAcAlpha2-3)GalBeta1-4GlcBeta1-1'-ceramide (GM1) at aqueous interfaces of thermotropic LCs. We note that GM1 binds the bacterial toxin produced by Vibrio cholerae and, consequently, that the biophysical properties of GM1 have been studied in detail within Langmuir monolayers and lipid bilayers.

Gangliosides, in general, are lipids with head groups comprised of oligosaccharides containing one or more N-acetylneuraminic acid (sialic acid) residues.14 GM1 contains a pentasaccharide (Figure 1). As noted above, the self-organization of gangliosides within in vitro mimics of biological membranes have been widely studied15–22 as have the interactions of protein toxins with these models of cell membranes.23–35 In the context of the former investigations, the physicochemical characteristics of GM1-laden interfaces have been investigated with atomic force microscopy (AFM),16, 19–21, 24 light scattering techniques,17, 18 surface plasmon resonance (SPR),25 and fluorescence microscopy.36 In this paper, we report the results of an investigation that sought to create a new class of GM1-decorated interfaces that are prepared by self-assembling GM1 at the interfaces of LCs. By analogy to phospholipid-decorated LCs, we hypothesized that the orientational ordering of the LC would be closely coupled to the formation of the GM1-decorated interface, and thus that the LC ordering behavior could be used to report on the interfacial behavior of the GM1. Although certain similarities in the orientational ordering of the LC in the presence of phospholipids and GM1 are noted in our paper, the results of our study also reveal striking differences in the dynamics of these two systems. We end our introduction by noting that the development of methods that lead to formation of GM1-laden interfaces of LCs, and an understanding of the equilibrium and dynamic properties of the interfaces, is a prerequisite to exploring their potential use as biomolecular interfaces at which protein toxin interactions with GM1 can be reported via ordering transitions in LCs. In future studies, we will investigate the influence of protein toxins on the ordering of GM1-decorated interfaces of LCs.

Figure 1.

Figure 1

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Structures of GM1, BODIPY FL GM1, DLPC and DPPC. Gal = Galactose, Glc = Glucose, GalNAc = N-Acetylgalactosamine, Neu5Ac = N-Acetylneuraminic acid.

MATERIALS & METHODS

Materials

GalBeta1-3GalNAcBeta1-4(NeuAcAlpha2-3)GalBeta1-4GlcBeta1-1'-Cer (GM1) was obtained from Avanti Polar Lipids, Inc.(Alabaster, AL). Trizma-hydrochloride (Tris[hydroxymethyl]aminomethane hydrochloride, Tris HCl), sodium azide, ethylenediaminetetraacetic acid (EDTA), and chloroform were obtained from Sigma-Aldrich (St. Louis, MO). Octadecyltrichlorosilane (OTS), sodium chloride, methanol, methylene chloride, sulfuric acid, hydrogen peroxide (30% w/v), 2-propanol, and heptane were obtained from Fisher Scientific (Pittsburgh, PA). BODIPY FL C5-GalBeta1-3GalNAcBeta1-4(NeuAcAlpha2-3)GalBeta1-4GlcBeta1-1'-Cer (BODIPY FL-GM1) was purchased from Molecular Probes (Eugene, OR). Sodium hydroxide was obtained from LabChem Inc. (Pittsburgh, PA). The LC 4'-pentyl-4-cyanobiphenyl (5CB) was obtained from EM Sciences (New York, NY). All chemicals were used as obtained. Deionization of a distilled water source was performed with a Milli-Q system (Millipore, Bedford, MA) to give water with a resistivity of 18.2 MΩ•cm. Glass microscope slides were Fisher’s Finest Premium Grade obtained from Fisher Scientific. Gold specimen grids (20 µm thickness, 50 µm wide bars, and 283 µm grid spacing) were obtained from Electron Microscopy Sciences (Fort Washington, PA).

Preparation of LC-filled grids

Detailed descriptions of procedures used to prepare LC-filled grids can be found in our previous publications.1–13, 37 Briefly, glass microscope slides were cleaned according to published procedures38 and coated with OTS.8 The quality of the OTS layer was assessed by checking the alignment of 5CB confined between two OTS-coated glass slides. Any surface not causing homeotropic anchoring (perpendicular alignment of 5CB) of 5CB was discarded.39, 40 Gold specimen grids that were cleaned sequentially in methylene chloride, ethanol, and methanol were placed onto the surface of OTS-coated glass slides. Approximately 1 µL of 5CB was dispensed onto each grid and then excess LC was removed by contacting a capillary tube with the droplet of 5CB.1–13, 37 We estimated the variation in 5CB thickness to be approx ±10% based on interference colors generated when viewing the 5CB with white light. Each LC-filled grid was equilibrated at ambient temperature and subsequently immersed in or contacted with the aqueous solution of interest at the desired temperature.

Preparation of Aqueous Dispersions of GM1

Dispersions of GM1 were prepared following published procedures.5, 9, 32, 35 Briefly, GM1 was dissolved in a mixture of chloroform and methanol (4:1 by volume) and dispensed into glass vials. The chloroform:methanol mixture was evaporated under a stream of N2, and the vial containing the lipids was then placed under vacuum for at least 2 hours. The dried lipid was resuspended in an aqueous solution consisting of 50 mM Tris HCl, 200 mM NaCl, 3 mM NaN3, and 1 mM Na2EDTA adjusted using NaOH to a pH of 7.5 (denoted as pH 7.5 TRIS buffer in the remainder of this paper). This procedure resulted in a slightly cloudy solution. Before contacting with LC interfaces, the aqueous dispersion of GM1 was equilibrated at ambient temperatures for at least 1 hour. Experiments utilizing the aqueous GM1 dispersions were typically initiated within 4 hours of their preparation.

Determination of the Orientation of Liquid Crystals by Polarized Light Microscopy

The orientation of 5CB was observed by using an Olympus BX60 microscope with crossed polarizers (transmission mode). LC-filled grids were placed on a rotating stage located between the polarizers. Orthoscopc examinations were performed with the source light intensity set to 50% of full illumination and the aperture set to 10% to collimate the incident light. Homeotropic (perpendicular) alignment of a LC was determined by first observing the absence of transmitted light during a 360° rotation of the sample. Next, insertion of a condenser below the stage and a Bertrand lens above the stage allowed conoscopic examination of the specimen. An interference pattern consisting of two crossed isogyres confirmed the homeotropic alignment.41 Images were captured with a microscope-mounted digital camera (Olympus C-4000 Zoom) set to an f-stop of 2.8 and a shutter speed of 1/320 s. Temperature was controlled with a heat stage (INSTEC, Inc., Boulder, CO) or a water bath (ISOTEMP 202, Fisher Scientific, Pittsburgh, PA).

Epifluorescence Imaging of GM1-Laden Aqueous-LC Interfaces

Lipid layers comprised of mixtures of BODIPY FL-GM1 and GM1 were formed by incubating the LC interface against aqueous dispersions of GM1 that contained 1 mol% BODIPY FL-GM1. Before performing the epifluorescence measurements, the LC-filled grid was sequentially moved from the aqueous dispersion of BODIPY FL-GM1 and GM1 to 2mL and then subsequently 400mL of the pH 7.5 TRIS buffer (see above for composition) before a final transfer into a third volume of the pH 7.5 TRIS buffer solution. During each transfer of the sample between solutions, care was taken to ensure that a thin film of the water covered the lipid-laden interface of the 5CB hosted within the grid.4, 42 GM1-laden interfaces were then imaged by epifluorescence microscopy using an Olympus IX71 inverted microscope equipped with a 100 W mercury lamp. A fluorescence filter cube with excitation at 480 nm and emission at 535 nm (Chroma, Rockingham, VT) was used to visualize BODIPY FL fluorescence. Images were collected with a Hamamatsu 1394 ORCA-ER CCD camera (Bridgewater, NJ) connected to a computer and controlled through SimplePCI imaging software (Compix, Inc., Cranberry Twp., NJ).

Fluorescence imaging was performed using an objective power of 4X and an exposure time of 0.3 seconds. After each epifluorescence measurement, the sample was discarded. At least 4 replicate LC-filled grids were measured at each time point. To account for effects of fluorophore degradation/bleaching encountered when using long incubation times (>72 hours), we normalized all fluorescence measurements to the intensity measured using an OTS-treated slide immersed in the same aqueous dispersion of BODIPY FL-GM1/GM1. All fluorescence intensity measurements were corrected for background fluorescence, measured using lipid-free buffer solutions. Intensity values were determined using SimplePCI and Adobe Photoshop.

Quantification of Interfacial Concentration of BODIPY FL-GM1/GM1 Adsorbed at Aqueous-LC Interfaces via Fluorimetric Measurements

The interfacial concentration of BODIPY FL-GM1/GM1 adsorbed at the aqueous-LC interface was determined by using the following procedure. First, a calibration plot of fluorimetric intensity versus known BODIPY FL-GM1 concentration in bulk solution prepared using 2mL of a 2-propanol solution containing 1 µL of 5CB, known amounts of BODIPY FL-GM1 and GM1 (at molar ratio of BODIPY FL-GM1 to GM1 of 1:99), and 5 µL of pH 7.5 TRIS buffer solution. This matrix was used so as to mimic the solution composition that resulted when BODIPY FL-GM1/GM1-decorated 5CB hosted within grids was extracted and then dissolved in a 2-propanol solvent (see below). Fluorimetric measurements were performed using a FluoroMax-3 fluorimeter (Instruments S. A/Jobin Yvon/Spex Horiba Group, Edison, NJ) with an excitation wavelength of 480 nm (0.5 nm excitation slit) and an emission wavelength range of 490–850 nm (5 nm emission slit) for the detection of BODIPY fluorescence. The fluorimeter was connected to a computer and controlled using DATAMAX software (Instruments S. A./Jobin Yvon/Spex Horiba Group).

To determine the interfacial concentration of BODIPY FL-GM1/GM1 adsorbed at an aqueous-LC interface, the LC sample (LC hosted in grid, volume of LC was ∼1µL) was physically transferred from the aqueous BODIPY FL-GM1/GM1 dispersion to 2 mL and then 400 mL of pH 7.5 TRIS buffer solution to successively dilute BODIPY FL-GM1/GM1 in the bulk solution contacting the surface of the LC sample. Each sample was then removed from the pH 7.5 TRIS buffer, and a blunt-tip 10 µL micro-syringe was used to withdraw the lipid-decorated 5CB from each grid The 5CB extracted using the micro-syringe was dissolved in 2 mL 2-propanol for fluorimetric measurements to determine the concentration of BODIPY FL-GM1 in the sample. We illuminated the solutions at 480 nm and recorded the emission intensity from 490 to 850 nm to ensure that only monomer emission was observed with a peak around 510 nm. With a known BODIPY FL-GM1 concentration versus fluorimetric intensity calibration plot (Figure S1), this measurement permitted quantification of the interfacial concentration of GM1 adsorbed at the interface as a function of time during the LC ordering transition. Polarized light microscopy was used to confirm that all LC was extracted from each grid. Control experiments not containing BODIPY FL-GM1 were performed to determine the background fluorescence. Furthermore, to ensure that fluorescence intensities resulted only from assemblies of BODIPY FL-GM1/GM1 adsorbed at the aqueous-5CB interface (and did not include any contributions from the fluorophore-containing bulk solution), we confirmed that the 400 mL pH7.5 TRIS buffer solution used to dilute excess BODIPY FL-GM1/GM1 at the LC sample surface did not possess significant fluorescence intensity after the last dilution step (data not shown). As noted above, we observed the fluorimetric intensity of BODIPY-FL in the bulk aqueous solution to decrease in experiments that involved long incubation times (>72 hours) due to photobleaching/degradation. To correct for this process of degradation, fluorimetric measurements of the bulk solution intensity were recorded at each time point. All raw fluorimetric intensities measured after ∼ 72-hour of incubation were corrected for this process of degradation. To minimize degradation processes, light exposure was minimized in all steps of the experiments.

The above measurements, when combined with knowledge of the areas of the aqueous-LC interfaces within each grid, were used to calculate the areal density (area/molecule) of the GM1 molecules assembled at each interface. All experiments were repeated 4 times using the same batch of reagents (e.g., 2-propanol, BODIPY FL-GM1, and GM1 from the same lot number). Error bars were calculated as the standard deviation of these replicates. As described in our Results section, the above-described procedure for quantification of the interfacial concentration of lipid was validated by determining the interfacial concentration of l-DLPC at saturation coverage (for which values have been reported in the literature).

Preparation of GM1 Monolayers at Aqueous-LC Interfaces by using the Langmuir-Schaefer Method

Langmuir monolayers of GM1 were prepared using a NIMA 602A film balance (Coventry, England) equipped with a paper Wilhelmy plate for surface pressure measurements. A 4:1 v/v chloroform:methanol solution of GM1 (∼0.3 mg/mL) was spread at the aqueous-air (pH 7.5 TRIS buffer) interface at 25 °C. Temperature was controlled with a water bath (ISOTEMP 1006D, Fisher Scientific, Pittsburgh, PA). The solvent was allowed to evaporate for 20 minutes at 25 °C before monolayer compression was initiated. Symmetric monolayer compression was performed at a rate of 100 cm2/min (26.0 ∼ 34.6 Å2/molecule·min). Once the desired surface pressure was reached, the LC sample was lowered horizontally into contact with the monolayer at the aqueous-air interface using tweezers and immediately submerged into the pH 7.5 TRIS buffer sub-phase for analysis using polarized microscopy. Detailed descriptions regarding the experimental setup used for the Langmuir-Schaefer transfer can be found elsewhere.10

RESULTS and DISCUSSION

Spontaneous Adsorption of GM1 onto Aqueous-LC Interfaces

Our initial experiments sought to determine if GM would adsorb spontaneously from bulk aqueous solution onto the interface of a LC and thereby trigger an ordering transition in the LC. We also sought to determine whether or not the dynamic and equilibrium characteristics of LC ordering transitions triggered by GM1, if observed, would be similar to those reported previously for phospholipids such as l-DLPC.3, 5, 6, 9, 11, 12 To this end, we first contacted nematic 5CB hosted within grids (see Materials and Methods for details) with aqueous dispersions of 10 µM GM1 (pH 7.5 TRIS buffer) at 25 °C. As shown in Figures 2A–H, we recorded the optical appearance of the LC under crossed polars for 336 hours (14 days). Immediately following contact with the aqueous dispersion of GM1, the optical appearance of the LC appeared colorful and bright (Figure 2A). This optical appearance of the LC is caused by orientations of the LC that are illustrated in the cartoon shown in Figure 3A. In brief, as described in past studies, 3, 5, 6, 9, 11, 12 in the absence of adsorbates at the aqueous-LC interface, the orientation of 5CB is close to parallel to the aqueous-LC interface. At the interface of the OTS-treated glass, the LC assumes a perpendicular orientation. These two boundary conditions, when combined, result in the introduction of splay and bend distortions into the LC as the film of LC accommodates the two boundary conditions. The LC,2, 4 when viewed under white light illumination, possesses pale yellow-green and pink hues due to interference effects caused by the birefringence of the LC. Variation in the azimuthal orientation of the 5CB results in the dark brush patterns evident in Figure 2A within each compartment of the grids.2

Figure 2.

Figure 2

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(A–P) Optical micrographs (crossed polars) of dynamic LC ordering transitions triggered by spontaneous adsorption of (A–H) GM1 and (I–P) L-DLPC from aqueous solution at 25°C. Times are indicated in units of hours, and t = 0 hrs corresponds to the time of contact of the 10 µM GM1 or 10 µM DLPC aqueous dispersion with the interface of the nematic 5CB. The GM1 and DLPC dispersions were prepared in pH 7.5 TRIS buffer. Scale bar is 300 µm.

Figure 3.

Figure 3

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(A) Schematic illustration corresponding to Figure 2A and 2I, showing the orientation of the nematic LC the aqueous-LC and OTS-LC interfaces, and the deformation of the LC induced by these boundary conditions. (B) Schematic illustration corresponding to Figure 2H and 2P, showing the homeotropic orientation of the LC at the interface of the LC decorated with GM1 or DLPC. (C and D) Effective tilt angles of 5CB at the aqueous-LC interface estimated from interference colors generated by the 5CB with white-light illumination. The tilt angles are plotted as a function of time of adsorption of (C) GM1 and (D) DLPC, with each lipid present at a concentration of 10 µM in pH 7.5 TRIS buffer.

Inspection of Figures 2A–H reveals that the optical appearance of the LC changed gradually from bright to dark during incubation against the aqueous dispersion of 10 µM GM1 for ∼ 240 hours. Orthoscopic and conoscopic examination of the sample shown in Figure 2F (see Methods for procedures) indicated that the LC had slowly transitioned to a homeotropic orientation at the aqueous-LC interface during the 240 hours (see Figure 3B for a schematic illustration). To further characterize the nature of the transition, we used the interference colors observed under white light illumination to calculate the tilt angle of the LC (orientation of the LC at the aqueous-LC interface measured with respect to the surface normal, with 0° corresponding to homeotropic alignment; detailed descriptions of methods can be obtained from our past study4) during incubation against the aqueous dispersion of GM1 (Figure 3C). Inspection of Figure 3C reveals that the change in the orientation of the LC occurred in two steps. Within the first 24 hours, the tilt of the LC changed to 65°, and subsequently, between 100 hours and 200 hours, a second continuous transition in the orientation to the homeotropic state occurred. Incubation of the LC against the aqueous dispersion of GM1 for times longer than 240 hours did not result in any further changes in the orientation of the LC (see Figures 2F–H, and Figure 3C).

We interpret the results described above to indicate that GM1 does transfer spontaneously from aqueous dispersions onto the aqueous-LC interface, and that the GM1 present at the aqueous-LC interface does cause an ordering transition in the LC. The final state of the interface corresponds to the homeotropic orientation of the LC. Although the final homeotropic state of the GM1-laden interface of the LC is similar to that observed in past studies of the orientation of phospholipid-decorated interfaces of LCs5, 9 and thus suggests that the hydrophobic tails of the GM1 play a central role in defining the interactions of GM1 with the nematic LC, we note here two important differences between the behaviors of GM1 and phospholipids. First, past studies of the self-assembly of l-DLPC (10 µM in 10mM Tris (hydroxymethyl)-aminomethane and 100 mM NaCl at pH 8.9, referred hereafter to as pH 8.9 TBS buffer) at the aqueous-LC interface have reported that l-DLPC will trigger an ordering transition of 5CB within 2 hours.5, 9 In contrast, the dynamics of the 5CB ordering transition induced by contact with the dispersion of GM1 (Figures 2A–H) was surprising slow. Second, past observations of LC ordering transitions driven by l-DLPC have revealed that it can occur through a process that involves the nucleation and growth of domains of oriented LC: In contrast, from inspection of Figures 2A–H, it is evident that the ordering transition driven by GM1 is a continuous one.9, 10 We note, however, that the above-described measurements with GM1 and l-DLPC were performed in different aqueous buffers.

In order to provide a more precise comparison of the characteristics of the ordering transitions of the LC induced by GM1 and l-DLPC, we performed measurements of the ordering of nematic 5CB incubated against aqueous dispersions of l-DLPC prepared using the same buffer as used in our measurements with GM1 (pH 7.5 TRIS buffer; Figures 2A–H). Optical micrographs of the LC incubated against the l-DLPC are shown in Figures 2I–P, and the tilt angle of the LC at the aqueous-LC interface is shown in Figure 3D. We make two principal observations from these measurements. First, the time-scale of the ordering transition induced by l-DLPC when dispersed in pH 7.5 TRIS buffer is much shorter (∼4 hours) than that observed with GM1 (240 hours). Second, consistent with past measurements of the self-assembly of l-DLPC at the aqueous-LC interface from pH 8.9 TBS buffer5, 9 the ordering transition of the LC induced by l-DLPC in TRIS pH 7.5 buffer involves domains of oriented LC. Figure S3 of Supporting Information presents a direct comparison of the ordering transition of the LC induced by l-DLPC dissolved in the two buffers. Inspection of this Figure S3 reveals relatively minor effects of the buffer, the most pronounced effect being a change in the rate of the ordering transition (the rate changes by a factor of approximately 2).

The most striking difference between the behavior of GM1 and l-DLPC that emerges from the above-reported study is the slower dynamics of the ordering transition induced by GM1, as compared to l-DLPC. To provide a first perspective on the possible origins of the slow ordering transition induced by GM1, we calculated the time for GM1 molecules present in bulk solution at a concentration of 10 µM to diffuse to the aqueous-LC interface in quantity sufficient to saturate the interface with GM1 molecules. The calculated diffusion time was ∼ 4 minutes, estimated as L2/2Dm where L= Γ/C and corresponds to the diffusion distance, Dm is the diffusion coefficient of GM1 aggregates (micelles) obtained from light scattering measurements43 (Dm = 3.7 × 10−6 cm2/s), Γ is the surface concentration of GM1 molecules at the interface, and C is the concentration of GM1 in the bulk solution. For this calculation, we estimated the density of GM1 molecules at the aqueous-LC interface to be ∼ 40 Å2/GM1.26 We note that the conclusion extracted from this calculation is not sensitive to the exact value of the interfacial density that we assumed. The main conclusion is that the ordering transition of 5CB driven by GM1 is not controlled by diffusion-limited transport of GM1 to the interface.

Quantification of the Interfacial Concentration of BODIPY FL-GM1/GM1 at Aqueous-LC Interfaces via Fluorescence Measurements

We hypothesized that the slow ordering transition of the LC induced by spontaneous adsorption of GM1 from the bulk aqueous dispersion onto the aqueous-LC interface (∼240 hours) was due to slow kinetics of adsorption of GM1 from the bulk dispersion adjacent to the interface. We note that GM1 dispersed under the aqueous solution conditions used in our study is known to form micellar aggregates, and evolution of the aggregate structure and size distribution has been reported to occur slowly (over days) at room temperature43, 44. To test the above-described hypothesis, we incorporated 1% BODIPY FL-GM1 (Figure 1) into a mixture of BODIPY FL-GM1 and GM1, and used epifluorescence intensity measurements, as described in the Methods section, to record the rise in fluorescence of the aqueous-LC interfaces at various time intervals following contact with the BODIPY FL-GM1/GM1. Inspection of Figure 4A confirms BODIPY FL-GM1/GM1 adsorption at the interface of the LC. Surprisingly, however, inspection of Figure 4A suggests that the interfacial concentration of the mixture of BODIPY FL-GM1 and GM1 reached near-saturation coverage (within experimental uncertainty) within ∼20 hours. In contrast, as noted in Figure 2 and Figure 3, polarized light microscopy revealed the ordering transition of the LC to occur over ∼240 hours following contact with the dispersion of GM1. In particular, inspection of Figure 3C shows that the orientation of the LC was nearly parallel to the interface after ∼20 hours of incubation in the aqueous dispersion of GM1.

Figure 4.

Figure 4

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Quantification of BODIPY FL-GM1/GM1 adsorbed at aqueous-5CB interfaces by fluorescence measurements at 25 °C (10µM GM1 in pH 7.5 TRIS buffer). (A) Plot of epifluorescence intensity generated by mixed BODIPY FL-GM1/GM1 assemblies adsorbed at the interface of nematic 5CB as a function of time. (B) Plot of fluorimetric intensity generated by mixed BODIPY FL-GM1/GM1 assemblies adsorbed at the LC interface, and subsequently dissolved into 2-propanol for quantification. (C) Plot of the corresponding area per GM1 molecule at the LC interface as a function of time, obtained using data shown in part B.

Although the above-described measurements lead to the suggestion that the ordering transition of the LC lags substantially behind the adsorption of the GM1 onto the aqueous-LC interface, quantitative interpretation of fluorescence intensity measurements during adsorption processes should be interpreted cautiously because past studies have shown that BODIPY-labeled lipids possess concentration-dependent emission properties that are consistent with dimer formation in both monolayer45, 46 and unilamellar vesicle47, 48 systems. For example, Dahim et al have demonstrated that if the BODIPY monomers are sufficiently close (critical radius is 25.9 ± 1.8 Å) within a fluid monolayer at the time of excitation, excited dimers of BODIPY will form and the monomer emission at 515 nm will be progressively replaced by the dimer emission at ∼ 620 nm.45 To address possible artifacts associated with dimer formation in the epifluoresence measurements shows in Figure 4A, we pursued a second methodology based on fluorescence measurements wherein the lipids adsorbed at the aqueous-LC interface were extracted, and quantified after dissolution into an organic solvent (see Methods for the details). This method had two advantageous attributes. First, dilution of BODIPY FL-GM1 into the organic solvent avoided the potential for dimer formation. Second, the methodology led to an estimate of the absolute density of BODIPY FL-GM1 molecules at the aqueous-LC interface.

To validate the second methodology, we first used it to quantify the interfacial concentration of l-DLPC within monolayers adsorbed at the aqueous-5CB interface, as determined previously by Brake and coworkers4–6, 9 (see Supporting Information). The fluorimetric results revealed that at saturation coverage, the areal density of the l-DLPC at the interface was 48± 10 Å2/l-DLPC. This value is in reasonable agreement with the area occupied by l-DLPC at the air-water interface within condensed monolayers.10, 49–53 In addition, recent experiments by Meli and coworkers used the Langmuir-Schaefer method to transfer monolayers of l-DLPC from the aqueous-air interface onto the aqueous-5CB interface. These experiments revealed that monolayers of l-DLPC with an area density between 45 and 56 Å2/L-DLPC molecule caused homeotropic ordering of l-DLPC (consistent with our experimental results).10

Figure 4B shows a plot of the fluorimetric intensity of mixtures of BODIPY FL-GM1 and GM1 extracted from the aqueous-LC interface, as a function of time of incubation of the LC against the GM1 dispersion (at 25 °C). As can be seen in Figure 4B, the fluorimetric intensity of the extracted BODIPY FL-GM1/GM1 reached a saturation value within 24 hours of incubation. This result is consistent with epifluorescence measurements shown in Figure 4A, suggesting that the saturation of the epifluoresence intensity in Figure 4A was not an artifact associated with dimer formation. We note that the uncertainties in the values of the fluorescence intensity shown in Figure 4B are substantial, limiting the precision with which we can define the time at which saturation coverage of the interface takes place. We suspect that variation in the shape of the LC-aqueous meniscus leads to variation in the LC interfacial area, and thus capacity of the interface to adsorb the lipid. By combining the calibration plot shown in Figure S1 with the mean value of the fluorimetric intensity shown in Figure 4B in the limit of long adsorption times, we calculate the limiting area density of GM1 at the aqueous-LC interface to be 50 ± 12 Å2/GM1. This mean area density is similar to the density of GM1 at the air-aqueous interface at which surface pressure-area isotherms indicate densely-packed, condensed monolayers of GM1 to form.17, 54–56. Past studies have reported that GM1 forms a densely-packed monolayer at ∼ 50–62 Å2/GM1 molecule and a tilted, condensed monolayer at ∼ 62–71 Å2/GM1 molecule within Langmuir films (26 °C and a subphase pH of 5.6),56 following the definitions described by Kaganer et al.57.

The results above lead us to conclude that a relatively high surface concentration of GM1 (52± 14 Å2/GM1) at the aqueous-LC interface is achieved within ∼ 24 hours of placement of the LC into contact with the aqueous dispersion of GM1; further incubation of the LC against the GM1 dispersion does not result in a measurable increase in the surface concentration of GM1 (50± 12 Å2/GM1), whereas the ordering of the LCs changes over 9 days of subsequent incubation. The above described observations led us to consider several possible physical scenarios. First, we considered it possible that the slow ordering transition of the LC was triggered by the very final stages of adsorption of the GM1, which leads to changes in surface concentrations of GM1 that lie beyond the precision of the methods we have developed to determine the surface concentrations. Second, we considered it possible that the adsorbed GM1 undergoes a slow ordering process at the interface, and that an ordered state of the GM1 triggers the ordering transition in the LC. Below, we report experiments that were performed to distinguish between these two scenarios.

GM1 Adsorption and LC Anchoring Transitions at the Aqueous-LC Interface

The first experiment we performed sought to find evidence for the second physical scenario, in which ordering of the adsorbed GM1 controls the rate of the LC ordering transition. To this end, we incubated the LC against an aqueous dispersion of 10 µM GM1 at 25 °C for 24 hours and then physically transferred the LC into a lipid-free buffer solution. Through transfer of the GM1-laden aqueous-5CB interface into the pH 7.5 TRIS buffer, we eliminated the possibility of additional GM1 adsorption. We hypothesized that if the slow ordering of the LC was due to a process that involved the ordering of adsorbed GM1 at the interface, the ordering transition of the LC in the above experiment would be similar to that shown in Figure 2. It was not. After transfer of the GM1-decorated LC interface to the lipid-free buffer, we observed the LC to remain in an orientation that was nearly parallel to the interface (at 25°C) for the subsequent ∼240 hours. This result suggests that additional adsorption of GM1 to the interface of the LC occurs during the prolonged incubation of the LC interface against the dispersion of GM1, and that the final stages of the adsorption process leading to saturation of the GM1-laden interface is a slow process that regulates the dynamics of the LC ordering transition.

The above-mentioned results suggest that the rate of adsorption of GM1 is much lower than for l-DLPC. We note that past studies of gangliosides and phospholipids have demonstrated that they differ substantially in their self-assembly behavior in bulk solution and at interfaces. The sialic acid moiety of the head group of GM1 gives the ganglioside a negative charge near neutral pH.14, 58 The resulting electrostatic interactions influence the phase behavior of GM1 both in aqueous dispersions and in monolayers formed at air-water interfaces (as evidenced in surface pressure-area isotherms and measurements of bulk phase transition temperatures (Tm, from a gel phase to a fluid phase).59 When compared with DPPC (with two saturated C16 chains),60 dilauroylphosphatidylcholine (DLPC, with two saturated C12 chains),10, 49–53 and dioleoylphosphatidylcholine (DOPC, with two symmetric unsaturated C18 chains),61 the surface pressure-area isotherm of GM1 has a larger collapse pressure and the liquid expanded (LE)/liquid condensed(LCD) phase coexistence region is observed at higher surface pressure.54 The higher collapse and LE/LCD phase coexistence pressures of the GM1 isotherm suggests that intermolecular GM1 interactions within a well-packed monolayer are more repulsive than with phospholipids, and thus the kinetics of adsorption of GM1 at the aqueous-LC interface may be slower than for DLPC and other zwitterionic phospholipids.

Langmuir-Schaefer Transfer of Monolayers of GM1 from the Aqueous-Air Interface to the Aqueous-LC Interface

The results above suggest that the slow ordering transition of the LC induced by GM1 is due to a slow rate of adsorption of GM1 to the aqueous-LC interface during spontaneous transfer from an aqueous dispersion of GM1. To further test this proposition, we hypothesized that processes that would lead to accelerated rates of transfer of GM1 onto the aqueous-LC interface would lead to more rapid ordering transitions of the LC. Here we report the use of the Langmuir-Schaefer transfer technique, as described recently by Meli et al, 10, 11 to rapidly transfer preformed, high density monolayers of GM1 onto the aqueous interface of the LC. Figure 5A shows the surface pressure-area isotherm for GM1 within a Langmuir monolayer, with data points indicating the areal densities of GM1 at which monolayers of GM1 were transferred to the interface of the LC. Figures 5B–J show optical micrographs (crossed polars) of the LC after transfer of the preformed GM1 monolayers onto the interface of the LC. Inspection of Figures 5B–E reveals that liquid-condensed monolayers of GM1 (< 53 Å2/GM1 molecule) cause homeotropic ordering of the LC within 1 min of transfer onto the interface of the LC. Tilted states of the LC were observed when the density of GM1 at the interface of the LC was between 53–58 Å2/GM1 (Figures 5F and G). Finally, liquid-condensed/liquid-expanded monolayers of GM1 (> 58 Å2/GM1 molecule) resulted in near-planar alignment of the LC. Because homeotropic ordering of the LC was observed almost immediately following transfer of the preformed densely-packed monolayer of GM1 onto the LC, these results support our hypothesis that the slow ordering transition of the LC observed during spontaneous adsorption of GM1 is due to a slow adsorption process leading to completion of the GM1 monolayer.

Figure 5.

Figure 5

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(A) Surface pressure-area isotherm measured at 25°C for a Langmuir film of GM1 formed on an aqueous subphase comprising pH 7.5 TRIS buffer. Squares depict surface pressure-area conditions under which Langmuir-Schaefer transfer to the LC interface was performed. (B to J) Polarized light micrographs of nematic 5CB after Langmuir-Shaefer transfer of GM1 monolayers (prepared at the indicated surface pressure) to the interface of the LC. Scale bar is 300 µm.

Influence of Temperature on the Adsorption of GM1 to Aqueous-LC Interfaces

Past studies have demonstrated by using differential scanning calorimetry (DSC) measurements that hydrated GM1 undergoes a broad endothermic phase transition at 26 °C 59, 62, 63 In multilayer systems formed by GM1 below the phase transition temperature, the mobility of the GM1 was low. Above the phase transition temperature, the tails of the GM1 showed increased degrees of freedom, as is typically for the so-called Lα phase.64 Because the measurements described above lead to the conclusion that the slow ordering transition of GM1 is due to a slow adsorption process, and given that our measurements were performed at 25 °C (below the above-mentioned phase transition temperature of GM1), we investigated the influence of temperature on GM1 adsorption at the LC interface. We sought to determine if the phase state of GM1 in the aqueous dispersions played a central role in the slow adsorption process that we noted above. Figure 6 reveals that the temperature of the GM1 dispersion does play a key role in the dynamics of the ordering transition. Specifically, we observed a threshold time that characterized the planar-to-homeotropic ordering transition (defined as the time required for LC within at least 6 of 40 grid squares in the Au grid to turn homeotropic) decreased dramatically above 25°C (Figure 6). We further note that the liquid crystal 5CB is nematic at 25°C, and that it undergoes a bulk phase transition from a nematic phase to an isotropic phase at 35 °C.65, 66 Because of this phase transition, the data reported in Figure 6 at temperatures above 35°C was obtained by cooling the 5CB to room temperature just before observation of the orientation of the LC. Overall, the results in Figure 6 strongly support our proposition that the phase state of GM1 plays a central role in the rate of adsorption of GM1, and thus the dynamics of the ordering transition of the LC. This result is consistent with past reports of the adsorption of phospholipids. Whereas \ dispersions of l-DLPC (Tm= −1 °C) caused nematic 5CB to undergo an ordering transition from planar to homeotropic (vesicle dispersion in pH 8.9 TBS buffer) within ∼ 2 hours, DPPC (Tm= 41 °C) (in pH 8.9 TBS buffer) did not spontaneously formed densely packed monolayers at interfaces of LCs within the experimental times investigated (days).5, 9 In this context, the dynamics of the anchoring transition of 5CB upon contact with GM1 were relatively slow compared to l-DLPC, but faster than that of pure DPPC. We conclude that the differing rates of the LC ordering transitions induced by l-DLPC, DPPC, and GM1 correlate closely with the phase states of the lipids within the lipid vesicles/micelles.5, 9

Figure 6.

Figure 6

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(A) Time required for GM1 adsorption to trigger an ordering transition in nematic 5CB, plotted as a function of the temperature of the system. The GM1 was adsorbed from a 10 µM GM1 dispersion in pH 7.5 TRIS buffer. (B) Expanded view of A at the high incubation temperatures.

CONCLUSIONS

The principal conclusions of this paper are threefold. First, by using polarized light microscopy, we have demonstrated that self-assembly of the glycolipid GM1 at the aqueous-LC interface triggers an anchoring transition in the LC that results in homeotropic ordering of the LC. Relative to ordering transitions induced by l-DLPC, however, the dynamics of the LC transition induced by GM1 are very slow. Second, although both epifluorescence microscopy and fluorimetric measurements show that a relatively high surface concentration of GM1 (52 ± 14 Å2/GM1) at the aqueous-LC interface is achieved within 24 hours of contact with aqueous dispersions of GM1, our results suggest that a slow process that leads to completion of the GM1 interfacial assembly must take place in order to trigger the ordering transition in the LC. This conclusion is supported by experiments in which the Langmuir-Schaefer technique is used to rapidly transfer preformed monolayers of GM1 to the aqueous-LC interface: rapid delivery of a densely-packed GM1 monolayer (<53Å2/GM1) of GM1 to the aqueous-LC interface led to an almost immediate transition of the LC to the homeotropic orientation. Third, we demonstrate the time-dependent ordering transitions of LCs induced by GM1 are strongly influenced by the phase state of GM1 within micelles in aqueous dispersions. At temperatures above approximately 30°C, the LC ordering transitions induced by GM1 take places on a time-scale comparable to that observed when using l-DLPC. Overall, the measurements reported in this paper provide a quantitative characterization of the dynamic and equilibrium characteristics of ordering transitions induced by GM1-decorated aqueous-LC interfaces. These results provide a foundation of knowledge that can be used to guide the design of biomolecular interfaces based on LCs at which interactions involving GM1 can be studied.

Supplementary Material

01. Supporting Information Available.

Additional experimental detail regarding the methodology used to quantify interfacial concentrations of l-DLPC, calibration plot of fluorimetric intensity versus concentration of BODIPY FL GM1/ GM1 in 2-propanol, polarized light micrographs of LC decorated with spontaneously adsorbed l-DLPC (in pH 7.5 TRIS buffer and in pH 8.9 TBS buffer), polarized light micrographs of LCs prepared at elevated incubation temperatures, and polarized light micrographs of LCs upon contact with 40µM GM1 (in pH 7.5 TRIS buffer).

ACKNOWLEDGMENTS

This research was supported by the National Science Foundation (DMR-0520527, CTS-0553760), the National Institutes of Health (CA108467), and the Quebec nature and technology research fund (Fonds québécois de la recherche sur la nature et les technologies) (M.V.M.). We also thank Nathan A. Lockwood, Kun-Lin Yang, Yan-Yeung Luk and Gary M. Koenig Jr. for their helpful discussion in preparing this report.

ABBREVIATIONS

5CB

4’-pentyl-4-cyanobiphenyl

l-DLPC

l-α-dilauroylphosphatidylcholine

DPPC

1,2-dipalmitoyl-sn-Glycero-3-phosphocholine

GM1

monosialoganglioside GalBeta1-3GalNAcBeta1-4(NeuAcAlpha2-3)GalBeta1-4GlcBeta1-1'-Ceramide

BODIPY FL-GM1

BODIPY FL- GalBeta1-3GalNAcBeta1-4(NeuAcAlpha2-3)GalBeta1-4GlcBeta1-1'-Cer.

Footnotes

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

01. Supporting Information Available.

Additional experimental detail regarding the methodology used to quantify interfacial concentrations of l-DLPC, calibration plot of fluorimetric intensity versus concentration of BODIPY FL GM1/ GM1 in 2-propanol, polarized light micrographs of LC decorated with spontaneously adsorbed l-DLPC (in pH 7.5 TRIS buffer and in pH 8.9 TBS buffer), polarized light micrographs of LCs prepared at elevated incubation temperatures, and polarized light micrographs of LCs upon contact with 40µM GM1 (in pH 7.5 TRIS buffer).

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