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Langmuir 2000, 16, 3385-3390

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Thermodynamic Characteristics of Mixed DPPC/DHDP Monolayers on Water and Phosphate Buffer Subphases Tzung-Han Chou and Chien-Hsiang Chang* Department of Chemical Engineering, National Cheng Kung University, Tainan, Taiwan 70101, R.O.C. Received May 12, 1999. In Final Form: November 4, 1999

The mixed monolayer behavior of dipalmitoylphosphatidylcholine (DPPC) and dihexadecyl phosphate (DHDP), two of the major components in the preparation of liposomes, on both water and phosphate buffer subphases was investigated from the measurements of surface pressure-area per molecule (Π-A) isotherms. The Π-A isotherms indicated that the two components were miscible at the air/water interface. The miscibility and nonideality of the mixed monolayers were examined by calculating the excess area as a function of composition, and deviations from ideality were observed, which suggests that the existence of attractive interactions between DPPC and DHDP molecules in the mixed monolayers on a water subphase. Nevertheless, the buffer subphase environment enhanced the dissociation of DHDP, and the presence of ions in the subphase may also disturb molecular interactions and packing in the mixed monolayers, resulting in more expanded monolayers and complicated excess area behavior. Furthermore, the excess free energies of mixing and free energies of mixing were evaluated from the isotherms, and the most stable state of the mixed monolayers on a water subphase was found with XDHDP ) 0.5 or 0.6. However, the tendency for DPPC and DHDP to form a mixed monolayer was less signficant on the phosphate buffer subphase than on a water subphase.

Introduction Liposomes or artificial vesicles are formed by dispersing lipids in water as spherical self-enclosed systems, in which an inner aqueous phase is surrounded by a lipid bilayer structure. They have long been used as models for studying the biological membranes. For the past three decades, it has been proven that liposomes have potential as drug delivery carriers, since both hydrophobic and hydrophilic drugs might be carried simultaneously within the lipid bilayer and aqueous phase compartments of liposomes, respectively.1-3 For pharmaceutical applications, it is important that a liposome product remains stable for a reasonable period of time under ambient storage conditions. However, liposomes are susceptible to aggregate and/or fusion, resulting in particle size changes during storage. In some cases, the substances encapsulated by liposomes may produce side effects, such as impairing immunological defenses, on the human body. Under such circumstances, liposomes are obviously necessary to keep stable until they reach the destination for the purpose of controlling drug delivery. Thus, the stability of liposomes is always of great interest in the pharmaceutical and pharmacological aspects in vivo and in vitro. From the viewpoint of liposome structure, the stability of liposomes depends on their physicochemical properties, especially the molecular interactions between the constituents in the bilayer structures.4,5 Since a monolayer study is a fundamental method to provide the information on * To whom correspondence should be addressed. Tel (+) 8866-2757575, ext. 62671; Fax (+) 886-6-2344496; E-mail changch@ mail.ncku.edu.tw. (1) Kellaway, I. W.; Farr, S. T. Adv. Drug Deliv. Rev. 1990, 5, 149. (2) Crommelin, D. J. A.; Schreier, H. In Colloidal Drug Delivery Systems; Kreuter, J., Ed.; Marcel Dekker: New York, 1994; Chapter 3. (3) Jones, M. N.; Chapman D. Micelles, Monolayers, and Biomembranes; Wiley-Liss, Inc.: New York, 1995; Chapter 4. (4) Sekiguchi, A.; Yamauchi, H.; Manosroi, A.; Manosroi, J.; Abe, M. Colloids Surf. B: Biointerfaces 1995, 4, 287. (5) MacDonald, R. C. In Vesicles; Rosoff, M., Ed.; Marcel Dekker: New York, 1996; Chapter 1.

molecular interactions in an oriented system, the study of mixed monolayer behavior of liposome components may be an easier and useful method to give important implications of liposome stability. Dipalmitoylphosphatidylcholine (DPPC) is often used as the main phospholipid to prepare liposomes for a wide range of applications due to its neutral charge and inertness. Nevertheless, there will be other additional materials, such as cholesterol and dihexadecyl phosphate (DHDP), introduced during synthesizing liposomes or artificial vesicles to enhance their stability.4,6-12 Due to the effects of cholesterol on the lateral interactions of hydrocarbon chains of lipids,13 it is one of various methods to increase the stability of liposomes by introducing significant amounts of cholesterol into the liposomes.3,14-16 Regarding the incorporation of DHDP in liposomes, it is to inhibit the aggregation of liposomes and hence help to improve their stability, since negative charges of DHDP (6) Pierce, N. F.; Sacci, J. B., Jr.; Alving, C. R.; Richardson, E. C. Rev. Infect. Dis. 1984, 6, 563. (7) Gregory, R. L.; Michalek, S. M.; Richardson, G.; Harmon, C.; Hilton, T.; McGhee, J. R. Infect. Immun. 1986, 54, 780. (8) Alving, C. R.; Richard, R. L.; Moss, J.; Alving, L. I.; Clements, J. D.; Shiba, T.; Kotani, S.; Wirtz, R. A.; Hockmeyer, W. T. Vaccine 1986, 4, 166. (9) Audera, C.; Ramirez, J.; Soler, E.; Carreira, J. Clin. Exp. Allergy 1991, 21, 139. (10) Florence, A. T. In Liposome Technology: Liposome Preparation and Related Techniques, 2nd ed.; Gregoriadis, G., Ed.; CRC Press: Boca Raton, FL, 1993; Vol. I, Chapter 10. (11) Taylor, K. M. G.; Farr, S. J. In Liposome Technology: Liposome Preparation and Related Techniques, 2nd ed.; Gregoriadis, G., Ed.; CRC Press: Boca Raton, FL, 1993; Vol. I, Chapter 11. (12) De Hann, A.; Geerligs, H. J.; Huchshorn, J. P.; Van Scharrenburg, G. J. M.; Palache, A. M.; Wilschut, J. Vaccine 1995, 13, 155. (13) Baszkin, A.; Boissonnade, M. M.; Rosilio, V.; Kamyshny, A.; Magdassi, S. J. Colloid Interface Sci. 1999, 209, 302. (14) Kirby, C.; Gregoriadis, G. Biochem. J. 1981, 199, 251. (15) Khand, L.; Rogerson, A.; Halbert, G. W.; Baillie, A. J.; Florence, A. T. J. Pharm. Pharmacol. 1987, 39 (Suppl.), 41P. (16) Woodle, M. C.; Newman, M. S.; Working, P. K. In Stealth Liposomes; Lasic, D., Martin, F., Eds.; CRC Press: Boca Raton, FL, 1995; Chapter 10.

10.1021/la990581+ CCC: $19.00 © 2000 American Chemical Society Published on Web 02/11/2000

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molecules can provide electrostatic repulsive forces between the suspended vesicles.10,16,17 The molecular interactions between DPPC and cholesterol in mixed monolayers have been extensively examined in the literature.18-21 There have been few reports on the DHDP monolayer behavior at the air/liquid or liquid/liquid interfaces.18,22-25 However, no research has been done on revealing the thermodynamic characteristics of mixed DPPC/DHDP monolayers in order to provide the information on intermolecular interactions between DPPC and DHDP molecules. In this paper, a study examining the miscibility and thermodynamic properties of mixed DPPC/DHDP monolayers is presented. The effects of Na2HPO4/NaH2PO4 buffer subphase with a pH value of 7 on the mixed monolayer behavior at the interface were also investigated by comparing with the results obtained on a water subphase. Experimental Section Materials. L-R-Dipalmitoylphosphatidylcholine (DPPC) (>99%) and dihexadecyl phosphate (DHDP) (99%) were purchased from Sigma Chemical Company, USA, and were used without further purification. Ethyl alcohol (∼99.5%) was supplied by Seoul Chemical Industry Co., Ltd., Korea, and n-hexane of HPLC grade (>99%) was obtained from Tedia Company, USA. An ethanol/ n-hexane (1:9, v/v) mixture was used as the spreading solvent for monolayer-forming materials. A buffer solution of NaH2PO4/ Na2HPO4 was used for a subphase with a pH value of 7. Only highly pure water, which was purified by means of a Milli-Q plus water purification system, with a resistivity of 18.2 MΩ‚cm was used in all experiments. Methods. The isotherm trough system (model minitrough, KSV Instrument Ltd., Finland) has been described in previous papers.26,27 The Teflon trough was 320 mm long and 75 mm wide. Temperature regulation of the trough was controlled by circulating constant temperature water from an external circulator through the tubes attached to the aluminum-based plate of the trough. The trough was placed on an isolated vibration-free table and was enclosed in a glass chamber to avoid contaminants from the air. A computer with an interface unit obtained from KSV Instrument Ltd. was used to control the Teflon barriers. One of the important features of the trough system is that two barriers confining a monolayer at the interface are driven symmetrically during the compression of the monolayer. The surface pressure was measured by the Wilhelmy plate method. The resolution for surface pressure measurement is 0.004 mN/m, and the inaccuracy of surface area regulation is less than 1%, according to the specifications of the instrument. A surface pressure-area per molecule (Π-A) isotherm was obtained by a continuous compression of a monolayer at the interface by two barriers. Before each isotherm measurement, the trough and barriers were cleaned with an ethanol solution and then rinsed by purified water. The sandblasted platinum plate used for surface pressure measurements was also rinsed with purified water and then flamed before used. Besides, all glassware was cleaned prior to use in the same manner as the trough and barriers. (17) Martin, F. J.; Heath, T. D.; New, R. R. C. In Liposomes: A Practical Approach; New, R. R. C., Ed.; Oxford University Press: New York, 1990; Chapter 4. (18) Shah, D. O.; Schulman, J. H. J. Lipid Res. 1967, 8, 215. (19) Ladbrooke, B. D.; William, R. M.; Chapman, D. Biochim. Biophys. Acta 1968, 150, 333. (20) Gershfeld, N. L.; Pagano, R. E. J. Phys. Chem. 1972, 76, 1244. (21) Mu¨ller-Landau, F.; Cadenhead, D. A. Chem. Phys. Lipids 1979, 25, 315. (22) Shah, D. O.; Schulman, J. H. J. Lipid Res. 1965, 6, 341. (23) Hunt, E. C. J. Colloid Interface Sci. 1969, 29, 105. (24) Claesson, P.; Carmona-Ribeiro, A. M.; Kurihara, K. J. Phys. Chem. 1989, 93, 917. (25) Zhou, N.-F.; Neuman, R. D. Colloids Surf. 1992, 63, 201. (26) Chen, K.-B.; Chang, C.-H.; Yang, Y.-M.; Maa, J.-R. J. Chin. Inst. Chem. Eng. 1996, 27, 455. (27) Chou, T.-H.; Chang, C.-H. Colloids Surf. B: Biointerfaces 2000, 17, 71.

Chou and Chang To start the experiment, the freshly cleaned trough was placed into position in the apparatus first, and then it was filled with purified water as the subphase with a temperature controlled at 37 ( 1 °C. The clean platinum plate was hanged in the appropriate position for surface pressure measurements. The surface pressure fluctuation was checked to be less than 0.2 mN/m during the compression of the entire trough surface area range. Then, the two barriers were moved back to their initial positions, and a sample containing monolayer-forming materials was spread on the subphase by using a 25 µL microsyringe (Hamilton Co., USA). At least 10 min was allowed for ensuring complete solvent evaporation. After the solvent was evaporated, the monolayer was compressed continuously at a rate of 1 Å2/(molecule min) to obtain a single Π-A isotherm. The experiments for each monolayer isotherm were run three times to check the reproducibility of the isotherm measurements to be (1-3 Å2/(molecule min) depending on the composition and surface pressure. Similarly, the measurements of Π-A isotherm for the mixed monolayers on the phosphate buffer subphase were also carried out.

Results and Discussion Behavior of Mixed Monolayers on Water Subphase. Miscibility of Mixed Monolayers. The surface pressure-area per molecule (Π-A) isotherms for mixed DPPC/DHDP monolayers at various DHDP molar fractions at 37 °C on a water subphase are demonstrated in Figure 1a. Curves 1 and 7 were the isotherms for the monolayers of pure DPPC and pure DHDP, respectively, and the other curves showed that the lift-off values of mean molecular area decreased with increasing DHDP ratio in mixed monolayers. The shape of the Π-A isotherm for a pure DHDP monolayer indicates that the DHDP monolayer on a water subphase may be considered as a condensed monolayer. A DPPC monolayer distinctively showed a phase transition between liquid-expanded and liquid-condensed states at Π = 35 mN/m. The presence of a phase transition in a DPPC monolayer was eliminated as 20 mol % of DHDP was added, which is similar to the condensing effect of cholesterol on a DPPC monolayer.27 According to the phase rule, if there is only one surface phase that is in equilibrium with a bulk phase at the gas/liquid interface, there are three degrees of freedom, i.e., temperature, external pressure, and surface pressure. Therefore, if the two components in the mixed monolayers are miscible at the interface, the collapse surface pressure should vary with composition.28,29 It is obviously shown in Figure 1a that the collapse surface pressures of mixed monolayers varied with composition. Therefore, it can be concluded that DPPC and DHDP were miscible at the air/water interface. To proceed with a quantitative analysis of the mixed monolayer behavior at the air/liquid interface, the excess area (Aex) should be calculated, which can be an examination of miscibility for a mixed monolayer in another way. At a given surface pressure, the excess area can be represented by comparing the average area per molecule of a mixed monolayer consisting of components 1 and 2 with that of an ideal mixed monolayer.30-32

Aex ) A12 - Aid ) A12 - (X1 A1 + X2 A2 )

(1)

(28) Crisp, D. J. Res. (London) Suppl. Surf. Chem. 1949, 17. (29) Wu, S.; Huntsberger, J. R. J. Colloid Interface Sci. 1969, 29, 138. (30) Gaines, G. L., Jr. Insoluble Monolayers at Liquid-Gas Interfaces; Interscience Publishers: New York, 1966; Chapter 6. (31) Birdi, K. S. Lipid and Biopolymer Monolayers at Liquid Interfaces; Plenum Press: New York, 1989; Chapter 4. (32) Jones, M. N.; Chapman D. Micelles, Monolayers, and Biomembranes; Wiley-Liss, Inc.: New York, 1995; Chapter 2.

Mixed DPPC/DHDP Monolayers

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Figure 1. Surface pressure-area per molecule isotherms of mixed DPPC/DHDP monolayers of various compositions on (a) water and (b) phosphate buffer subphases at 37 °C.

Figure 2. (a) Area per molecule and (b) Aex/Aid as a function of composition for mixed DPPC/DHDP monolayers on a water subphase at various surface pressures.

where A12 and Aid are the mean and ideal areas per molecule of the mixed monolayer at a given surface pressure, respectively, X1 and X2 imply the mole fractions of components 1 and 2, respectively, and A1 and A2 are the areas per molecule of each pure monolayers at the same surface pressure. When the binary components form an ideal mixed monolayer or the two components are immiscible, the excess area will be zero and A12 will be linear in X1 at a given surface pressure. Any deviation from linearity between A12 and X1 would indicate that the two components are miscible and form nonideal monolayers. Thus, this area additivity rule at a constant surface pressure allows one to decide on the ideality of mixed monolayer behavior. Figure 2a illustrates the mean area per molecule as a function of composition at different surface pressures for the mixed DPPC/DHDP monolayers on a water subphase. Since the linearity between mean molecular area and XDHDP was not observed, DPPC and DHDP were considered to be miscible and expressed nonideal monolayer behavior at the air/water interface. However, it cannot be totally ruled out that DPPC and DHDP might

be immisible or form ideal mixed monolayers when XDPPC or XDHDP is close to 1. Figure 2b shows the Aex/Aid versus the mole fraction of DHDP in mixed monolayers. The error for the data was estimated to be (5% based on the reproducibility of the isotherm measurements. Negative deviations for all compositions at different surface pressures were observed. The deviations from the area additivity rule indicated in Figure 2b and the variations of collapse pressure with composition demonstrated in Figure 1a imply that DPPC and DHDP were miscible on a water subphase. It is also shown in Figure 2b that the most significantly negative deviation appeared at XDHDP ) 0.6 at a constant surface pressure. This result suggests that the incorporation of DHDP into a DPPC monolayer may increase the interactions between molecules in the mixed monolayers, and the influence of intermolecular interaction on molecular packing was significant, especially at XDHDP ) 0.6. Furthermore, the extent of negative deviations decreased with increasing surface pressure, which can be expected since at higher surface pressures the monolayers were more compact and the effects of intermolecular interaction on molecular packing would be less significant.

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Chou and Chang

Stability of Mixed Monolayers. Once it has been established that the constituents of a mixed monolayer are miscible, a more detailed examination of the thermodynamic properties of the mixed monolayer system can provide useful information on monolayer stability. The stability of a mixed monolayer compared with a monolayer with separation between individual components can be investigated by the Gibbs free energy of mixing, ∆Gmix, and the extent of deviation from the ideal mixing can be represented by the excess free energy of mixing, ∆Gex.30,33 The ∆Gex is expressed as

∆Gex )

∫0Π[A12 - (X1A1 + X2A2 )] dΠ

(2)

Hence, the values of ∆Gex can be calculated from the Π-A isotherms of mixed monolayers. The ∆Gmix is then expressed by the following formula.

∆Gmix ) ∆Gex + ∆Gid

(3)

where ∆Gid is the ideal free energy of mixing, which can be evaluated from

∆Gid ) RT(X1 ln X1 + X2 ln X2)

(4)

where R is the universal gas constant and T is the absolute temperature. Treatments of data by the approach described above gave the ∆Gex of mixed DPPC/DHDP monolayers as a function of composition at various surface pressures, which is illustrated in Figure 3a. Examining the relation of ∆Gex with composition at a constant surface pressure, one can find that at all compositions the values of ∆Gex were negative, and there was a minimum that occurred in each curve, which corresponded to XDHDP ) 0.6. The negative values of ∆Gex indicate the mutual attractions of the molecules in mixed monolayers, and the occurrence of a minimum suggests that the influence of molecular interaction on monolayer stability was more significant for the mixed monolayer with a composition of XDHDP ) 0.6 than for mixed monolayers with other compositions. Besides, the negative deviation of ∆Gex was enhanced with increasing surface pressure, which implies that the intermolecular interaction was stronger when the mixed monolayer was in a more condensed state. Figure 3b shows the ∆Gmix versus mole ratio of DHDP on a water subphase at different surface pressures at 37 °C. It is shown in Figure 3b that the values of ∆Gmix were negative for all compositions in mixed monolayers, and a phase separation did not occur across the entire composition range in mixed DPPC/DHDP monolayers at various surface pressures. This implies that the mixed monolayers were more stable than the monolayers with separation between individual components, especially at XDHDP ) 0.5 or 0.6. In addition, the more negative values of ∆Gmix were obtained at higher surface pressures. That is, the extent for the monolayer-forming components to form a mixed monolayer has a tendency to increase at a higher surface pressure. Behavior of Mixed Monolayers on Buffer Subphase. Miscibility of Mixed Monolayers. Measurements of Π-A isotherms for the mixed DPPC/DHDP monolayers on the Na2HPO4/NaH2PO4 buffer subphase with a pH value of 7 were also performed. Similarly, a thermodynamic analysis of the isotherms obtained on the buffer (33) Goodrich, F. C. In Proceedings of the Second International Congress on Surface Activity; Schulman, J. H., Ed.; Butterworth Press: London, 1957; Vol. 1, p 85.

Figure 3. (a) Excess free energy of mixing and (b) free energy of mixing as a function of composition for mixed DPPC/DHDP monolayers on a water subphase at various surface pressures.

subphase was carried out by following the approach described in the previous section. The shapes of the Π-A isotherms for mixed DPPC/ DHDP monolayers apparently depended on the subphase environment. This is illustrated for the case of Na2HPO4/ NaH2PO4 buffer subphase in Figure 1b. On the phosphate buffer subphase, all mixed DPPC/DHDP monolayers showed a phase transition region between liquid-expanded and liquid-condensed states. However, the phase transition of a DPPC monolayer occurred at Π = 25 mN/m, which was about 10 mN/m lower than that observed on a water subphase. In addition, a DHDP monolayer distinctively showed a phase transition region at Π = 5 mN/m, which was not observed for this monolayer on a water subphase. Nevertheless, it is also demonstrated in Figure 1b that the collapse pressures of mixed monolayers varied with composition. Thus, it appears that DPPC and DHDP were also miscible on the phosphate buffer subphase. In Figure 4, the area per molecule and Aex/Aid versus the mole fraction of DHDP in a mixed monolayer is shown at different surface pressures at 37 °C. However, one

Mixed DPPC/DHDP Monolayers

Figure 4. (a) Area per molecule and (b) Aex/Aid as a function of composition for mixed DPPC/DHDP monolayers on the phosphate buffer subphase at various surface pressures.

should note that the extent of the effect of buffer subphase on the behavior of a monolayer-forming component in a mixed monolayer was assumed to be similar to that in a pure monolayer. Apparently, a nonlinear relationship existed between molecular area and XDHDP except at lower surface pressures, at which a linear relationship seemed to occur. Furthermore, at all compositions, the mean areas per molecule showed negative deviations from ideality except at lower surface pressures. Thus, DPPC and DPDH might be immiscible or form an ideal mixed monolayer on the buffer subphase at lower surface pressures at 37 °C. Besides, the mixed monolayers exhibited two different types of behavior in Figure 4b. Below the surface pressure of 20 mN/m corresponding to the region including liquidexpanded, transition, and liquid-condensed phases in Π-A isotherms, the deviations from the ideal behavior became significant on increasing surface pressure. However, when the mixed monolayers were compressed to more condensed states (Π > 20 mN/m), the deviations were all negative for all compositions, and their magnitude seems to decrease with increasing surface pressure. Stability of Mixed Monolayers. A plot of the ∆Gex versus XDHDP for mixed DPPC/DHDP monolayers on the phos-

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Figure 5. (a) Excess free energy of mixing and (b) free energy of mixing as a function of composition for mixed DPPC/DHDP monolayers on the phosphate buffer subphase at various surface pressures.

phate buffer subphase at various surface pressures is displayed in Figure 5a. One can see that the ∆Gex changed from a value close to zero to negative values with increasing surface pressure, since the intermolecular interaction was stronger as the monolayer was more condensed. Moreover, the mixing of the mixed monolayer with XDHDP ) 0.8 was most affected by the intermolecular interaction compared with the monolayers with other compositions. As shown in Figure 5b, the values of ∆Gmix for mixed DPPC/DHDP monolayers on the buffer subphase were always negative at different surface pressures. It appears that the mixed monolayers were thermodynamically more stable than the monolayers with separation between DPPC and DHDP, especially at XDHDP ) 0.4 or 0.5. Nevertheless, at the air/buffer-solution interface, a partially miscible behavior in the mixed DPPC/DHDP monolayers may occur. This situation is demonstrated between XDHDP ) 0.5 and XDHDP ) 0.8 in Figure 5b. It can be seen that, over a portion of the composition range XDHDP ) 0.5-0.8, the free energy of the mixed monolayer system may be minimized if two phases of compositions XDHDP ) 0.5 and XDHDP ) 0.8 were present instead of a single phase of any

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intermediate composition, such as XDHDP ) 0.6. That is, two equilibrium phases would exist for any overall composition between XDHDP ) 0.5 and XDHDP ) 0.8. Similar behavior was also observed between XDHDP ) 0.2 and XDHDP ) 0.5. Comparison of Mixed Monolayer Behavior on Water and Buffer Subphases. According to the measurements of Π-A isotherms and the thermodynamic analysis for the mixed monolayer behavior on both water and phosphate buffer subphases, one can find that the intermolecular interaction of mixed DPPC/DHDP monolayers depended upon the subphase environment. The differences between two subphases were the presence of ions (Na+, HPO42-, and H2PO4-) in the buffer subphase and pH = 7 for the buffer subphase compared with pH = 5.6 for a water subphase. By contrast with the Π-A isotherms obtained on the phosphate buffer subphase, hardly any liquid-expanded phase and transition region were present in the Π-A isotherms of mixed DPPC/DHDP monolayers on a water subphase. It is evident that the molecular packing of the mixed monolayers became more expanded and complicated on the buffer subphase. The data of Aex also suggest that the effects of intermolecular interaction on molecular packing of mixed monolayers were less significant on the buffer subphase than on a water subphase and became more complicated in the presence of buffer subphase. It has been shown that zwitterionic lipids such as phosphatidylcholines were not very sensitive to pH or ions, at least over the pH range 3-8.34 It was also believed that the ionization status of common phosphatidylcholines remained unchanged, and the surface potentials were constant in the pH range of approximately 6-8.32 Thus, it is likely that the ion instead of pH may play a more important role in the influence on DPPC status in the mixed monolayers. For a DHDP monolayer, the limiting molecular area showed no perceptible change over the pH range of 1.4-8.7 on pure perchlorate solutions.23 However, it has been demonstrated that the DHDP surface pressure-area isotherm apparently changed as the pH was adjusted from 3 to 9 with or without the addition of NaCl due to the change in the extent of DHDP dissociation.24 At pH 5.2-5.7 with a NaCl concentration of 2 × 10-4 M, less than 2% of the DHDP molecules were estimated to be charged. Thus, one may expect that the dissociation of DHDP on pure water was insignificant, which explained the observed condensed DHDP monolayer behavior in Figure 1a. An increase of pH or subphase ionic strength induced a monolayer expansion originating from the dissociation of DHDP at the interface,24,35 as observed in Figure 1b. Moreover, the DHDP monolayer hydration was expected to increase through the binding of hydrated sodium ion existed in the buffer subphase, resulting in repulsive intralayer hydration forces and expanded monolayer behavior.24 Therefore, the presence of the buffer subphase strongly affected the geometric accommodation of molecules or the intermolecular forces in the mixed monolayers. Obviously, the results of ∆Gex also showed that the deviations from ideal mixing were dependent on the (34) Papahadjopoulos, D. Biochim. Biophys. Acta 1968, 163, 240. (35) El Mashak, E. M.; Lakhdar-Ghazal, F.; Tocanne, J. F. Biochim. Biophys. Acta 1982, 688, 465.

Chou and Chang

subphase environment, and the existence of ions resulted in smaller excess free energy of mixing. Furthermore, at the same surface pressure, the absolute values of the free energy of mixing obtained on the buffer subphase were less than those obtained on a water subphase. This implies that the tendency for DPPC and DHDP to form mixed monolayers was less significant thermodynamically on the buffer subphase than on a water subphase. Besides, a markedly partially miscible behavior may occur at the air/buffer interface as the mixed monolayers were disturbed by ions. The surface pressure-composition diagram of the mixed monolayers of the single negatively charged dimyristoylphosphatidic acid (DMPA) with DPPC on a pure water subphase has been investigated by Albrecht et al.36 The free energy was found to be minimal for the mixture at XDMPA = 0.5. This was explained in terms of the shielding of the electrostatic repulsive forces between the charged DMPA headgroups by the zwitterionic DPPC. Similar behavior may also be observed for the mixed DPPC/DHDP monolayers on the buffer subphase. However, the situation for DPPC/DHDP was more complicated due to the influence of ionic strength in the subphase and the strong interations between sodium ions and DHDP molecules. Conclusions From an analysis of surface pressure-area per molecule (Π-A) isotherms for the mixed DPPC/DHDP monolayers, it seems that two components were miscible on both water and phosphate buffer subphases at 37 °C, except at lower surface pressures on the buffer subphase. The mean areas per molecule of mixed monolayers on a water subphase apparently exhibited negative deviations from ideality due to molecular interactions, and the molecular packing was influenced significantly, especially at XDHDP ) 0.6. According to the results of ∆Gex and ∆Gmix obtained on a water subphase, the stability of a mixed monolayer with XDHDP ) 0.6 was most affected by molecular interaction, and the mixed monolayers, especially with XDHDP ) 0.5 or 0.6, were more stable thermodynamically than the monolayers with separation between individual components. The Π-A isotherms obtained on the phosphate buffer subphase demonstrated the dissociation of DHDP at pH ) 7, which resulted in more expanded monolayers and complicated excess area behavior. Moreover, it seems that the presence of a phosphate buffer subphase strongly influenced the molecular packing of molecules and intermolecular interactions in the mixed DPPC/DHDP monolayers. Comparing the results of ∆Gex and ∆Gmix obtained on the buffer subphase with those obtained on a water subphase, it was shown that the mixed monolayers were less liable to form on the buffer subphase than on a water subphase. Acknowledgment. This study was supported by the National Science Council of the Republic of China through Grants NSC 87-2214-E006-016 and NSC 88-2214-E006021. LA990581+ (36) Albrecht, O.; Gruler, H.; Sackmann, E. J. Colloid Interface Sci. 1981, 79, 319.