Catanionic Vesicle−PEG−Lipid System: Langmuir Film and Phase

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Catanionic Vesicle-PEG-Lipid System: Langmuir Film and Phase Diagram Study Amir Berman,*,†,‡ Meir Cohen,† and Oren Regev*,‡,§ Departments of Biotechnology Engineering-Institute for Applied Biosciences and Chemical Engineering and Ilse Katz Center for Nanoscale Science and Technology, Ben-Gurion University of the Negev, 84105 Beer-Sheva, Israel Received November 2, 2001. In Final Form: May 7, 2002 Catanionic surfactant systems containing PEG-lipid molecules are studied at the air-solution interface and in bulk. It is found that, upon introduction of the optimum amount of PEG-lipid molecules, the region of vesicle stability in the bulk is increased. At the air-solution interface, the system forms an equimolar (salt) film upon compression. The transition to the salt structure in the presence of PEG-lipid took place at higher surface pressure, thus supporting the results of increased stability observed with the bulk samples. The presence of PEG-lipid molecules induces film buckling, resulting in significantly smaller areas per molecule. The combined results are discussed in terms of electrostatic and steric repulsion forces.

1. Introduction Novel means of drug delivery, including slow drug release, targeting, gene therapy, and others, are based on encapsulation of the active ingredients in vesicles.1-3 The vesicle stability in the body environment and its life span are prime parameters for the effectiveness of such treatments. Phospholipid vesicles doped with PEG-bearing lipids (termed PEG-lipids or lipopolymers) have been demonstrated to evade the immune system for long times; thus, they have been dubbed “stealth liposomes”.4,5 Stealth liposomes have attracted considerable attention as potential drug delivery agents because of their low or inert immune response, originating from their PEG coating, which makes them almost indistinguishable from the body aqueous environment. Although these liposomes are already in routine and exploratory clinical use, various aspects of their stability and performance are still under investigation. These liposomes can be prepared with various lipid compositions, PEG lengths, and PEG surface densities. The presence of PEG chains on the vesicle surface resembles grafted polymers on a rigid surface on one hand, while cooperatively affecting the phase behavior of the lipid-based system, on the other. The performance of mixed-surfactant systems is often superior to that of single-component surfactant systems. Hence, in industrial applications surfactant mixtures are almost always used. Catanionic mixtures are aqueous mixtures of oppositely charged surfactants that display an ability to spontaneously form stable vesicles at high dilution.6-8 * E-mails for correspondence: [email protected] and [email protected]. † Department of Biotechnology Engineering-Institute for Applied Bioscience. ‡ Ilse Katz Center for Nanoscale Science and Technology. § Department of Chemical Engineering. (1) Langer, R. AIChE J. 2000, 46 (7), 1286. (2) Barenholz, Y. Curr. Opin. Colloid Interface Sci. 2001, 6 (1), 66. (3) Cohen, M. The effect of attached PEG substitution on aggregation forms of surfactants. M.Sc. Thesis, Ben-Gurion University of the Negev, Beer-Sheva, Israel, 1998. (4) Allen, T. M.; Hansen, C.; Rutledge, J. Biochim. Biophys. Acta 1989, 981 (1), 27. (5) Allen, T. M. Trends Pharm. Sci. 1994, 15, 215. (6) Herrington, K. L.; Kaler, E. W.; Miller, D. M.; Zasadzinski, J. A.; Chiruvolu, S. J. Phys. Chem. 1993, 97, 13792.

In this study, we explore the effect of the PEG-lipid molecules on the catanionic system in the bulk and at the air-solution interface as a model system for stealth drug delivery. A possible advantage of using such a model system is its sensitive phase response to changes in various conditions such as concentration, composition, and ionic strength, which is manifested by complex phase diagrams (Figure 1). In the bulk, we studied the effect of the addition of PEGlipid molecules on the phase behavior and the stability of the vesicular region. At the air-solution interface, we formed monolayers of an analogous system and observed the changes in compression isotherms upon partial substitution of the surfactants by PEG-lipid molecules at different cationic-to-anionic molar ratios. 2. A Model System Sodium dodecyl sulfate (SDS), a single-chain anionic surfactant, and didodecyldimethylammonium bromide (DDAB), a double-chained cationic surfactant, were used in this study. The PEG-lipid DPPE-PEG2000 (where DPPE is dipalmitoylphosphatidylethanolamine) was added to the catanionic system at various molar ratios. The phase behavior of the catanionic mixture of SDS and DDAB in water has been studied in detail.9-11 Two lobes of isotropic vesicular phases exist near the water apex in the triangular phase diagram (Figure 1A). The upper and lower lobes with excess DDAB and SDS,9 correspond to regions of positive and negative net charge, respectively. We chose to study the relatively larger area of the SDS-rich lobe, where polydispersed small vesicles are found (Figure 1A).9 The phase diagram for the PEG-lipid-free bulk system depicts the domains in which stable vesicles are present (Figure 1A). In particular, the lobe marked V (also known as L412) is made up exclusively of vesicles. In the present (7) Kaler, E. W.; Herrington, K. L.; Kamalakara, M.; Zasadzinski, J. A. J. Phys. Chem. 1992, 96, 6698. (8) Khan, A.; Marques, E. Catanionic Surfactants; Robb, I. D., Ed.; Blackie Academic and Professional: London, 1997; p 37. (9) Marques, E.; Regev, O.; Khan, A.; Lindman, B.; Miguel, M. J. Phys. Chem. B 1998, 102, 6746. (10) Marques, E.; Regev, O.; Khan, A.; Miguel, M.; Lindman, B. J. Phys. Chem. B 1999, 103, 8353. (11) Regev, O.; Marques, E. F.; Khan, A. Langmuir 1999, 15, 642. (12) Regev, O.; Guillemet, F. Langmuir 1999, 15, 4357.

10.1021/la011633+ CCC: $22.00 © 2002 American Chemical Society Published on Web 06/26/2002

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Figure 1. Phase diagrams of the dilute region of the SDSDDAB system: (A) triangular phase diagram of PEG-lipidfree system,9 (B) detailed orthogonal phase diagram of PEGlipid-free system, (C) 5 mol % substituted DPPE-PEG2000, and (D) 8 mol % substituted DPPE-PEG2000 at 25 °C. V ) vesicle phase, LR ) lamellar phase, S ) catanionic crystals, and M ) micelles. Bars above and below correspond to lower- and higherdensity phases, respectively. When no bar is used, the precipitate phase is dispersed.

study, PEG-lipids were added to mixed-surfactant system of various compositions within and in the vicinity of the vesicle phase (V). The different phases were assigned by ocular inspection. Expansion or contraction of the vesicle phase boundaries in the phase diagram upon addition of PEG-lipid is a manifestation of enhancement or reduction in vesicle stability, respectively. Analogous monolayer system was used to gain quantitative information on organization through the area per molecule and phase transition during isothermal compression. Conclusions based on both the “bulk” and the “interface” situations are drawn. 3. Experimental Section 3.1. Materials. SDS, DDAB, and DiTAB (ditetradecyldimethylammonium bromide) was purchased from Fluka, and chloroform was obtained from Aldrich. DPPE-PEG2000 [1,2dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-poly(ethyleneglycol)2000] was purchased from Northwestern Lipids, Inc. All compounds were used without further purification. Ultrapure water was used (18 MΩ). 3.2. Bulk Sample Preparation. Stock solutions of DDAB and SDS were prepared. Samples were then mixed by volume, depending on the required composition. The PEG-lipids were then added and stabilized at 25 °C. 3.3. Phase Diagram Determination. Samples were equilibrated for several days at 25 °C prior to the examination of the phases. Samples were checked between two crossed polarizers to identify the existence of anisotropy precipitates (lamellar phase). Precipitates were found either floating on top or sunk at the bottom of the test tube, reflecting the different relative densities of the separated phases. Similar behavior was reported for systems without PEG-lipid.12 Bluish samples in the vesicular region were monitored for a few months to check for any phase separation phenomena, such as flocculation or crystal precipitation. 3.4. Phase Diagram Presentation. The conventional triangular representation of the dilute region in the ternary phase diagrams (Figure 1A) was modified to an orthogonal representation for simplicity (Figure 1B). Here, the origin is the water corner in the ternary phase diagram. The weight ratios in the triangular diagram were transformed to molar concentrations. This gives a more accurate presentation, especially when high-molecularweight PEG-lipid molecules are used.

Berman et al. 3.5 Monolayer Studies at the Air-Solution Interface. Monolayer films were prepared on a Langmuir film balance (NIMA, U.K., with symmetric compression, model 601, dimensions 500 × 70 mm2) having a liquid volume of 230 mL. DiTAB (25 nmol, 1.5 × 1016 DiTAB molecules) was spread from a 2 mM chloroform solution. Compression isotherms were measured at 25 °C at 20 cm2/min. Aqueous subphase solutions were prepared with SDS. All experiments were allowed to stand for 15 min for solvent evaporation and relaxation before compression. The kinetic aspects of such systems have recently been extensively studied in terms of pressure and area relaxation.13 In monolayer studies, DDAB was replaced by DiTAB. These compounds share similar molecular features, with DiTAB being less water-soluble, thus forming more stable films at the airwater interface.14 The effect of the slightly longer hydrocarbon chains does not change the limiting area upon 2D compression.14 DiTAB was spread over the SDS-containing subphase. The SDS concentration in the subphase ranged from pure water to 50 000 nM SDS. The upper limit concentration was chosen to be well below the critical micelle concentration (CMC) of SDS. Because of its higher water solubility, SDS is partitioned between the monolayer and the subphase; hence, it is impractical to spread lipid mixture in the desired final ratio. For the same reason, the area in the isotherm was calculated and expressed per DiTAB molecule (almost insoluble in water), as the amount of DTAB at the air-solution interface is known. In the present work, we do attempt not to report the exact value of the SDS concentration in the monolayer,15 but rather to show the qualitative effect of increased SDS amount in the monolayer. Throughout this study, a fixed total amount of (DiTAB + PEG-lipid) was spread. In experiments where PEG-lipid was used, the DiTAB amount was lowered accordingly. Each compression experiment was repeated at least three times. The results were found to be reproducible.

4. Results and Discussion 4.1. Bulk Phase Diagrams. The documented SDSDDAB-water phase diagram is used as a reference for our observations (Figure 1A). Our phase diagram determination (Figure 1B) for the PEG-lipid-free system reproduced the reported phase behavior of Marques et al.9 (Figure 1A). In this phase diagram, diagonal lines originating from the origin in Figure 1 are of constant molar fraction, defined as XDDAB ) CDDAB/(CSDS + CDDAB). CSDS and CDDAB are the molar concentrations of SDS and DDAB, respectively. SDS colorless solution (with no DDAB) consists of spheroidal micelles.16 With increasing DDAB concentration, a phase transition from spheroidal micelles to disklike micelles was reported below CSDS ) 17 mM.9 The bluish vesicle phase (V) extends in the region of 0.13 < XDDAB < 0.34 and CSDS < 40mM. At higher SDS concentrations, a phase separation occurs, with precipitation of a lamellar phase at the bottom (LR) or at the top (LR) of the test tube, below or above the vesicle lobe, respectively. For 0.34 < XDDAB < 0.39, a three-phase region of vesicle, lamellar, and catanionic crystals (S) is found. At higher DDAB mole fraction and below the equimolar line, catanionic crystal precipitation occurs, and vesicle solutions are found. For a given phase Y, Y h and Y are defined as precipitate at the top and bottom of the test tube, respectively. The phase diagrams of 5 mol % (Figure 1C) and 8 mol % (Figure 1D) substituted PEG-lipid were compared with the phase diagram of the PEG-lipid-free system (Figure (13) Viseu, M. I.; da Silva, A. M. G.; Costa, S. M. B. Langmuir 2001, 17, 1529. (14) Dynarowicz, P.; Romeu, N. V.; Trillo, J. M. Colloids Surf. A 1998, 131, 249. (15) da Silva, A. M. G.; Viseu, M. I.; Campos, C. S.; Rechena, T. Thin Solid Films 1998, 320, 236. (16) Su¨ss, D.; Cohen, Y.; Talmon, Y. Polymer 1995, 39, 1809.

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1B). It was found17,18 that the effect of PEG-lipids on similar bulk vesicular system is negligible below substitution of about 10 mol % of PEG-lipid. Because our highest PEG-lipid substitution is 8%, we do not expect substantial supramolecular changes in vesicle structure. It should be mentioned that, above 10 mol % substitution, the PEGlipid molecules induce collapse of the vesicles to disks and globular micelles. It is observed that the phase diagrams are strongly affected by the substitution of the catanionic surfactants by PEG-lipid. This is expressed by shifted boundaries between phases. The region of stable vesicles for the 5 mol % PEG-lipid system is increased by 20% with respect to that of the PEG-lipid-free system. For the 8 mol % PEG-lipid system, this region is decreased by 50% with respect to the PEG-lipid-free system. For the 5 mol % PEG-lipid-containing system, the area of the vesicle phase grows at the expense of the V + LR +S h region at lower total surfactant concentration [Ct () CSDS + CDDAB) < 40 mM]. The presence of PEG-lipids induces steric repulsion, thus hindering the formation of solid precipitates. Moreover, above CSDS ) 28 mM, V + LR and V + LR regions substitute part of the V + LR + S h phase, which further indicates that (PEG-lipid)-catanionic complex stabilizes the lamellar structure. The overall conclusion is that PEG-lipid molecules stabilize the lamellar structure and reduce the formation of solid precipitates In the 8 mol % PEG-lipid-containing system, the trend of increasing the vesicle region is reversed, and the area of the vesicle region is decreased. We assume that an optimum is obtained for the PEG-lipid content in the mixed system. 4.2 Langmuir Film Study. 4.2.1. PEG-Lipid-Free System. In this work, the π-A isotherms are plotted as pressure vs area per DiTAB molecule (instead of vs the mean molecular area as is usually done) to avoid uncertainty because of the high water solubility of SDS, as mentioned before. Characteristic compression isotherms on subphases ranging from 0 to 50 000 nM SDS are shown in Figure 2. Compression isotherms show one phase transition. They change over a range of SDS subphase concentrations. The observations are discussed according to three SDS concentration regions: Region I. Low SDS Concentration: 0-450 nM SDS. From 0 to 450 nM SDS in the subphase, all isotherms exhibit liquid-expanded-type surface pressure isotherms with no clear transition from expanded to condensed monolayer (Figure 2a). The limiting area is determined at the calculated point of minimum copressibility [C ) -(1/A)∂A/∂π], with the area per molecule axis (Figure 2b). The results show a small shift, from 120 to 115 Å2/molecule, with increasing SDS concentration, in agreement with the compression isotherms reported by Dynarowitcz and co-workers.14 A slight increase in SDS subphase concentration results in a lower area per DiTAB molecule because the SDS molecules intercalate into the liquid-expanded DiTAB monolayer and screen the electrostatic repulsion between the charged monolayer molecules19 (Figure 2a). Increased stability of the monolayer with increasing SDS concentration in the subphase can be deduced from the increased slope of the isotherms around 40 mN/m. (17) Edwards, K.; Johnsson, M.; Karlsson, G.; Silvander, M. Biophys. J. 1997, 73, 258. (18) Woodle, M. C.; Lasic, D. D. Biochim. Biophys. Acta 1992, 1113, 171. (19) da Silva, A. M. G.; Viseu, M. I. Colloids Surf. A 1998, 144, 191.

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Figure 2. (a) π-A isotherms of DiTAB monolayers compressed over subphases containing 0-50 000 nM SDS at 25 °C: (b) pure water, (0) 50 nM SDS, (4) 400 mM SDS, (O) 500 nM SDS, ()) 625 nM SDS, (2) 750 nM SDS, (1) 1250 nM SDS, (() 5000 nM SDS, (9) 50 000 nM SDS. (b) π-A isotherm of DiTAB over 750 nM SDS (top), illustrating the determination of the limiting areas AS and AL. The intercepts of the tangents to the compression isotherm at the compressibility minima (bottom) were defined as the limiting areas AS and AL.

Region II. Intermediate SDS Concentration: 500-1250 nM SDS. Upon increasing the SDS concentration to between 500 and 1250 nM, a transition to a “solid” phase (DTA-DS salt) is observed at about 20 mN/m, resulting in a steeper slope in the compression isotherm. Compression isotherms in this region (Figure 2a) present first a liquidlike phase with a high limiting area per molecule, AL, preceded by a transition zone and a solid phase with a lower limiting area, AS (Figure 2a). The limiting areas were determined as described before, with the difference that two minima in compressilbility were used (Figure 2b). The solid-phase limiting area (AS) ranges from 95 Å2/DiTAB molecule at 500 nM SDS to 76 Å2/DiTAB for SDS concentrations of 1250 nM and higher. A shoulder manifests the “liquid-solid” phase transition. This shoulder is likely a result of a process in which excess SDS (above an equimolar SDS/DiTAB ratio) molecules are continuously being excluded from the mixed monolayer upon compression. AL gradually increases with increasing SDS subphase concentration. Region III. High SDS Concentration: >2500 nM SDS. The liquid-phase limiting area (AL) increases gradually with increased SDS concentration in the subphase up to 130 Å2/DiTAB molecule at 50 000 nM SDS (Figure 2). The

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Figure 3. Summary of expanded, AL (O), and condensed, AS (∇), phase limiting areas for the entire range of SDS subphase concentrations for the PEG-lipid-free system. The lines are a guide to the reader eyes. The error bars were obtained from the results of at least three independent measurements.

solid-phase limiting area (AS), however, remains nearly constant at 75 Å2/DiTAB molecule, corresponding to the salt structure.19 The three regions mentioned above for the PEG-free system are shown in Figure 3, where AL and AS values derived from Figure 2 are plotted vs the SDS subphase concentration. In the absence of SDS as a countercharge surfactant, electrostatic repulsion dominates the interaction between DiTAB molecules. Increasing SDS concentration is manifested first in a decrease in the AL value (region II), along with the introduction and reduction of AS until a minimum value of 75 Å2/DiTAB molecule is reached (Figure 3). This is followed by a slight increase in AL at higher SDS concentration (region III), indicating that more SDS is accommodated in the film at low surface pressure. The lower limiting area (AS) corresponds to 1:1 mixture of DiTAB and SDS. With further increases in the SDS concentration in the subphase, the AS values remain unchanged. In region I, one finds only values for AL, because no phase transition takes place and the films retain their fluid character. The transition to region II is characterized by the appearance of AS. A decrease in AS values in this region is attributed to more effective screening at the interface. Region III is therefore defined as a closely packed monolayer for which no change in AS is obtained. We show that the area per DiTAB molecule decreases with increasing SDS concentration for region I (Figure 3). This trend is attributed to an electrostatic screening effect. At higher SDS concentrations, the penetration of SDS molecules into the monolayer becomes dominant, which results in a slight increase in the area per DiTAB molecule for regions II and III. Interpretation of the increase in AL in regions II and III is as follows. At low surface pressure, two opposite processes in the liquid phase are likely to occur. On one hand, SDS molecules from the subphase penetrate into the expanded DiTAB monolayer and increase the apparent area per DiTAB molecule. On the other hand, the addition of SDS molecules increases the ionic strength of the system and drives the monolayer into more condensed packing. Therefore, the observed slight increase in AL (in regions II and III) of ca. 10% [(130 - 115)/115 ) 0.13] is likely the outcome of a small advantage of the former over the latter opposing processes.

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For CSDS > 1250 nM (region III), a solid behavior is observed at surface pressures higher than ca. 20 mN/m (Figure 2). This is likely the salt DTA-DS in a stoichiometric 1:1 ratio. The measured limiting area in the solid phase (AS), 75 Å2/DiTAB molecule (Figure 3), includes two DiTAB chains and one SDS chain. The area per alkyl chain is therefore 25 Å2/chain. This results in a slightly higher value than reported for fully compressed fatty acids measured from the compression isotherm and grazing incidence diffraction to be 19 Å2/molecule.20,21 Onset of the Solid Phase at Low SDS Concentration. The following calculation illustrates that an equimolar film could indeed form even at very low SDS concentration (e.g., region II): The number of SDS molecules, NSDS, in the subphase is NSDS ) VCSDSNA, where V is the trough volume, CSDS is the SDS concentration in the subphase, and NA is Avogardo’s number. NSDS ) 6.9 × 1015 for CSDS ) 50 nM and 6.9 × 1016 for CSDS ) 500 nM. The compressed monolayer occupies only ca. 30% of the trough surface. Therefore the number of SDS molecules below the film for 50 < CSDS < 500 nM is [(6.9 × 1016) × 0.3 )] 2 × 1015 < 0.3NSDS < 2 × 1016. We spread 1.5 × 1016 DiTAB molecules at the interface to form the monolayer. Hence, the assumption of an equimolar film is indeed reasonable for CSDS > 500 nM. Our results suggest that, above this concentration, a condensed monolayer is, indeed, formed. Da Silva et al.15 studied the analogous system of oppositely charged DODAB (dioctadecyl bromide) and SHS (sodium hexadecyl sulfate). Using different spreading techniques and relaxation times, they demonstrated that the film composition converged to the salt (equimolar) composition. 4.2.2. PEG-Lipid-Containing System. PEG-lipid molecules were added to DiTAB in the chloroform spreading solution. The area was recorded per known amount of (PEG-lipid + DiTAB) mixture solution. The PEG-lipid and DiTAB solutions were compressed on subphases with various SDS concentrations (Figures 4 and 5). The analysis of the results in this section describes the effect of the PEG-lipid concentration on the structure of the compression isotherm in different pressure regions. Low Pressure: Interchain PEG Interaction. Upon addition of PEG-lipid to the monolayer, we observe (Figure 4) the emergence of a new shoulder at a low π value (about 10 mN/m, in the liquid-expanded phase of the compression isotherm) in addition to the one shown in Figure 2. This shoulder originates from an adjacent “pancake-like” PEG conformation, which undergo a transition to a “mushroom-like” structure.22 The shoulder position shifts to higher area with increasing PEG-lipid content in the film for all subphase concentrations (see, for example, the arrows in Figure 4d). Comparing the SDS-free isotherms of films with different PEG-lipid contents (Figure 4a), we find that, at low pressure (π < 10 mN/m), the isotherm of the 5 mol % PEG-lipid system overlaps with the pure DiTAB isotherm but forms a shoulder at π ) 12 mN/m and A ) 110 Å2. Therefore, it occupies a significantly lower area per DiTAB or PEG-lipid molecule compared with that of the PEGlipid-free system. In contrast, the 8 mol % PEG-lipid isotherm presents a shoulder at higher area per molecule (π ) 10 mN/m, A ) 130 Å2). This confirms the observation (20) Wennerstro¨m, H.; Evans, F. The Colloidal Domain; VCH: New York, 1994. (21) Bohm, C.; Leveiller, F.; Jacquemain, D.; Mohwald, H.; Kjaer, K.; Als-Nielsen, J.; Weissbuch, I.; Leiserowitz, L. Langmuir 1994, 10, 830. (22) Majevski, J.; Kuhl, T. L.; Gerstnberg, M. C.; Israelachvili, J. N.; Smith, G. S. J. Phys. Chem. B 1997, 101.

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Figure 5. π-A isotherms of DiTAB and (a) 0, (b) 5, and (c) 8 mol % PEG-lipid monolayer compressed over subphase concentration (CSDS) of 0 (no symbol), 500 (O), 1250 (0), and 50000 ()) nM SDS at 25 °C.

Figure 4. π-A isotherms of DiTAB and 0, 5 and 8 mol % PEG-lipid-substituted monolayer compressed over subphase containing (a) 0, (b) 500, (c) 1250, and (d) 50 000 nM SDS at 25 °C. The arrows in d indicate the transition of PEG to a mushroom-like conformation for 5 and 8 mol % PEG-lipidsubstituted monolayers. PEG-lipid concentration: 0 (4), 5 mol % PEG-lipid (0), and 8 mol % PEG-lipid (O).

that neighboring PEG chains are interacting.22 Because the area per DiTAB molecule is about 130 Å2 at π ) 10 mN/m (for the 8 mol % system) and the surface dilution is about 1:12.5 (8:100), the neighboring PEG chains start to interact with each other when a PEG chain occupies an area of 12.5 × 130 ) 1625 Å2. This simplistic calculation is in rough agreement with an area calculation based on the PEG2000 radius of gyration measured by Kuhl and coworkers23 and Wiessenthal,24 who found similar values for area per polymer molecule in a pure PEG-lipid system. When PEG-lipid molecules are diluted to 5 mol %, the available area per PEG molecule is correspondingly larger, and therefore, under these conditions, the molecules do not interact at low pressure, as seen from the overlap with the pure isotherm at π < 10 (Figure 4a). (23) Kuhl, T. L.; Leckband, D. E.; Lasic, D. D.; Israelachvili, J. N. Biophys. J. 1994, 66, 1479. (24) Wiesenthal, T.; Baekmark, T. R.; Merkel, R. Langmuir 1999, 15 (20), 6837.

Intermediate Pressure: Transition from an Expanded to a Condensed Phase. The addition of SDS in the subphase (Figures 4 and 5) resulted in the same sequence of events as for the PEG-free system (Figures 2 and 5). A slight increase in the pressure of the liquid-to-solid transition shoulder (e.g., from 18 to 23 mN/m for CSDS ) 1250 nM) is detected with increasing PEG-lipid concentration from 0 to 5 mol % (Figure 4c,d). This could originate from additional steric repulsion induced by the PEG chains. At 8 mol % PEG-lipid, the pressure is slightly reduced to 21 mN/m for CSDS ) 1250 nM. The increase in the surface pressure at which the transition to salt takes place is indicative of the stabilizing effect of the PEG-lipid molecules on the monolayer. High Pressure: Buckling. The most evident effect in the isotherms is the reduction of the area per DiTAB molecule in the PEG-lipid-containing systems for all subphase compositions. For the SDS-free system, we observe that isotherms containing PEG-lipid molecules coincide at lower area per DiTAB or PEG-lipid molecule compared to the PEGfree system (Figure 4). This could indicate that either PEG-lipid molecules were extruded into the subphase or the monolayer was deformed. The area per DiTAB or PEG-lipid molecule for the PEG-lipid-containing systems is reduced by about 25% with respect to a PEGlipid-free system (Figure 4). Kuhl et al. and others22,23,25 have reported film protrusion for systems containing DSPE/PEG-lipid and pure (25) Majevski, J.; Kuhl, T. L.; Gerstnberg, M. C.; Als-Nielsen, J.; Israelachvili, J. N.; Smith, G. S. J. Am. Chem. Soc. 1998, 120 (7), 1469.

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PEG-lipid, based on surface force apparatus and X-ray reflectivity studies. In their studies, the limiting area (AS) in the presence or absence of PEG-lipid was not changed. In contrast, in our system, a substantial reduction in AS was detected in monolayers when PEG-lipid was present. Possible reasons for the observed differences between our work and Kuhl et al.’s might arise from differences in the lipid alkyl chain length (reflected in Tm, the chain melting temperature) and the experimental temperature: 12/14/16-hydrocarbons at 25 °C (above Tm, liquidcrystalline state) for our work vs 18-hydrocarbons at 21 °C (below Tm, gel/frozen state) for the Kuhl et al. work. In addition, we used a catanionic system, whereas Kuhl et al. used a zwitterionic system. The outcome of these differences is a more flexible and compliant monolayer, which could maintain a higher amplitude of undulation. This is most probably expressed by the 25% reduction (Figure 4) in the area per molecule. Considering this, we argue that the PEG-lipid molecules are retained in the monolayer because its exclusion would have decreased the area per molecule by only 5% or less (because of the presence of SDS). We note that there is a small but consistent difference in the solid-phase limiting area between the 5 and 8 mol % PEG-lipid. The slightly higher area for the 8 mol % system originates from the steric repulsion between the PEG chains at the respective concentrations. It also corroborates our argument that PEG-lipid is not excluded from the monolayer: Had it been excluded, AS for 8 mol % PEG-lipid would have been smaller than that for the 5 mol % PEG-lipid. 5. Concluding Remarks In this paper, we have presented a complex lipid system that encompasses various elements that have been studied

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elsewhere, namely, catanionic surfactant mixtures and PEG-lipids. Catanionic surfactants and PEG-lipidcontaining systems have been studied separately before in the bulk or at the air-solution interface. Here, we present a combined approach that brings together both geometries. It was found that the incorporation of the PEG-lipid with the catanionic surfactants increases the system stability through a delicate interplay between steric, hydrophobic, and electrostatic interactions. In the bulk, the increased stability induced by the presence of the PEG-lipid is manifested by a larger vesicle region on the phase diagram (Figure 1). At the interface, it is observed that the transition from the liquid-expanded phase to the solid monolayer phase takes place at higher pressure (Figure 4). In this study, we present a combined approach of measurements of comparable systems in the bulk and at the interface. Lessons learned from the interface part can help in understanding the bulk behavior. The superposition of the PEG-lipid system with the catanionic system, separately studied before by others, makes a more stable system because of electrostatic stabilization of PEG-lipid vesicles.

Acknowledgment. This research was supported by The Israel Science Foundation founded by the Israeli Academy of Science and Humanities. Kareem Albador is acknowledged for excellent technical help. LA011633+