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Phospholipid Diffusion at the Oil-Water Interface Robert B. Walder, Andrei Honciuc, and Daniel K. Schwartz* Department of Chemical and Biological Engineering UniVersity of Colorado, Boulder, Colorado 80309 ReceiVed: June 11, 2010; ReVised Manuscript ReceiVed: July 22, 2010
Fluorescence recovery after photobleaching was used to characterize the diffusion of fluorescently labeled phospholipids at the oil/water interface for oil viscosities that varied over four orders of magnitude. Measurements were performed over a range of surface concentrations corresponding to molecular areas of 40-130 Å2/molecule. As expected, the interfacial diffusion coefficient increased with molecular area, saturating at an area of ∼100 Å2/molecule. At molecular areas below ∼80 Å2/molecule, macroscopic domains of a condensed monolayer phase were observed; the diffusion of these domains was characterized by direct tracking and trajectory analysis. For oils with viscosity e1500 cP, the diffusion coefficients of both individual probe molecules and condensed domains were consistent with a mechanism where the objects moved within the interface, experiencing drag from the adjacent bulk phases. Because this drag was dominated by the oil viscosity, the diffusion coefficients decreased proportionally to the inverse of the oil viscosity. However, for oils with higher viscosity, the diffusion coefficient of individual probe molecules decreased much more slowly. These observations suggested that two diffusive mechanisms are involved: one where surfactant molecules move within the interface and one that is analogous to the activated “hopping” processes that occur at the solid/ liquid interface. This latter mode becomes significant only for very viscous oil phases. Introduction The liquid-liquid interface is a defining characteristic for many interesting materials, including food colloids and personal care products as well as substances that are relevant to drug delivery, oil recovery, and many other applications. Rheology has traditionally been an important tool for characterizing the macroscopic mechanical properties of the liquid-liquid interface1,2 and of high interfacial-area materials such as emulsions. Microrheology has also recently emerged as a useful way to measure the micrometer scale mechanical properties of individual droplets in Pickering emulsions3 and biological cells.4 Microscopy methods, such as fluorescence microscopy, complement these mechanical studies by providing direct information about molecular mobility and interfacial structure. Biologists have used fluorescence microscopy methods to probe the dynamics of a wide variety of molecules and structures in biological cells. For example, fluorescence recovery after photobleaching (FRAP) has been successfully used to determine the diffusion rates in biological membranes.5-8 FRAP has also been used to study dynamics in surfactant multibilayers9 and monolayers10-12 at the air-water interface. However, studies aimed at understanding the dynamics of molecules at the oil-water interface are exceedingly rare. In one, Adalsteinsson and Yu9 studied the effects of interfacial concentration on the mobility of the lipid DLPC at the heptane-water interface. In the other study of which we are aware, Negishi and coworkers measured lipid diffusion at the interface of phospholipid-coated microdroplets13 using a variety of naturally occurring oils whose viscosity varied over the approximate range 1-1000 cP. They found that the interfacial diffusion coefficient decreased as η-0.85 and concluded that drag from the oil phase dominated the interfacial lipid diffusion. They suggested that the anomalous exponent (as opposed to η-1) represented a minor correction. * To whom correspondence should be addressed. E-mail: Daniel.Schwartz@ colorado.edu. Tel: 303-735-0240. Fax: 303-492-4341.
Complicating their analysis was the fact that the relatively high interfacial concentration of lipids in their experiments left the effect of nearest neighbor interactions on the measured diffusion rate as an open question. Nevertheless, these intriguing results motivated us to pursue these questions in greater detail. In the experiments reported here, we have performed FRAP experiments where the interfacial lipid concentration was varied from 40 Å2/molecule to 130 Å2/molecule, and we extended the range of oils used to encompass viscosities as large as 300 000 cP, enabling a careful analysis of the effect of viscosity and interfacial concentration on interfacial diffusion. The motion of condensed lipid domains was also observed, and diffusion constants were calculated from the mean squared displacement (MSD) versus time. By comparing the ensemble-averaged diffusion coefficient of individual molecules, from FRAP experiments, to the diffusion coefficients of micrometer scale lipid domains, we were able to develop new insight into the mechanisms of diffusion at the liquid-liquid interface. Experimental Methods Materials. N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (NBD-DPPE) was purchased from Avanti Lipids. The immersion oil FF was purchased from Cargille Laboratories. All other oils were synthetic polyalphaolefin or polybutene viscosity standards (Table 1) purchased from the Cannon Instrument Company. The oil viscosity for the immersion oil FF was measured using an ARES rheometer (TA Instruments). Sample Preparation. We cleaned a coverslip by scrubbing the surfaces with a solution of Micro-90 detergent purchased from Electron Microscopy Sciences using KimWipes purchased from Fisher Scientific. After thorough rinsing in Millipore water (18.2 MΩ cm), the coverslip was further rinsed in HPLC grade isopropyl alcohol purchased from Fischer Scientific. The isopropyl alcohol was then removed using a stream of high purity dry nitrogen gas that had been passed through molecular sieves.
10.1021/jp1053869 2010 American Chemical Society Published on Web 08/13/2010
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TABLE 1: Viscosity at 30 °C for the Various Oils Used in Samples oil name
oil viscosity (cP) at 30 °C
chemical type
N190,000 N30,000 N4,000 N1,000 immersion oil FF N26
300 000 50 000 7000 1500 120 30
polybutene polybutene polybutene polyalphaolefin aliphatic/alicyclic hydrocarbon polyalphaolefin
A small drop of oil was then deposited on the glass surface using a capillary tube, and a 100 Mesh Square TEM grid purchased from Electron Microscopy Sciences was placed on top of the oil surface using tweezers and pushed through to the glass surface using a capillary tube. In several recent studies, TEM grids have been used to stabilize hydrophobic phases (such as nematic liquid crystals) by capillarity in the presence of an aqueous phase to achieve a flat and stable interface.14-17 The lipid layer was created by Langmuir-Schaefer (LS) horizontal deposition onto the oil surface.15 A monolayer of NBD-DPPE was deposited onto the air-water interface of a Langmuir trough (model no. 312D, Nima Technology). The monolayer was left at room temperature for 20-30 min to allow for solvent evaporation. The monolayer then underwent one complete compression and decompression of the monolayer to ensure homogeneity. The monolayer was then compressed to a target area per molecule (which ranged from 40 to 130 Å2) for LS deposition. The coverslip with the oil was then loaded oil side down onto a PTFE coverslip holder, with the coverslip oriented parallel to the surface of water. The sample was then passed through the surface until the coverslip was fully submerged. Using stainless steel tweezers, we then moved the coverslip into the dipping well of the trough and flipped it so that the coverslip was oil side up. The coverslip was then moved underneath the barrier on the PTFE surface of the trough. Oil was applied to the bottom of a nylon ring, and the ring was pressed oil side down onto the glass to form a water tight seal. The sample was then moved to an inverted fluorescence microscope for the FRAP measurements. FRAP Measurements. After equilibration, the sample was placed on a heating/cooling stage (model Biostage 600, 20/20 Technology) of a Nikon inverted fluorescence microscope (model Ti-E Inverted Microscope); all samples were held at 30 °C using a standalone temperature controller (model TC-500, 20/20 Technology) for the FRAP measurements. The front aperture of the microscope was closed to form a small hole, and the sample was translated vertically until the light transmitted through the aperture formed a focused image on the fluorescent lipid layer. The sample was then exposed to light from a 120W Mercury Vapor Short Arc lamp (model C-HGFIE, Nikon), photobleaching the fluorescent lipid for ∼5 min until the intensity of fluorescence in the exposed region stopped decreasing. The front aperture was then fully opened, and a time series of images was taken for 20-90 min, depending on the time required for full recovery. Nikon Elements software was used to determine the fluorescence intensity inside and outside the photobleached region for the time series of images. A custom written Mathematica program was then used to calculate the fractional recovery curve R(t)
R(t) )
Finside(t) - Finside(0) Foutside(t) - Finside(0)
(1)
Figure 1. Representative images from fluorescence recovery time series for increasing surface concentration (top to bottom) and increasing time duration of the experiment (left to right). The darkened disk in the center of panels (A), (D), and (G) is the initial photobleached region. As time elapses after photobleaching (left to right), the region appears to grow brighter as the surrounding fluorescent lipid diffuses into the initially photobleached region. Surface concentrations were (A-C) 105, (D-F) 80, and (G-I) 40 Å2/molecule. The times were: for 105 Å2/ molecule: (A) 0, (B) 84, and (C) 360 s; for 80 Å2/molecule: (D) 0, (E) 120, and (F) 500 s; and for 40 Å2/molecule: (G) 0, (H) 840, and (I) 3840 s.
where Finside(t) is the fluorescence intensity inside the photobleached region as a function of time t and Foutside(t) is the fluorescence intensity in the region surrounding the photobleached spot as a function of time t obtained from the image data. This data was then fit to the equation18
R(t) ) A e-2τ/t[I0(2τ/t) + I1(2τ/t)]
(2)
where A is a constant, t is time, τ is the time constant, I0 is a zero-order modified Bessel function of the first kind and I1 is a first-order modified Bessel function of the first kind. The time constant τ from the fit is then used in the equation
D)
r2 4τ
(3)
to calculate the diffusion coefficient D, where r is the radius of the photobleached region. Results Figure 1 shows a representative series of images illustrating the recovery of fluorescence in a photobleached NBD-DPPE layer at various surface concentrations. At 105 Å2/molecule (Figure 1a-c), the monolayer appears to be a uniform fluorescent layer. At 80 Å2/molecule (Figure 1d-f), dark areas within the monolayer are present that appear to be condensed phase domains. These regions of closely packed lipid appear dark in fluorescence images because of self-quenching effects associated with the NBD fluorophore.19 The 40 Å2/molecule images (Figure
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Figure 3. Interfacial diffusion coefficient (O) of NBD-DPPE versus the oil phase viscosity. The expected Stokes-Einstein diffusion is represented by the dashed line. The diffusion constant for the 120 cP oil is for a surface concentration of 80 Å2/molecule. All other diffusion constants are for a surface concentration of 105 Å2/molecule. The extrapolated diffusion constant for a monolayer on 120 cP oil with surface concentration of 105 Å2/molecule is denoted by the asterisk.
Figure 2. (A) Isotherm of NBD-DPPE at the air-water interface. (B) Diffusion coefficient versus molecular area for lipid monolayers at the interface between water and 7000 cP oil.
1g-i) show a mostly condensed monolayer with some fluid phase around domain structures. These observations of condensed phases are consistent with the expected monolayer phase properties from the isotherm of NBD-DPPE at the air-water interface in Figure 2a. Qualitatively, the fluorescence recovered from the edges, with the center of the “hole” recovering last, as expected for interfacial diffusion for surface concentrations of 105 and 80 Å2/molecule. Whereas full recovery occurred for a 105 Å2/ molecule monolayer, an 80 Å2/molecule monolayer showed recovery only around the condensed phase domains, which remained dark, as expected because of self-quenching. As described above, these data were analyzed to extract the effective diffusion coefficient for each experiment. However, a monolayer deposited at 40 Å2/molecule showed only partial recovery. The recovery into the photobleached area occurred only within the “cracks” between solid-phase domains. Full recovery never occurred on the time scale of the experiments (120 min) into all of cracks that had been photobleached. These qualitative observations suggested that the local microenvironment of the monolayer is an important factor in determining the diffusion constant for the lipid monolayer. The FRAP technique did not provide useful quantitative information for the 40 Å2/molecule because of the high degree of structural heterogeneity. However, for lower surface concentrations, such as 80 Å2/molecule or 105 Å2/molecule, FRAP provided quantitative measurements (the ensemble-averaged diffusion coefficient for the lipid monolayer) to complement these qualitative observations.
FRAP experiments were performed for NBD-DPPE monolayers that were deposited at molecular areas in the range of 60-130 Å2/molecule. Figure 2B shows a plot of the measured interfacial diffusion coefficient of NBD-DPPE versus the area per molecule of NBD-DPPE for the 7000 cP oil phase. The variation in diffusion coefficient versus molecular area showed that intralayer molecular interactions had a significant impact on lipid transport at the interface. This is consistent with previous measurements of interfacial diffusion as a function of interfacial lipid concentration.9 This decrease in diffusion coefficient with increasing surface concentration is likely due to molecular crowding and to an effectively longer path length for diffusion due to the presence of condensed phase domains. For molecular areas g95 Å2, the diffusion coefficient saturated at a maximum value within experimental uncertainty. Similar behavior was previously observed.9 FRAP experiments performed as a function of oil viscosity yielded unexpected results. In Figure 3, we plot the diffusion versus viscosity for five different oil viscosities. For viscosities >120 cP, we measured diffusion constants for a monolayer with a surface concentration of 105 Å2/molecule. For the 120 cP oil viscosity, however, the fast rate of diffusion at 105 Å2/molecule made complete photobleaching problematic. However, a monolayer at 80 Å2/molecule for the 120 cP oil was successfully photobleached, and a FRAP diffusion constant was measured. We calculated an estimated value of the diffusion coefficient at 105 Å2/molecule based on the trends observed with surface concentration in experiments with more viscous oils. Specifically, we used the ratio of diffusion coefficients measured at 80 and 105 Å2/molecule, respectively, from the data in Figure 2B. This extrapolated diffusion coefficient is plotted with an asterisk in Figure 3, whereas all measured data points are plotted as open circles with error bars. The Saffmann-Delbruck model20,21 is often used to estimate enhanced drag coefficient for monolayers between two aqueous phases by the addition of the in-plane drag from the monolayer to the interfacial drag from the surrounding aqueous phases. However, this expression is appropriate in the limit where the monolayer viscosity is large compared with the viscosity of the adjacent bulk phases, which is not the case in our experiments, particularly those performed at low surface concentrations. A
Phospholipid Diffusion at the Oil-Water Interface modified hydrodynamic drag term λ ) 8(η1 + η2)R for the Einstein relation D ) kBT/λ has previously been experimentally verified22 to be an accurate model for lipid objects with radius R in a lipid monolayer between two viscous media with viscosities η1 and η2. For our case, where ηoil . ηwater, we simplify this expression to D ) kBT/8ηoilR. This expression is plotted as a dashed line in Figure 3. Interestingly, while this calculated value is in satisfactory agreement with the measured diffusion coefficient for lower oil viscosities (e.g.,
