Surface Properties of Dioleoyl - American Chemical Society

Feb 11, 2006 - EVanston, Illinois 60208-3500, and Hormel Institute, UniVersity of Minnesota, Austin, Minnesota. ReceiVed September 8, 2005. In Final F...
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Langmuir 2006, 22, 2770-2779

Surface Properties of Dioleoyl-sn-glycerol-3-ethylphosphocholine, a Cationic Phosphatidylcholine Transfection Agent, Alone and in Combination with Lipids or DNA Robert C. MacDonald,*,† Alex Gorbonos,†,‡ Maureen M. Momsen,§ and Howard L. Brockman§ Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern UniVersity, EVanston, Illinois 60208-3500, and Hormel Institute, UniVersity of Minnesota, Austin, Minnesota ReceiVed September 8, 2005. In Final Form: December 31, 2005 Long-chain cationic amphipaths are routinely used for transfecting DNA into cells, although the mechanism of DNA delivery by these agents is poorly understood. Since their interfacial properties are undoubtedly involved at some stage in the process, a comprehensive study of the surface behavior of at least one of these compounds is highly desirable. Hence, the behavior of the cationic transfection agent EDOPC (dioleoyl-sn-glycerol-3-ethylphosphocholine or O-ethyldioleoylphosphatidylcholine), has been characterized at the air-water interface, by itself and in mixtures with other phospholipids. Surface pressure-molecular area isotherms obtained at the argon-buffer interface revealed that EDOPC is considerably (5-10 Å2) more expanded than the parent phosphatidylcholine (DOPC) and even more expanded than the corresponding phosphatidylglycerol (DOPG), which has a similar charge density (of opposite polarity) as EDOPC. A 1:1 mixture of EDOPC and DOPG is very slightly condensed relative to DOPG and considerably condensed relative to EDOPC. The surface/dipole potential of this mixture is the mean of those of EDOPC and DOPG and is almost the same as that of DOPC. When the composition of EDOPC mixtures was varied, several surface parameters, including surface dipole moment, collapse pressure, and compressibility, exhibited discontinuities at a 1:1 mole ratio. EDOPC is unusually surface-active; the equilibrium surface tension of its dispersion was lower and the rate of fall of the surface tension (dynamic surface activity) of a dispersion with an initially clean surface was more than an order of magnitude greater than that for dispersions of DOPG. A 1:1 mixture of the cationic lipoid and phosphatidylglycerol had lower surface activity than DOPC in water but similar surface activity in 0.1 NaCl. Analysis, in terms of surface concentration, of the formation of EDOPC monolayers at the air interface of vesicle dispersions revealed a simple exponential rise to a maximum, at least for higher concentrations. Addition of a small proportion of DNA to EDOPC increased its dynamic surface activity even though DNA alone has no detectable surface activity at the concentrations used. This enhancement by DNA is presumably due to the disruption of the continuity of the bilayer and creation of defects from which lipoid spreads readily. The surface properties of this cationic compound, both alone and in combination with anionic lipids, provide insight into the previously described nonbilayer phase preferences of cationic-anionic lipid mixtures. In addition, they provide critical data (area condensation of mixed cationic-anionic monolayers) supporting a previously proposed mechanism of fusion of cationic bilayers with anionic bilayers. Such a process, involving anionic cellular membranes, is believed to be required for release of DNA from lipoplexes and is therefore a key stage of transfection.

Introduction

* To whom correspondence should be addressed: tel (847) 491-5062; e-mail [email protected]; fax (847) 467-1380. † Northwestern University. ‡ Present address: Loyola University Medical Center, Maywood, Illinois 60153. § University of Minnesota.

air/water interface of lipid dispersions as a function of time. Recently, however, some new analyses and methods for such investigations have been published,6,7 and a careful study of several methods of spreading monolayers revealed modest but significant differences in surface structure, depending upon the method of spreading.8 Lipid adsorption to the air/water interface is a subject of relevance to the function of lung surfactant, and investigations of monolayer formation also appear in the literature on pulmonary function (for a current review, see ref 9). As will be described in the Discussion, kinetic analyses indicate that the formation of monolayers from bilayer vesicles involves more than simple collision with the interface. There are almost no natural cationic lipids, but recently, cationic phosphatidylcholine derivatives, as DNA transfection agents, have been described. Preliminary investigations indicated that these compounds have quite unusual properties relative to natural phospholipids, especially when present in mixtures with anionic

(1) MacDonald, R. C. The relationship and interactions between lipid bilayer vesicles and lipid monolayers at the air/water interface. In Vesicles; Rossoff, M., Ed.; Marcel Dekker: New York, 1996; pp 1-48. (2) MacDonald, R. C.; Simon, S. A. Proc. Natl. Acad. Sci. U.S.A 1987, 84, 4089-4093. (3) Mohwald, H. Annu. ReV. Phys. Chem. 1990, 41, 441-476. (4) Vollhardt, D. Mater. Sci. Eng., C 2002, 22, 121-127. (5) Brockman, H. L. Chem. Phys. Lipids 1994, 73, 57-79.

(6) Lee, S.; Kim, D. H.; Needham, D. Langmuir 2001, 17, 5544-5550. (7) Lee, S.; Kim, D. H.; Needham, D. Langmuir 2001, 17, 5537-5543. (8) Lawrie, G. A.; Barnes, G. T.; Gentle, I. R. Colloids Surf., A: Physicochem. Eng. Aspects 1999, 155, 69-84. (9) Veldhuizen, R.; Nag, K.; Orgeig, S.; Possmayer, F. Biochim. Biophys. Acta, - Mol. Basis Dis. 1998, 1408, 90-108.

Biological lipids and related synthetic compounds have been quite well characterized in terms of monolayer properties. These investigations have usually focused on how surface pressure and electrostatic properties depend on molecular area. Such studies have typically been done in order to extract information useful in understanding biological membranes,1,2 and a number of reviews of this literature are available, for example, refs 3-5. Less commonly studied [see ref 1 for a review] is the dynamic surface activity, that is, the lowering of surface tension at the

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Surface Properties of Cationic Lipoids

phospholipids. In particular, the cationic derivatives rapidly give rise to monolayers at the air/water interface of their dispersions,10 their mixtures with anionic lipids reveal the formation of an unusually rich variety of nonlamellar phases, both in the presence and absence of DNA,11 and vesicles prepared from cationic lipoid fuse with anionic vesicles.12,13 These properties, along with the fact that the cationic phosphatidylcholine derivatives are efficient DNA transfection agents with low toxicity,14 indicated that an investigation of the surface properties of cationic phospholipoids and their mixtures with normal phospholipids could reveal some unusual aspects of surface behavior. Of particular interest was the possibility (indeed, likelihood) that the interaction of positive and negative charges of such amphipaths would introduce a new dimension into the surface behavior of their mixtures. The P-A behavior and dynamic surface activity of cationic detergents and related surfactants have been studied intensively.15 In contrast, investigations of cationic surfactants that have applications in biology (as transfection agents) have been rather few, and they have tended to have been focused on the monolayer as an adsorption surface for DNA or other polyanions, for example, refs 16-20. The only studies of cationic monolayers that are related to natural lipids have been on sphingosine.21-24 The surface chemistry of mixtures of cationic and anionic surfactants (also known as catanionic systems) has received considerable attention, largely because of the significant effects of electrostatic interactions. Both the properties of catanionic monolayers at the air-water interface and those of dispersed colloids have been investigated, although the vast majority of such investigations have involved soluble, single-chain compounds.25 Of significance is that, at a 1:1 charge ratio, the monolayers are maximally condensed.26,27 Similarly, catanionic dispersions most commonly generate aggregates at equimolar compositions28-30 and there is usually a strong synergism in depression of the surface tension.31 Occasionally, mixtures have been studied by Langmuir trough techniques in which one of the (10) MacDonald, R. C.; Ashley, G. W.; Shida, M. M.; Rakhmanova, V. A.; Tarahovsky, Y. S.; Pantazatos, D. P.; Kennedy, M. T.; Pozharski, E. V.; Baker, K. A.; Jones, R. D.; Rosenzweig, H. S.; Choi, K. L.; Qiu, R.; McIntosh, T. J. Biophys. J. 1999, 77, 2612-2629. (11) Koynova, R.; MacDonald, R. C. Biophys. J. 2003, 85, 1-17. (12) Pantazatos, D. P.; Pantazatos, S. P.; MacDonald, R. C. J. Membr. Biol. 2003, 194, 129-139. (13) Pantazatos, D. P.; MacDonald, R. C. J. Membr. Biol. 1999, 170, 27-38. (14) MacDonald, R. C.; Rakhmanova, V. A.; Choi, K. I.; Rosenzweig, H. S.; Lahiri, M. K. J. Pharm. Sci. 1999, 88, 896-904. (15) Rubingh, D. N.; Holland, P. M. Cationic Surfactants: Physical Chemistry; Marcel Dekker: New York, 1990. (16) Tanaka, K.; Okahata, Y. J. Am. Chem. Soc. 1996, 118, 10679-10683. (17) Kago, K.; Matsuoka, H.; Yoshitome, R.; Yamaoka, H.; Ijiro, K.; Shimomura, M. Langmuir 1999, 15, 5193-5196. (18) Bordi, F.; Cametti, C.; De Luca, F.; Gili, T.; Gaudino, D.; Sennato, S. Colloids Surf., B: Biointerfaces 2003, 29, 149-157. (19) Subramanian, M.; Holopainen, J. M.; Paukku, T.; Eriksson, O.; Huhtaniemi, I.; Kinnunen, P. K. J. Biochim. Biophys. Acta, Biomembr. 2000, 1466, 289-305. (20) Matti, V.; Saily, J.; Ryhanen, S. J.; Holopainen, J. M.; Borocci, S.; Mancini, G.; Kinnunen, P. K. J. Biophys. J. 2001, 81, 2135-2143. (21) Smaby, J. M.; Brockman, H. L. Langmuir 1992, 8, 563-570. (22) Ryhanen, S. J.; Saily, M. J.; Paukku, T.; Borocci, S.; Mancini, G.; Holopainen, J. M.; Kinnunen, P. K. J. Biophys. J. 2003, 84, 578-587. (23) Saily, V. M. J.; Alakoskela, J. M.; Ryhanen, S. J.; Karttunen, M.; Kinnunen, P. K. J. Langmuir 2003, 19, 8956-8963. (24) Koiv, A.; Mustonen, P.; Kinnunen, P. K. J. Chem. Phys. Lipid 1994, 70, 1-10. (25) Rosen, M. J. ACS Symp. Ser. 1992, 501, 316-326. (26) Corkill, J. M.; Goodman, J. F.; Harrold, S. P.; Tate, J. R. Trans. Faraday Soc. 1967, 63, 247-&. (27) da Silva, A. M. G.; Viseu, M. I. Colloids Surf., A: Physicochem. Eng. Aspects 1998, 144, 191-200. (28) Tomasic, V.; Stefanic, I.; Filipovic-Vincekovic, N. Colloid Polym. Sci. 1999, 277, 153-163. (29) Tondre, C.; Caillet, C. AdV. Colloid Interface Sci. 2001, 93, 115-134. (30) Khan, A.; Marques, E. F. Curr. Opin. Colloid Interface Sci. 1999, 4, 402-410. (31) Cui, Z. G.; Canselier, J. P. Colloid Polym. Sci. 2000, 278, 22-29.

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molecules is two-tailed, usually the cationic component, and in that case, any excess of the one-tailed compound tends to be squeezed out of the 1:1 cation-anion mixture.32 These behaviors indicate that there is a strong electrostatic interaction in such surfactant systems and that a similar but perhaps even stronger interaction would be expected with mixtures of oppositely charged, double-chain molecules. There has been a report of vesicles forming in such a mixture;33 however, there have been no reports that we are aware of on mixtures of anionic and cationic phospholipids or their derivatives. Greater understanding of the association of anionic lipids with cationic compounds (doublechained) of the type used to transfect DNA into cells is desirable because mixing of cellular anionic lipids with the transfection complex is almost certainly required for release of the DNA inside the cell.34 This mixing is generally thought to be due to fusion of cell membranes with the lipoplex.35 As shown previously, cationic and anionic bilayers fuse readily.13,36-39 Surface pressure-molecular area-interfacial potential (πA-V) studies of oppositely charged lipid mixtures, in particular, have become of interest because mixtures of the cationic phosphatidylcholine derivative EDOPC (phosphate triester of ethanol and dioleoylphosphatidylcholine, also termed O-ethyldioleoylphosphatidylcholine) with anionic phospholipids exhibit an unusual array of mesomorphic and polymorphic phases, ranging from inverted hexagonal through cubic to lamellar, depending on the charge ratio.11,40,41 The phase stability depends on intrinsic curvature and, in these cases, the latter depends on electrostatic interactions, which are partly accessible by π-A∆V measurements.5 Importantly, it has recently been found that the phase generated upon mixing different cationic lipoids with an anionic lipid is well correlated with the efficiency of transfection by the former; those cationic lipoids that gave the largest negative curvature were those that both released DNA from their lipoplexes most effectively and transfected cells most efficiently (Koynova, Wang, Tarahovsky, and MacDonald, to be published). This suggests that transfection depends on electrostatic interactions that maximally condense the headgroups in cationic-anionic mixtures. Moreover, electrostatic interactions between headgroups occurring when cationic bilayers come into contact with anionic bilayers are expected to lead to pronounced changes in area per molecule. That cationic lipoids can be condensed electrostaticallysa key postulate in published hypotheses for the mechanism for fusion of oppositely charged bilayers42swas an important result of the present study. Thus, a number of phenomena suggest that transfection by cationic lipoids is strongly influenced by the area per molecule and that this is likely to be significantly altered upon interaction with anionic lipids. Surface properties of transfection lipoids thus seem important for their biological effectiveness, and this paper provides the first characterization of these properties for such a (32) Viseu, M. I.; da Silva, A. M. G.; Costa, S. M. B. Langmuir 2001, 17, 1529-1537. (33) Chung, Y. C.; Regen, S. L. Langmuir 1993, 9, 1937-1939. (34) Bhattacharya, S.; Mandal, S. S. Biochemistry 1998, 37, 7764-7777. (35) Noguchi, A.; Furuno, T.; Kawaura, C.; Nakanishi, M. FEBS Lett. 1998, 433, 169-173. (36) Anzai, K.; Masumi, M.; Kawasaki, K.; Kirino, Y. J. Biochem. (Tokyo) 1993, 32, 487-491. (37) Pantazatos, S. P.; MacDonald, R. C. J. Membr. Biol. 2003, 191, 99-112. (38) Bailey, A. L.; Cullis, P. R. Biochemistry 1997, 36, 1628-1634. (39) Du¨zgu¨nes, N.; Goldstein, J. A.; Friend, D. S.; Felgner, P. L. Biochemistry 1989, 28, 9179-9184. (40) Tarahovsky, Y. S.; Arsenault, A. L.; MacDonald, R. C.; McIntosh, T. J.; Epand, R. M. Biophys. J. 2000, 79, 3193-3200. (41) Lewis, R. N.; McElhaney, R. N. Biophys. J. 2000, 79, 1455-1464. (42) Chanturiya, A.; Scaria, P.; Kuksenok, O.; Woodle, M. C. Biophys. J. 2002, 82, 3072-3080.

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compound, in this case for a cationic lipoid that has otherwise been well characterized physically and biologically. Materials and Methods Materials. EDOPC was synthesized by reacting DOPC with ethyl trifluoromethanesulfonate (Aldrich, Milwaukee, WI) and purified on silica gel by elution with chloroform-methanol (9:1), as previously described.43 This procedure generates the trifluoromethanesulfonate salt. The chloride salt is available from Avanti Polar Lipids (Alabaster, AL), from which all lipids other than EDOPC were purchased. A closely related synthesis of EDOPC has also been described.44 Methods: Measurement of Monolayer Characteristics. Stock solutions were prepared by dissolving lipids at 1-2 mM in hexane (Burdick Jackson Laboratories, Muskegon, MI)/ethanol (95:5) or, for POPG (palmitoyloleoylphosphatidylglycerol) and its mixtures with other lipids, benzene. Solvent and buffer purity were verified by interfacial potential measurements with the 210Po ionizing electrode also used to record interfacial potential during compression of lipid monolayers, as previously described.45 Water for the subphase buffer was purified by reverse osmosis, activated charcoal adsorption, and mixed-bed deionization (Elix 3, Millipore Corp., Bedford, MA) and then passed through a Milli-Q UV Plus System (Millipore Corp., Bedford, MA) and filtered through a 0.22 µm Millipak 40 membrane. The aqueous subphase supporting the lipid monolayers consisted of pure water to which ethylenediaminetetraacetic acid (EDTA) was added to 10 µM (EDOPC, DOPC, and mixtures containing DOPG) or 100 µM (EDOPC and mixtures containing POPG) adjusted to pH 7.0 with NaOH. EDTA was present to minimize potential perturbation by divalent inorganic cations. Control experiments showed that EDTA at the concentrations used had a negligible effect on the π-A isotherms compared to a simple water subphase. EDTA-dependent changes were noted for the surface potential. These were manifested primarily as a decrease, ∼25 or ∼43 mV for the cationic and anionic lipids, respectively, of the value of the area-independent component (∆V0, see below) of the interfacial potential (∆V). The other component of the potential, the dipole moment (µ⊥), was essentially unaffected. Glassware was acid-cleaned and rinsed thoroughly with deionized water and then with hexane/ethanol (95:5) before use. Surface pressure-molecular area-potential (π-A-∆V) isotherms were measured on a computer-controlled, Langmuir-type film balance. Surface pressure was calibrated according to the equilibrium spreading pressures of known lipid standards.46 The change in interfacial potential relative to a lipid-free interface (∆V) was measured with a 210Po electrode relative to a calomel reference electrode in the aqueous phase. Lipids were mixed and then spread automatically (51.67 µL aliquots), or in some cases manually (50.0 µL aliquots), from mixtures of their stock solutions (see above). Films were compressed at a rate of 0.999), which was then used to convert tension-time data to surface concentrationtime data.

Results π-A Isotherms of EDOPC and Its Mixtures with Other Dioleoylphosphatides at the Argon-Water Interface. Figure 1 compares the π-A isotherm of EDOPC with those of DOPC and DOPG, as well as with the 1:1 (mol/mol) mixture of EDOPC and DOPG. The solid line passing through each set of selected experimental data points (shown as symbols; from a total of ∼2000/isotherm) shows the excellent fit of the data over the range used for parameter determination. EDOPC is significantly more expanded at all pressures than any of the others. DOPG is more expanded than DOPC and the mixture at low pressures, but above about 25 mN/m the curves are quite similar. Also, at higher pressures, the EDOPC isotherm roughly parallels that of the other lipids but is displaced toward higher areas by ∼7-10 Å2. Another difference is that the collapse pressure for EDOPC is lower than that of the other three monolayers. The isotherm of the neutral mixture and that of DOPC are quite similar throughout. The area measured for DOPC is in excellent agreement with previously published data.53,55 Figure 2 depicts the measured interfacial potential-area (∆VA) isotherms of the same lipids as shown in Figure 1 measured over the same range of molecular areas. Note that the measured ∆V includes contributions arising from both water and lipid dipoles, that is, the dipole potential, and from any formal charges of the lipids, with their associated subphase counterions, that is, the surface potential.5 The 1:1 mixture and DOPC have very similar ∆V values throughout the liquid-expanded monolayer state (essentially the area range used for π-A, as well as ∆V-A, analysis as denoted by the solid fit line in each isotherm). The anionic lipid and cationic lipoid have potentials displaced by equal amounts but in opposite directions from the potential of the neutral monolayers, DOPC and the EDOPC-DOPG mixture. Although the data of Figures 1 and 2 suggest that the surface properties of the neutral cationic-anionic lipid mixture are clearly different from those of the compounds separately, they do not indicate how those properties change with composition. Are the changes gradual or abrupt, and are there special properties of the (55) Harmony, J. A. K.; Jackson, R. L.; Ihm, J.; Ellsworth, J. L.; Demel, R. A. Biochim. Biophys. Acta 1982, 690, 215-223.

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MacDonald et al.

Figure 2. Surface potential-molecular area isotherms of EDOPC and dioleoyl lipids. Conditions and symbols are as described for Figure 1. Solid lines show the calculated isotherms obtained by fitting the data, over the range shown, to an equation of state as referenced under Materials and Methods.

Figure 4. Surface characterization of EDOPC-DOPG mixtures as a function of composition. Parameters derived from surface pressuresurface potential-molecular area isotherms or their fitted curves, as exemplified in Figures 1 and 2, were (A) collapse surface pressure; (B) modulus of compression at selected surface pressures of 10, 20 ([), and 30 (2) mN/m; (C) monolayer molecular area extrapolated to infinite surface pressure; (D) activity coefficient of water at isotherm liftoff; (E) area-independent component of the interfacial potential; and (F) dipole moment perpendicular to the interface. Solid lines show values expected for ideal mixing of the monolayer components.

Figure 3. Molecular area-composition isobars of EDOPC-DOPG mixtures. Conditions for data acquisition were as described for Figure 1. Molecular areas were determined from surface pressure-molecular area isotherms at 10 (b), 20 (1), and 30 (9) mN/m.

neutral mixture? To answer these questions, we further investigated the properties of mixtures in 0.1 mole fraction steps. Surface molecular area, which is probably the most important property, is described by Figure 3. As is clear from Figure 1, the average area per molecule in the mixture is lower than that of each of the components, particularly for the cationic compound. What Figure 3 shows that could not be seen in Figure 1 is that the 1:1 mixture has the minimum area, particularly at the lower pressures; as the pressure was increased, the contraction relative to the DOPG became insignificant, although the area of EDOPC was always considerably higher than the average area per molecule in the mixture. Although the contraction was smaller as the pressure was increased, so too was the area, so that the percentage change was relatively constant; the difference between the area of EDOPC and the average area per molecule in the 1:1 mixture was 14%, 13%, and 12% at 10, 20, and 30 mN/m, respectively. Additional properties of the cationic-anionic mixtures are shown in the panels of Figure 4. As shown in Figure 4A, πc values are nearly constant with increasing EDOPC in DOPG until the mixture is neutral and then πc decreases toward that of pure EDOPC. It should be noted that collapse pressures obtained in dynamic monolayer compression experiments are not necessarily equilibrium values. However, in our experience53 that is the case, at least for liquid-expanded monolayers of phosphati-

dylcholines compressed at rates comparable to those used in this study. The behavior of πc deviates significantly from the ideal curve (line) predicted by assuming ideal mixing of the monolayer components,49 showing a maximal deviation at the neutral equimolar mixture. Similarly, the modulus of compression (Figure 4B) at each of the three π values shown, 10, 20, and 30 mN/m, deviates maximally and discontinuously at a 1:1 stoichiometry, consistent with the area condensation shown in Figure 3. The behavior of the fitting parameter A∞, the monolayer area extrapolated to infinite π and a reflection of the hard cylinder area of the dehydrated lipids, shows what appears to be a discontinuous change between 0.6 and 0.7 mole fraction EDOPC (Figure 4C). However, the absolute differences of 1-2 Å2 from ideal behavior (line) are small, considering the extrapolated nature of the parameter. In contrast, the generally progressive increase from ∼36 to ∼ 42 Å2 as DOPG is progressively replaced by EDOPC suggests that the larger area of EDOPC at lower π values reflects, in part, an incompressible area difference of ∼6 Å2 between the headgroups of these otherwise identical molecules. Figure 4D shows the activity coefficient of interfacial water at isotherm liftoff, that is, 0 mN/m, a fitting parameter that reflects the shape of the π-A isotherm. Whereas the pure anionic and cationic lipoids have similar values, charge neutralization progressively increases the parameter with the maximal deviation being again at a 1:1 composition. Panels E and F of Figure 4 show the fitting parameters derived from ∆V-A isotherms. In panel E, the value of the area-independent potential, ∆V0, for the 1:1 mixture falls on the ideal curve, yet examination of all the data shows that there is an abrupt shift in the parameter value as the net charge of the surface changes from negative to positive.

Surface Properties of Cationic Lipoids

Figure 5. Surface pressure-molecular area isotherms of EDOPC, POPG, and their 1:1 (mol/mol) mixture. Isotherms were determined at 24 °C on a subphase of 100 µM EDTA adjusted to pH 7.0 for EDOPC (O), POPG (4), and (1:1) EDOPC/DOPG (3). Solid lines show the calculated isotherms obtained by fitting the data, over the range shown, to an equation of state (eq 1).

This shows a tendency of this parameter, which is dominated by the headgroup and its hydration, to be dominated by one lipid species until the other charged species is in excess. Indeed, ∆V0 is nearly constant at EDOPC mole fractions greater than 0.5. A similar trend is exhibited by the concentration-dependent component of the interfacial potential, the dipole moment, denoted µ⊥, which changes little up to 0.5 EDOPC and then assumes ideal values in the positively charged interface. Isotherms of EDOPC, POPG, and Their Mixtures. To use π-A isotherms of EDOPC-POPG mixtures to analyze the formation of monolayers at the interface of dispersions of lipids of the same composition (see below), isotherms of these lipids were also determined. Figure 5 compares the π-A isotherm of EDOPC with that of POPG, as well as with the 1:1 (mol/mol) mixture of EDOPC and POPG (for comparison, the 1:1 EDOPCDOPG mixture is included). As may be seen in the figure, the area of POPG is considerably smaller than that of EDOPC and also smaller than that of the mixture. Throughout most of the isotherm, the area of the mixture is smaller than the mean of the two components, as would be expected, because the results shown in Figure 1 indicate that cationic component undergoes a large change in area upon neutralization with the negative component. The area of POPG is larger than that of the mixture at low pressures; this is again consistent with the data of Figure 1, showing that the intermolecular repulsion of the phosphoglycerol group has its most significant effect at low pressures. Figure 6 depicts the surface potential-area isotherms of the same monolayers as shown in Figure 5. As was the case with the DOPC-DOPG mixture, the positive and negative lipids have potentials displaced in opposite directions from the potential of the neutral EDOPC-DOPG mixture. Because the POPG molecule has a higher charge density due to its smaller area per molecule, the potential of the mixture is somewhat closer to that of the PG than to that of the cationic lipoid. Again, these data on potentials suggest an absence of abrupt changes in interactions between the molecules as a function of surface pressure, that is, packing density. Surface Activity of EDOPC and Its Mixtures with Other Dioleoylphosphatides. As indicated above, when the ethylphosphatidylcholines were first described, dispersions of these molecules were found to have a higher dynamic surface activity

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Figure 6. Surface potential-molecular area isotherms of EDOPC, POPG, and their 1:1 (mol/mol) mixture. Conditions and symbols are as described for Figure 5. Solid lines show the calculated isotherms obtained by fitting the data, over the range shown, to an equation of state (eq 1).

Figure 7. Time course of the change in surface tension of surfaces of dispersions of EDOPC (3), POPG (O), 1:1 mixture of EDOPC and POPG (1), and DOPC (b). The term “dynamic surface activity” is used in the text to describe the change in tension with time as shown here. Subphase: water. Concentration: 0.625 mg/mL.

than dispersions of phospholipids of native structure.10 Additional data on the rate at which the tension of an initially clean surface of EDOPC dispersions falls are presented here and compared with the dynamic surface activity of anionic lipid dispersions as well as of anionic-cationic mixtures. The formation of monolayers at the surface of lipid dispersions has been studied fairly extensively, although the results are not highly reproducible and the process remains incompletely understood (see, for example, ref 1). It is useful to recognize that the parameters that control the transfer of lipid molecules from their supramolecular structures to the air interface are multiple and probably interdependent; nevertheless, the differences in rate and extent of change of surface tension clearly reflect differences in the properties of the lipid molecules. Changes of Tension of Water Dispersions. Figure 7 depicts the time course of the change in surface tension of surfaces (initially swept clean) for EDOPC, POPG, their 1:1 mixture, and DOPC, all as dispersions in water at 0.625 mg/mL. The tension at the EDOPC surface fell so rapidly that the initial decline could not be recorded. Curiously, the tension at the POPG surface also initially dropped rapidly, but only by about 10 mN/m, after which the decline was very slow. The difference between the cationic and anionic dispersions is especially significant given that, by light microscopy, both are well hydrated and form largely unilamellar vesicles and, by dynamic light scattering, are about

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Figure 8. Time course of the change in surface tension of surfaces of dispersions of EDOPC (3), POPG (O), 1:1 mixture of EDOPC and POPG (1), and DOPC (b). The term “dynamic surface activity” is used in the text to describe the change in tension with time as shown here. Subphase: 0.1 M NaCl. Concentration: 0.625 mg/mL.

the same size.12 There was no change in tension at the surface of the DOPC suspension for nearly 5 min, after which the tension fell slowly. As will be seen below, this behavior is expected; until the surface concentration rose to the value corresponding to that at liftoff in a π-A curve, the tension remained constant at a value only slightly below that of the clean water surface. The cationic-anionic mixture was even less surface-active than DOPC, its surface tension falling by less than 5 mN/m over the course of 20 min. In both cases of the neutral dispersions, the electrostatic interactions must be attractive, in contrast to the repulsive forces in the dispersions of the separate cationic and anionic lipids. Changes in Tension of Dispersions in Electrolyte Solution. As can be seen in Figure 8, the dynamic surface activity of the four samples tested was much higher in 0.1 M NaCl than in water. In the case of EDOPC, the tension fell to a plateau value within 1 min and the minimum value was nearly 10 mN/m lower than that in water. The largest effect of an increase in ionic strength was on the mixture, which had the lowest dynamic surface activity in water but second highest (a poor second after EDOPC) in salt solution. The activity of DOPC was also higher in salt solution. POPG exhibited similar behavior in 0.1 M NaCl as in water in that there was an initial very rapid drop followed a gradual decline. Analysis of the Kinetics of Accumulation of Monolayer Molecules. Because the surface activity of the cationic derivatives of PC is higher than that of the phospholipids in general, and because the surface activity of its dispersions was observed to fall smoothly to a plateau value, it appeared that it might be possible to analyze the kinetics of EDOPC monolayer formation. For a kinetic analysis, it is necessary to convert surface tension data to surface concentration. A partial relationship between tension and surface concentration was available in the π-A curve (Figure 1; the region between liftoff and collapse). The tension as a function of time for different dispersion concentrations was readily available as described for the experiments of Figures 7 and 8. Accordingly, the curvilinear portion of the EDOPC π-A curve was fitted to a polynomial, which was then used to convert tension-time data to surface concentration-time data. Plots for three different concentrations (each differing by a factor of 4, from 0.0391 to 0.625 mg/mL) of EDOPC are shown as the symbols in Figure 9. At the lowest concentration, no symbols are shown below 164 pm/cm2 because this is the liftoff

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Figure 9. Changes in surface concentration of EDOPC dispersions as a function of time at different subphase concentrations. Subphase concentrations were 0.625 (O), 0.156 (0), and 0.0391 (]) mg/mL, in water. Surface concentrations, shown as symbols, were calculated from the EDOPC data in Figure 7, by use of the corresponding isotherm of Figure 1. The lines are least-squares fits to the experimental data by use of an exponential rise to a maximum function.

concentration and the surface tension changes below this concentration are not meaningful for this analysis (not only too small for us to measure but also essentially constant, as this is the liquid expanded-gaseous transition region of the monolayer). When the concentration of the dispersion was raised by a factor of 4, it took only about 1 min for the surface to acquire molecules up to the liftoff concentration, after which the surface concentration rose smoothly to a maximum. At the highest concentration, there was no “apparent” lag and, as was also seen in Figure 8, recording could not be started quite soon enough after the surface was swept clean to catch the initial values. Note that higher plateau values for surface concentration correspond to lower equilibrium surface tensions. Fitting the Data to a Kinetic Equation. The lines shown in Figure 9 correspond to best fits of the equation Γ(t) ) y) y0 + a[exp(-bt)]. As is seen, the fits are quite good (R > 0.99), indicating that the fall in surface tension of these dispersions is quite well described by an exponential rise in Γ to a maximum. The parameters corresponding to these fits were 218, 116, and 104 pmol/cm2 for y0; 67, 144, and 172 pmol/cm2 for a; and 1.8., 0.62, and 0.58 min-1 for b (all in order from high to low concentration). Similar fitting procedures were carried out for the other lipids and the cationic-anionic mixture. At least at the higher concentrations (>0.625 mg/mL) some of the fits were satisfactory, particularly for salt solutions, but generally, they were not as good as for EDOPC (results not shown), suggesting that, for dispersions of lower dynamic surface activity and/or at higher concentrations, monolayer formation follows a more complex time course than a single-exponential rise to a maximum. Small Proportions of DNA Dramatically Increase the Surface Activity of EDOPC. EDOPC and related cationic derivatives of phosphatidylcholine were developed initially as DNA transfection reagents. The surface activity of the lipoids in lipoplexes (complexes of cationic lipoid with DNA) may play a role in their facilitating the delivery of DNA to cells. It was therefore of some interest to determine how DNA affects the surface properties of EDOPC. As shown in Figure 10, the formation of a DNA complex by combining EDOPC and DNA in equimolar charge amounts (a composition similar to that used

Surface Properties of Cationic Lipoids

Figure 10. Surface tension as a function of time of lipoplexes (complex of DNA and EDOPC) (0, 4), EDOPC alone (3), and DNA alone (O). The term “dynamic surface activity” is used in the text to describe the change in tension with time as shown here. The EDOPC concentration was 0.08 mg/mL in all cases. Curves for lipoplexes with positive/negative charge ratios of 1:1 (0) and 6:1 (4) are shown. The DNA record is for a concentration that was 25% higher than the highest in lipoplexes. Subphase: water.

for transfecting cells) reduced the surface activity of the EDOPC; the rate of fall of the surface tension of the lipoid was reduced in the presence of the DNA. DNA at this overall concentration had no measurable surface activity. In the presence of a considerably smaller amount of DNA (a 1:6 DNAEDOPC charge ratio), on the other hand, the surface activity was even higher than for the lipoid alone.

Discussion Surface Pressure-Potential-Molecular Area Isotherms. The most striking feature of the π-A isotherms (Figures 1 and 5) is the fact that the EDOPC monolayer is considerably more expanded than the other monolayers, even at high pressure. DOPG (and POPG) were somewhat expanded, relative to DOPC, but only at lower pressures. The difference between the cationic and anionic molecules could suggest that there are cohesive interactions that operate between the phosphatidylglycerols (chosen because it is similar in size chemically to EDOPC and carries the same absolute value of electrostatic charge) at close packing that are absent for EDOPC. Hydrogen bonding is a possibility since it is precluded in EDOPC by the presence of the ethyl group. If that is correct, then we would assign the larger area of EDOPC to the pressure contribution from electrostatic repulsion, a quantity that can, in principle, be calculated according to the Poisson-Boltzmann equation. The electrostatic energy of a bilayer is equal to the energy required to charge it up from zero initial charge, and the electrostatic surface pressure is the derivative of that energy with respect to area.56 Typical calculations give a surface pressure of about 15 dynes/cm for a charge density of one unit electronic charge per lipid molecule (an approximation due to uncertainty in the dielectric constant of the interface57). Indeed, in the high pressure region of the isotherm, the surface pressure difference between EDOPC and DOPC at the same packing densities is about 15 dyn/cm (Figure 1). This may not be the whole story, however, for not only the DOPG monolayer, as pointed out, but also the A∞ parameter (Figure 4C) exhibits little expansion, which shows that the area (56) Jahnig, F. Biophys. Chem. 1976, 4, 309-318. (57) Cevc, G. Biochim. Biophys. Acta 1990, 1031-3, 311-382.

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difference between EDOPC and DOPC cannot be compressed out. This suggests that, at least at collapse, the steric contact of EDOPC headgroups also contributes significantly to its area. In that case, of course, the cationic-anionic mixture must undergo some kind of progressive interdigitation to account for its lower area. It appears, therefore, that the electrostatic pressure may be somewhat confounded by steric effects or other nonelectrostatic intermolecular interactions. The area contraction of EDOPC upon mixing with PGs is clear from the isotherms and is linearly related to the extent of charge neutralization in Figure 3. At low surface pressure, the EDOPC/POPG mixture also contracts somewhat, but less than the EDOPC/EDOPG mixture. That the EDOPC/DOPG mixture has a smaller area than DOPG may simply mean that, at collapse, these compounds are about as tightly packed as possible while still remaining liquid crystalline. In that case, the condensation in the mixture is probably due to the electrostatic attractions in the mixture; they are maximized and overall repulsion is minimized at 1:1 stoichiometry, where average area is a minimum. Although we have not identified π-A investigations of long, double-chain catanionic monolayer systems, there were several such investigations of soluble (largely single-chain) and micelleforming catanionic surfactant mixtures26,27 that included their dynamic surface activity;28,30,31 electrostatic interactions clearly lead to a minimum in both the average area per molecule and in the critical micelle concentration (cmc) and dynamic surface tension of solutions (see ref 29 for a recent review). Furthermore, the importance of electrical interactions of catanionic systems of soluble surfactants is evident from their role in theoretical calculations of minimum free energy.58 Thus, whether there are other interactions besides electrostatic in the case of the EDOPC/ DOPG mixture, electrostatic effects would be expected to lead to a significant area reduction in the 1:1 mixture of these twochain, insoluble compounds. The collapse pressure of EDOPC also stands out as being lower than that of the other lipids. As recently demonstrated,6,7 most naturally occurring phospholipid classes collapse at the same surface pressure, so the behavior of the cationic lipoid is anomalous. This behavior is also linearly related to charge neutralization, for, according to Figure 4A, πc becomes the same as that of DOPG at the point at which the EDOPC has been neutralized, suggesting that the weakness in the monolayer relative to collapsing is due to repulsive self-interactions of EDOPC. When these are replaced by attractive electrostatic interactions in the mixture with DOPG, the monolayer becomes more resistant to collapse. As expected, EDOPC and DOPG (and POPG) have very different ∆V values (Figure 2). At all areas in the liquid-expanded monolayer state, the 1:1 mixture exhibits potential values very close to the mean of the potentials of the molecules separately and also very close to that of DOPC. This indicates that the difference between the DOPC potential and that of the monolayers with a net charge is due essentially to the Gouy-Chapman effect, that is, the potential due to displacement, normal to the film, of the plane of fixed charges from the diffuse layer of counterions. For the equimolar mixture, we observed no significant changes in permanent dipoles due to either the electrostatic repulsion in the pure lipids or the electrostatic attraction in the mixed monolayer. Although the surface potentials seem to indicate additivity of charge effects for the 1:1 mixture (Figure 2), some unexpected features were exhibited by the mixtures as a function of mole fraction (Figure 4E,F). Both surface potential and dipole moment (58) Yuet, P. K.; Blankschtein, D. Langmuir 1996, 12, 3802-3818.

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changed relatively little until the center of the composition range; that is, a significant fraction of the total change occurred within 0.1 mole fraction unit of the 1:1 composition. This is not expected if the surface potential depends on the average surface charge density at each of these compositions and so indicates that some consideration should be given to the possible modes of mixing of these kinds of oppositely charged surface molecules. There are two general ways for neutralization to occur in this system. The first is complete and uniform mixing so that the surface charge over the whole area decreases uniformly toward neutrality as the 1:1 composition is approached. The second possibility is phase separation of patches of neutral ion pairs, leaving the excess charge as a separate phase. This would be expected if the electrostatic interaction were strong enough to counteract the mixing entropy that would tend to randomize all of the molecules. Phase separation of EDOPC from the neutral complex cannot be precluded by the behavior of monolayer collapse pressures up to 0.5 EDOPC mole fraction (Figure 4A), although there is strong evidence of miscibility, possibly of EDOPC with the neutral complex, above 0.5. A combination of partial contribution of both of these behaviors is also possible, and indeed there is some support for this in X-ray diffraction studies of the dipalmitoyl derivatives of PG and ethyl-PC at temperatures above the gelliquid phase transition temperature.11 These two modes of mixing differ in terms of how they would affect the surface potential as a function of composition because surface potential is a highly nonlinear function of surface charge (theoretically, as is well-known, but also experimentally59), and in particular, it changes most rapidly in the region of 10-20% surface charge. This is in fact what is seen, at least approximately, in Figure 4F, since most change in surface potential occurs between 0.4 and 0.6 mole fraction of EDOPC. If, neutralization occurred by the other mode of growth of phase-separated patches of ion pairs, then each patch would have a constant potential and only the relative areas would change, leading to a change closer to that shown by the ideal line. The data thus seem to favor random mixing of positive and negative surfactant molecules. Deviation in the parameter reflecting water activity (Figure 4D) toward more positive values as the interface approaches neutrality has been previously observed for mixtures of cationic (sphingosine) and anionic lipids.21 This is not surprising since, to the extent that the anion and cation reduce each other’s electrostatic field, their interaction with water would be reduced, thus increasing the activity of the surface-associated water. Dynamic Surface Activity and Its Analysis. The different rates of monolayer formation at the air/water interface of dispersions of lipid vesicles represent a composite of a variety of different influences. Both individual molecules and aggregates may contribute to monolayer formation. In the former case, the molecules must leave the aggregate and add to the monolayer, and in the latter case, the aggregates must rearrange to form an extended planar monolayer. Differences in size of the diffusing unit, amount of lipid in that unit, and differences in lipid interactions may all contribute to differences in dynamic surface activity; nevertheless, some important conclusions can be drawn from the dynamic surface tension data. One of the clearest findings of this aspect of the study was that the dynamic surface activity of EDOPC is at least an order of magnitude greater than that of the other lipids. The charge on the bilayer would certainly encourage release of individual molecules, but this itself is unlikely to account for the higher dynamic surface activity observed because all the concentrations investigated must have been above the CMCs and the aqueous (59) MacDonald, R. C.; Bangham, A. D. J. Membr. Biol. 1972, 7, 29-53.

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phase must also have been saturated with an essentially constant concentration of monomers. Nevertheless, increasing concentrations of total lipid led to a more rapid decline in surface tension, indicating that significant monolayer formation comes from liposomes or similar aggregates. A possible factor in this behavior is the looser packing of EDOPC (and lower cohesion), as reflected in its expanded packing in monolayers and lower collapse pressure. Also, the ethyl group may contribute enough hydrophobicity to the headgroup to favor fusion of vesicles with the developing monolayer. POPG has essentially the same surface charge density as EDOPC yet exhibits much lower surface activity than EDOPC. If electrostatic charge were the only factor involved, the two lipids should have similar behavior. As pointed out above, one obvious difference is that EDOPC has no potential for hydrogen bonding, in contrast to POPG, and this may contribute to lower areas per molecule as well as to lower dynamic surface activity. Although diffusion to the surface seems not to be the rate-limiting step in monolayer formation (see below), there is little difference between the positive and negative surfactants in terms of vesicle size,12 and in any case, diffusive flux could scarcely account for the differences in activity between the two lipids. (Consider two vesicles with radii r and 2r; the former will diffuse twice as fast at the latter. The latter, however, will carry 4 times as much lipid, so for a given diffusive flux of x particles/time, will deliver twice as much lipid to the surface. Even the effect of this large a difference in particle size is much smaller that the surface activity differences observed.) Both EDOPC and POPG had higher surface activity in salt solution than in water. These results are consistent with research in the surfactant literature, which experimentally60 and theoretically61 has recognized an effect of electrostatic interactions (stronger in water than in salt solution) on the rate of insertion of new molecules into partly formed monolayers. Although phospholipid monolayers may form by disintegration of vesicles at the surface, and not by single-molecule diffusion, electrostatic influences would still operate. The 1:1 mixture presumably spreads slowly in water because the intermolecular electrostatic interactions are strong at low ionic strength. Catanionic surfactant systems are known to exhibit low cmc valuess relative to the component molecules;62 this behavior is consistent with electrostatic attraction giving rise to stronger intermolecular interactions in the mixed-charge dispersion we have examined. Intrinsic curvature may also affect spreading,63 since cationic-anionic mixtures, at least at the high concentrations used for X-ray diffraction, tend to form nonlamellar phases.11 In any case, electrostatic interactions among the lipid headgroups would be weakened in 0.1 M NaCl, so it is not surprising that the mixture exhibits greater dynamic surface activity in salt solution. DOPC also exhibits higher dynamic surface activity in salt solution, and this may also reflect weakening of intermolecular charge interactions between phosphate anion and choline cation, but since the positive and negative charges cannot separate in phosphatidylcholine as they can in the EDOPC-POPG mixture, it is not surprising that the increase in ionic strength affected the mixture much more than it did the single-component dispersion of DOPC. Beyond rationalizing the different effects of ionic strength, not very much more can (60) Bonfillon, A.; Sicoli, F.; Langevin, D. J. Colloid Interface Sci. 1994, 168, 497-504. (61) Diamant, H.; Andelman, D. J. Phys. Chem. 1996, 100, 13732-13742. (62) Schulz, P. C.; Minardi, R. M.; Vuano, B. Colloid Polym. Sci. 1999, 277, 837-845. (63) Jordanova, A.; Lalchev, Z.; Tenchov, B. Eur. Biophys. J. 2003, 31, 626632.

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be inferred about the relative surface activity of the 1:1 mixture of cationic and anionic lipids and DOPC because the organization of the component lipids is likely different. The fact that the coverage of the air/water interface of EDOPC dispersions by an EDOPC monolayer follows an exponential rise to a maximum indicates that diffusion to the surface is not the rate-limiting step, for were that the case, the time would appear as a square root, not as an exponential. A number of groups of investigators have analyzed the formation of monolayers at the surface of lipid dispersions,61,64,65 and the analysis that we have found most useful66 involved adsorption of lipid from vesicles to an alkane-covered solid surface. At one extreme of their general model, where conversion of vesicles to monolayers at the interface is rate-limiting, Hubbard et al.66 found that the kinetics of monolayer formation are described by the equation Γ(t) ) Γm[1 - exp((-KC0/Γm)t)]. In this equation, Γm is the maximum or saturation value of the surface concentration and it corresponds to a in our fitting equation for Figure 9, and KC0/ Γm corresponds to b in our fitting equation, where K is the rate constant for formation of a monolayer from surface-associated vesicle and C0 is the bulk concentration of lipid. This equation, related to those that describe adsorption of polymers to interfaces, assumes that the adsorption is proportional, with a rate constant K, to the open or unoccupied area at the interface. Surface processes are assumed to be rate-limiting in this model, so there is no dependence of monolayer formation on diffusion to the surface and, correspondingly, there is no term in the square root of time. The fact that this model describes the formation of EDOPC monolayers in the surface concentration region from liftoff concentration to collapse pressure concentration suggests that even with these vesicles, which are evidently less stable than vesicles of natural phospholipids, the conversion of bilayer to monolayer at the surface is much slower than diffusion of vesicles to the surface. Still, the situation is somewhat more complicated than that implied by the Hubbard equation, for the data of Figure 9 do not correspond to a single value of K (although the difference did not exceed a factor of 2 even when the concentration varied by a factor of 16). A perhaps more serious problem with modeling the formation of monolayers from vesicles is that the plateau, or (apparent) equilibrium pressure, tends to increase with increasing lipid concentration. Even at relatively low total lipid concentrations, there is plenty of lipid to cover the surface, so if equilibrium is attained, the pressures for different concentrations should all be identical to the collapse pressure in a Langmuir trough measurement. In fact, this does not occur unless lipid concentrations are in the range of 1-5 mg/mL. The explanation may be in the reduction in entropy of the lipid molecules when they transfer from the vesicles to the surface monolayer. Whereas the twodimensional translational entropy increases when the molecules move from vesicles to monolayer (the area of the monolayer is much larger than the area of the vesicle), the three-dimensional entropy decreases even more (the volume of the monolayer is small compared to the total sample volume). The net effect would amount to approximately 10 kJ/mol at millimolar concentrations (about 1 mg/mL) in free energy. Now, the driving force to form the monolayer corresponds to the difference in the surface energy between the clean water surface and monolayer-covered (closepacked) surface, which is about 20 kJ/mol. Thus, the concentration (64) Ivanova, T.; Raneva, V.; Panaiotov, I.; Verger, R. Colloid Polym. Sci. 1993, 271, 290-297. (65) Zhdanov, V. P.; Keller, C. A.; Glasmastar, K.; Kasemo, B. J. Chem. Phys. 2000, 112, 900-909. (66) Hubbard, J. B.; Silin, V.; Plant, A. L. Biophys. Chem. 1998, 75, 163-176.

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(entropy) term is comparable to the surface energy term, so lipid concentrations, unless they are very high, should have measurable effects on equilibrium surface tensions. Effect of DNA on Surface Activity of EDOPC. EDOPC is unusually surface-active, but small amounts of DNA increased that activity even more, an effect that probably comes from the fact that DNA induces vesicle rupture (because of DNA-induced vesicle adhesion).67 Exposed edges of ruptured vesicles (evident in electron micrographs10) would be able to spread at the air/ water interface with a low activation energy because the hydrophobic core of the bilayer could contact the surface and spread directly. (It is tempting to suggest that lung surfactant might spread especially readily at the air-water interface for the same reason these lipoplexes do; surfactant contains cationic proteins,9 which might induce anionic bilayers to adhere strongly enough to one another to generate broken bilayer edges with a high propensity to release lipid to the surface.) That the neutral complex (1:1 DNA-lipoid charge ratio) is less surface-active than the 1:6 complex with a substoichiometric amount of DNA probably reflects the fact that all of the lipid in the former is electrostatically associated with DNA. This was essentially the same reason given above to rationalize the low surface activity of the 1:1 mixture of EDOPC and POPG. Implications for Membrane Fusion and Transfection. In order for DNA to be released from lipoplexes and enter the cell nucleus where it is transcribed, the cationic lipoid electrostatic charge must be neutralized. This can occur either by fusion of the lipoplex bilayer with cell membranes or by exchange of lipoid for cellular (anionic) lipid. It is not clear which of these process is most important, but the surface properties reported here are relevant to both. In the case of fusion between anionic and cationic bilayers, it has been postulated that a condensation of the oppositely charged contacting monolayers causes a rend or gap to form, at which point hemifusion commences.13,68 A similar mechanism for calcium-induced fusion of anionic lipid vesicles has also been proposed.69,70 The Langmuir trough data presented here confirm that such a condensation can occur and hence establish a critical element of such a mechanism. Although the trough data do not reveal a very large change in the area of PG monolayers upon neutralization (in contrast to that of EDOPC monolayers), a computer simulation of this type of mechanism indicates that the main requirement is for condensation of just one of the contacting monolayers.42 The surface properties of cationic lipoids used in lipoplexes are clearly important for their activity as transfection agents, and it may be hoped that manipulation of those surface properties will allow development of transfection agents with improved efficacy. Acknowledgment. We are grateful to Jaime Stearns for synthesis of EDOPC. We also thank the two reviewers who made numerous useful suggestions for improved presentation of this material. This research was supported by NIH research grants GM52329 (R.C.M.), GM57305 (R.C.M.), HL49180 (H.L.B.) and the Hormel Foundation (H.L.B.). LA0524566 (67) Kennedy, M. T.; Pozharski, E. V.; Rakhmanova, V. A.; MacDonald, R. C. Biophys. J. 2000, 78, 1620-1633. (68) Lei, G. H.; MacDonald, R. C. Biophys. J. 2003, 85, 1585-1599. (69) Chanturiya, A.; Scaria, P.; Woodle, M. C. J. Membr. Biol. 2000, 176, 67-75. (70) MacDonald, R. C. Mechanisms of membrane fusion in acidic lipid-cation systems. In Molecular Mechanisms of Membrane Fusion; Ohki, S., Doyle, D., Flanagan, T. D., Hui, S. W., Mayhew, E., Eds.; Plenum Press: New York, 1988; pp 101-112.