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Langmuir 2004, 20, 3284-3288
Monolayer Behavior of 1,2-Dipalmitoylgalloylglycerol, a Synthetic Lipid with Strong Cohesive Properties Rolf Schmidt and Christine E. DeWolf* Department of Chemistry and Biochemistry, Concordia University, 7141 Sherbrooke St. West, Montre´ al, Que´ bec, Canada H4B 1R6 Received November 7, 2003. In Final Form: January 14, 2004 Monolayers of 1,2-dipalmitoylgalloylglycerol (DPGG) were investigated at the air-water interface. The monolayers exhibit high rigidity which leads to the formation of surface tension gradients in the film. Transfer to solid substrate yields homogeneous Langmuir-Blodgett films with low surface roughness. Large numbers of aggregates were observed by Brewster angle microscopy and imaging ellipsometry at relatively high molecular areas. At all pressures, the DPGG molecules adopt conformations corresponding to low tilt angles. Constant area measurements result in a pressure increase as the film rearranges to maximize the intermolecular interactions. An optimal intermolecular distance required for the formation of a hydrogen-bond network between headgroups is proposed to explain the observed, highly cohesive monolayer behavior.
Introduction 1,2-Dipalmitoylgalloylglycerol (DPGG) is a diacyl glycerol-based lipid with a polyphenolic headgroup. There is a vast literature describing the ability of polyphenols to bind and precipitate proteins/peptides and to act as antioxidants (for a comprehensive review of natural and synthetic polyphenols, see reference 1 and references therein). It has been suggested that bilayers of this new synthetic lipid might be able to tether proteins, membranes, or cells to different substrates (such as glass slides or medical implants).2 Similarly, monolayers of DPGG could prove useful for the production of biocompatible coatings or biosensing applications; hence, it is important to understand the phase behavior of this synthetic lipid. Furthermore, it has been shown that bilayers of DPGG exhibit a high transition enthalpy (gel-to-lamellar) and temperature (Tm) as well as strong interbilayer adhesion,2 as also seen for phosphatidylethanolamines3 and cerebrosides.4 It has been proposed for DPGG that these properties likely arise from the strong attractive interactions between gallic acid headgroups. In addition to the strong interbilayer interactions (potentially through water bridges), there is a high potential for intermolecular hydrogen bonding in the plane of the bilayer.2 It has previously been suggested that these interactions could inhibit the lateral expansion of the bilayer required for the gel-to-lamellar transition, thus contributing to an increased Tm.5 In the current study, we used the monolayer phase behavior of DPGG at the air-water interface in order to understand the influence of polyphenolic headgroup interactions on the lateral cohesion present in lipid films. * To whom correspondence should be addressed. E-mail:
[email protected]. (1) Handique, J. G.; Baruah, J. B. React. Funct. Polym. 2002, 52, 163-188. (2) Pollastri, M. P.; Porter, N. A.; McIntosh, T. J.; Simon, S. A. Chem. Phys. Lipids 2000, 104, 67-74. (3) McIntosh, T. J.; Simon, S. A. Langmuir 1996, 12, 1622-1630. (4) Kulkarni, K.; Snyder, D. S.; McIntosh, T. J. Biochemistry 1999, 38, 15264-15271. (5) Nagle, J. F. Ann. Rev. Phys. Chem. 1980, 31, 157-195.
Figure 1. Schematic diagram showing placement of pressure sensors A and B.
Materials and Methods 1,2-Dipalmitoylgalloylglycerol (DPGG) with a purity >99% was obtained from Avanti Polar Lipids, Inc. (Alabaster, AL) and used without further purification. Ultrapure water (resistivity ) 18.3 MΩ‚cm) was obtained from an EasyPure II LF system (Barnstead, Dubuque, IA). Silicon wafers were purchased from Wafer World Inc. (West Palm Beach, FL) and cleaned using the following procedure: substrates were immersed in a 1:1 mixture of concentrated HCl and methanol for 30 min, rinsed abundantly with ultrapure water, immersed in concentrated H2SO4 for 30 min, rinsed abundantly with ultrapure water, and stored in ultrapure water. Storage times never exceeded 1 h. Silicon substrates were blown dry with clean air before use. Monolayers of DPGG were spread from chloroform solutions (1 mM) on a subphase of ultrapure water (pH ) 5.3-6.3 at 25 °C). Surface pressure-area isotherms were obtained on thermostated Langmuir film balances (Nima Technology Ltd., Coventry, U.K.) at 15, 20, 25, 30, and 35 °C with a compression speed of 5 cm2‚min-1 (equivalent to approximately 3 Å2‚ molecule-1‚min-1). Surface pressure measurements were made using a filter paper Wilhelmy plate. Monolayers were transferred at a constant pressure of 35 and 11.5 mN‚m-1 onto silicon wafers and freshly cleaved mica on the upstroke using the LangmuirBlodgett technique with a dipping speed of 0.2 cm‚min-1. All transfers were carried out under symmetric compression, and transfer ratios were always close to 1. To investigate surface pressure gradients in the spread film, a control experiment with two independent pressure sensors was performed. Pressure sensor A was positioned at the far end of the trough (i.e., furthest from the movable barrier), and pressure sensor B was positioned roughly in the middle of the trough (Figure 1). This enabled measurement of the pressure gradient while maintaining maximal film compression. Brewster angle microscopy (BAM) and ellispometry measurements were carried out with an I-Elli2000 imaging ellipsometer (Nanofilm Technologie GmbH, Go¨ttingen, Germany) equipped with a 50 mW Nd:YAG laser (ν ) 532 nm). All experiments were
10.1021/la036099f CCC: $27.50 © 2004 American Chemical Society Published on Web 03/19/2004
Monolayer Behavior of 1,2-Dipalmitoylgalloylglycerol
Langmuir, Vol. 20, No. 8, 2004 3285
Figure 3. Surface pressure-area isotherms for symmetric (s) and asymmetric (- - -) compression at 25 °C.
DPGG forms rigid monolayers at the air-water interface, and surface pressure-area isotherms for DPGG were recorded on water as the subphase at five different temperatures: 15, 20, 25, 30, and 35 °C (Figure 2). The overall shape of these isotherms is similar for tempera-
tures up to 30 °C with a critical area (corresponding to the onset of a pressure increase, to be referred to as the liftoff) in a range of 55-60 Å2‚molecule-1 and a condensed phase at all surface pressures above 0 mN‚m-1. The collapse for all temperatures lies in the range from 37 to 40 Å2‚molecule-1 and 44 to 53 mN‚m-1. At 35 °C, a plateau appears between 55 and 100 Å2‚molecule-1 corresponding to a phase transition between a liquid-expanded and a condensed phase. Surface pressure gradients have previously been reported for films displaying high rigidity.8,9 Figure 3 shows the comparison of DPGG isotherms for symmetric and asymmetric compression at 25 °C. The surface pressure-area isotherm of DPGG under symmetric compression exhibits a lift-off at 60 Å2‚molecule-1 and a collapse at 39 Å2‚molecule-1, whereas for asymmetric compression the lift-off occurs at 46 Å2‚ molecule-1 and is much steeper while the apparent ‘collapse’, beginning at about 41 Å2‚molecule-1, is more extended. Hence, for asymmetric compression, the true collapse area and lateral collapse pressure (30-40 mN‚m-1) are hard to determine. During the asymmetric film compression, the Wilhelmy plate is pushed out of the vertical position (to approximately 10° from the normal). This can already be detected at 0 mN‚m-1 at molecular areas nearing the critical area. This was not observed for the symmetric compression. Control experiments with two pressure sensors (Figure 4, see Methods and Materials section for details) clearly show that upon compression the surface pressure recorded varies with the position of the pressure sensor in the trough. The surface pressure begins to increase at pressure sensor B (see inset) before any surface pressure is registered at A, hence the sharper lift-off under asymmetric compression. Compression beyond this point yields higher surface pressures for a given molecular area nearest the point of compression (B) than further from the barrier (A). Thus, the compression creates a surface pressure gradient which persists over long time periods (compared to other lipids), due to the extreme rigidity of the film. In fact, upon compression of the film to 45 Å2‚molecule-1, a surface pressure gradient is still being registered 4000 s after stopping the compression (Figure 4). Furthermore, at the extreme, the differences in compression state can range up to 50 mN‚m-1 at either end of the film (as
(6) Blaudez, D.; Buffeteau, T.; Cornut, J. C.; Desbat, B.; Escafre, N.; Pezolet, M.; Turlet, J. M. Appl. Spectrosc. 1993, 47, 869-874. (7) Blaudez, D.; Buffeteau, T.; Cornut, J. C.; Desbat, B.; Escafre, N.; Pezolet, M.; Turlet, J. M. Thin Solid Films 1994, 242, 146-150.
(8) Dhathathreyan, A.; Mo¨bius, D. J. Colloid Interface Sci. 1995, 172, 358-360. (9) Alexandre, S.; Lafontaine, C.; Valleton, J.-M. J. Biol. Phys. Chem. 2001, 1, 21-23.
Figure 2. Surface pressure-area isotherms for DPGG monolayers at various temperatures under symmetric compression: 15 (- 2 -), 20 ° (- - -), 25 (‚ ‚ ‚), 30 (- / -), and 35 °C (s). Inset shows chemical structure of DPGG. performed using a 20× magnification with a lateral resolution of 1 µm. BAM experiments were performed at an incident angle of 53.15° (Brewster angle of water) and a laser output of 50% (analyzer, compensator, and polarizer were all set to 0). Ellipsometric measurements were carried out at an incident angle of 50.00° and a laser output of 100% with the analyzer and compensator set to 20.00°. To determine the optical thickness of the lipid monolayer at the air-water interface, the following two-box optical model was used: water as substrate (n ) 1.332, k ) 0), a generic organic film as the lipid monolayer (n ) 1.46, k ) 0). The reported thickness is an average of 10 measurements each taken at a different location on the same film and is consistent for multiple samples. The ellipsometric isotherm is reported in terms of δ∆, which is independent of the optical model (contrary to the film thickness). δ∆ is defined as the difference between the ellipsometric angle ∆ of the film on the subphase and the subphase alone (δ∆ ) ∆film - ∆subphase). For preliminary estimations of tilt angle changes in the monolayer, the refractive index was assumed to remain constant. Polarization modulationinfrared reflection absorption spectroscopy (PM-IRRAS) experiments at the air-water interface were carried out at the Universite´ Laval, Que´bec City, Canada. The experimental setup has been described elsewhere.6,7 Prior to taking the PM-IRRAS measurements, the film was held at constant pressure (8 and 25 mN‚m-1) for 30 min. The incident angle was set to 75° and the photoelastic modulator frequency to 1450 cm-1. Eight hundred scans were acquired at a resolution of 4 cm-1. The PM-IRRA spectra presented have been normalized (S(d) - S(0))/S(0). A Nanoscope IIIa (Digital Instruments, Santa Barbara, CA) was used to capture AFM images in air at room temperature using tapping mode at a scan rate of 1 Hz using etched silicon cantilevers (RTESP NanoProbe, Veeco, Santa Barbara, CA) with a resonance frequency of ∼300 kHz, a nominal spring constant of 20-80 N/m, and tip radius of
