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Interaction of a Partially Fluorinated Heptadecanoic Acid with Diacyl Phosphatidylcholines of Varying Chain Length Meenakshi Arora,‡ Paul M. Bummer,‡ and Hans-Joachim Lehmler*,† Department of Occupational and Environmental Health, The University of Iowa, Iowa City, Iowa 52242, and Graduate Center of Toxicology and College of Pharmacy, University of Kentucky, Lexington, Kentucky 40536 Received April 30, 2003. In Final Form: August 5, 2003
The interaction of 11-(perfluorohexyl)-undecanoic acid, a partially fluorinated carboxylic acid, with five phosphatidylcholines (PCs) of chain length varying from C14 to C18 was investigated in monolayers at the air-water interface and in fully hydrated bilayers. For the monolayer studies, the compression isotherms of mixtures of the acid with the respective PC were recorded at various compositions on hydrochloric acid (pH ) 1.9, 32 ( 2 °C) as a subphase. The mixing behavior was assessed by analyzing the concentration dependence of the average molecular area at constant film pressure (area/mole fraction or A-X diagram) and, where applicable, the concentration dependence of the breakpoint of the phase transition from the liquid-expanded to the liquid-condensed state. All five binary mixtures showed a negative deviation from ideality at low surface pressures (5 mN/m). At higher surface pressures, a negative deviation was observed for the even-numbered-chain PCs, whereas the odd-numbered-chain PCs showed nearly ideal behavior (g10 mN). The phase transition of the odd-numbered-chain PCs as well as the C16 PC (D16PC) was concentration dependent. The collapse pressures are in general concentration dependent over the entire concentration range with D17PC and D18PC being an exception. Mixtures of these two long-chain phospholipids display a concentration-independent collapse pressure at mid-to-high concentrations of the respective phospholipids. These observations suggest (partial) miscibility of the components at the airwater interface. For the bilayer studies, differential scanning calorimetry thermograms of fully hydrated acid-PC mixtures of varying mole fraction (XPC ranging from 1 to 0.6) were recorded and the influence of the acid on the pre- and main transition of the PC was analyzed. At no mole fraction was a peak corresponding to excess acid (melting point ) 62.5 °C) observed, suggesting that the acid is incorporated into the lipid bilayer, that is, not phase separated from the phospholipid. Similar to perhydrocarbon acids, the fluorinated acid eliminates the pretransition of all five PCs. However, in contrast to perhydrocarbon acid-PC mixtures, the temperature of the main phase transition hardly increases in the presence of the fluorinated acid although some peak broadening can be observed. The general differences in the phase behavior are attributed to a less favorable packing of the hydrophobic tails in the mono- and bilayer. The biological implications of the observed phase behavior of the fluorocarbon acid-PC mixtures are currently unknown, but immiscibilities in both mono- and bilayers suggest the presence of domains with high acid concentrations which may (adversely) impact the function of biological mono- and bilayers.
Introduction There is growing interest in partially fluorinated surface-active materials for use in cosmetics1 and pharmaceuticals2-4 such as pulmonary drug delivery.5 As a result of the high electronegativity of fluorine, the extreme thermal, chemical, and biological stability of the C-F bond, the high surface activity of fluorinated surfactants, and the nonideal mixing behavior of fluorinated with hydrocarbon materials, the introduction of a partially fluorinated tail into a surfactant often results in interesting and unpredicted properties. To utilize and/or further investigate these properties, partially fluorinated, odd-numbered carboxylic acids, such as 11-(perfluorohexyl)-undecanoic acid, have been incorpo* Corresponding author. Dr. H.-J. Lehmler, The University of Iowa, Department of Occupational and Environmental Health, 100 Oakdale Campus #124 IREH, Iowa City, IA 52242-5000. Phone: (319) 335-4414. Fax: (319) 335-4290. E-mail:
[email protected]. † University of Iowa. ‡ University of Kentucky. (1) Krafft, M. P. Cosmet. Sci. Technol. Ser. 1998, 19, 195. (2) Riess, J. G.; Krafft, M. P. MRS Bull. 1999, 24, 42. (3) Krafft, M. P.; Riess, J. G. Biochimie 1998, 80, 489. (4) Riess, J. G.; Krafft, M. P. Biomaterials 1998, 19, 1529. (5) Lehmler, H.-J.; Bummer, P. M.; Jay, M. CHEMTECH 1999, 29, 7.
rated into a large variety of surfactants of interest for biomedical applications.6,7 Little is known about the factors determining the phase behavior of mixtures of these partially fluorinated surfactants and (biologically relevant) perhydrocarbon surfactants in monolayers and bilayers. Similar to fluorocarbon-hydrocarbon bulk solvent mixtures,8 mixed binary systems containing a perfluorocarbon surfactant and a structurally related hydrocarbon surfactant are known to behave nonideally, that is, exhibit phase separation in insoluble monolayers at the air-water interface9-12 or form two types of micelles simultaneously, one type fluorocarbon-rich and the other hydrocarbonrich, in solution.13,14 This nonideal behavior at the airwater interface is thought to cause adverse effects at the lung surface after the inhalation of surface-active, per(6) Riess, J. G. Tetrahedron 2002, 58, 4113. (7) Riess, J. G. J. Fluorine Chem. 2002, 114, 119. (8) Patrick, C. R. Chem. Br. 1971, 7, 154. (9) Iimura, K.-i.; Shiraku, T.; Kato, T. Langmuir 2002, 18, 10183. (10) Imae, T.; Takeshita, T.; Kato, M. Langmuir 2000, 16, 612. (11) Ge, S.; Takahara, A.; Kajiyama, T. J. Vac. Sci. Technol., A 1994, 12, 2530. (12) Meyer, E.; Overney, R.; Lu¨thi, R.; Brodbeck, D.; Howald, L.; Frommer, J.; Gu¨ntherodt, H.-J.; Wolter, O.; Fujihira, M.; Takano, H.; Gotoh, Y. Thin Solid Films 1992, 220, 132. (13) Mukerjee, P. Colloids Surf., A 1994, 84, 1. (14) Mukerjee, P.; Yang, A. Y. S. J. Phys. Chem. 1976, 80, 1388.
10.1021/la034734b CCC: $25.00 © 2003 American Chemical Society Published on Web 09/16/2003
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fluorinated materials.15,16 These adverse effects are in contrast to the low toxicity of most partially fluorinated surfactants (for general reviews, see refs 2-4). At the air-water interface, binary systems of partially fluorinated surfactants and perhydrocarbon surfactants do not appear to exhibit such distinct nonideal behavior. For example, partial miscibility has been reported for mixtures of partially fluorinated17 and perfluorinated18 carboxylic acids with dipalmitoyl-phosphatidylcholine (D16PC) at the air-water interface. Similarly, partial miscibility was observed for mixtures of partially fluorinated carboxylic acids and their respective hydrocarbon analogues.19 Overall, mixtures of partially fluorinated, perfluorinated, and perhydrocarbon carboxylic acids with phosphatidylcholines have similar features, that is, show an attractive interaction at the air-water interface. Similarly, attractive interactions have been reported for the incorporation of fluorinated carboxylates20 and a large number of perhydrocarbon carboxylic acids (for a recent review, see ref 21) into bilayers of phosphatidylcholines. In extension of our previous work,17,19,22,23 the goal of the present study was to examine the phase behavior of mixtures of a typical partially fluorinated carboxylic acid, 11-(perfluorohexyl)-undecanoic acid, and a series of diacyl phosphatidylcholines. The hydrophobic tail of this surfactant consists of 10 methylene groups connected to 6 perfluorinated carbon atoms forming the end of the tail. As such, this partially fluorinated tail is representative for partially fluorinated surfactants. The phase behavior of the respective acid-phosphatidylcholine mixtures at the air-water interface was probed using pressure-area isotherms. In addition, differential scanning calorimetry (DSC) was employed to study alterations of the phase behavior of bilayers resulting from the addition of partially fluorinated acid. This technique was chosen to supplement our monolayer studies because the binary phase diagrams of several hydrocarbon acid-phosphatidylcholine mixtures have been recorded using DSC.21 A better understanding of interactions between partially fluorinated surfactants such as 11-(perfluorohexyl)-undecanoic acid and phosphatidylcholine will aid in our understanding of the factors determining the phase behavior of biological mono- and bilayers and, ultimately, the resulting biological effects. This will allow us to rationally design novel, fluorinated amphiphiles for a broad range of biomedical applications.6,7 Materials and Methods 11-(Perfluorohexyl)-undecanoic acid was synthesized as described earlier.17,22 The purity of the partially fluorinated acid was >99% (based on relative peak area) as determined by gas chromatography. Ditetradecanoyl- (D14PC), dipentadecanoyl(D15PC), dihexadecanoyl- (D16PC), diheptadecanoyl- (D17PC), and dioctadecanoyl-phosphatidylcholine (D18PC) were obtained (15) Tashiro, K.; Matsumoto, Y.; Nishizuka, K.; Shibata, K.; Yamamoto, K.; Yamashita, M.; Kobayashi, T. Intensive Care Med. 1998, 24, 55. (16) Okonek, S.; Reinecke, H. J.; Fabricius, W.; Preussner, K. Dtsch. Med. Wochenschr. 1983, 108, 1863. (17) Lehmler, H.-J.; Jay, M.; Bummer, P. M. Langmuir 2000, 16, 10161. (18) Yamamoto, S.; Shibata, O.; Lee, S.; Sugihara, G. Prog. Anesth. Mech. 1995, 3, 25-30. (19) Lehmler, H.-J.; Bummer, P. M. J. Colloid Interface Sci. 2002, 249, 381-387. (20) Inoue, T.; Iwanaga, T.; Fukushima, K.; Shimozawa, R. Chem. Phys. Lipids 1988, 46, 25-30. (21) Koynova, R.; Tenchov, B. Curr. Opin. Colloid Interface Sci. 2001, 6, 277. (22) Lehmler, H.-J.; Oyewumi, M. O.; Jay, M.; Bummer, P. M. J. Fluorine Chem. 2001, 107, 141. (23) Lehmler, H.-J.; Bummer, P. M. J. Fluorine Chem. 2002, 117, 17.
Arora et al. from Avanti Polar Lipids in >99% purity and used without further purification. 2-Propanol, toluene, chloroform, methanol, and n-hexanes were HPLC grade and were purchased from Fisher Scientific or VWR. Concentrated hydrochloric acid was also obtained from Fisher Scientific. Deionized water for the monolayer studies was distilled first from basic potassium permanganate followed by distillation from sulfuric acid.17,22 Deionized water for the DSC experiments was obtained from a PURELAB Plus water system and had a resistance of g18 Ω. Monolayer Experiments. All monolayer experiments were carried out in a rectangular Teflon trough (306 × 150 mm) held at 37 ( 2 °C (KSV-3000, Finland). The surface pressure was measured by the Wilhelmy plate method using paper plates (15 × 58 mm) as described previously.17,22 Every surface area-surface pressure isotherm was determined on a freshly poured subphase (hydrochloric acid, pH ) 1.9). The subphase was allowed to equilibrate for 10 min at 37 ( 2 °C. Surface-active impurities were removed from the air-water interface with a slight vacuum after compression of the barrier. Acid, phospholipid, and acidphospholipid solutions with a concentration of 1-2 mg/mL were freshly prepared every day in n-hexanes/2-propanol ) 9:1. A known quantity (30-80 µL) of solutions was spread on the surface, and 10 min at 37 ( 2 °C was allowed to elapse for solvent evaporation before the start of the compression. A constant compression speed of 15 cm2/min (10 mm/min) was used. Preparation of Samples for Differential Scanning Calorimetry. Calculated amounts of the phospholipids and fluorinated acid were dissolved in chloroform-methanol (3:1, v/v) at the appropriate mole fractions.24 The solvent was removed under a stream of nitrogen, and the mixtures were further dried under a vacuum for at least 3 h. The samples (mixture of phospholipid and fluorinated acid or pure phospholipid) were hydrated in an excess of water (3 times by weight). Samples were heated above the lipid transition temperature for 5 min and vortexed for 2 min. This process was repeated four times. Finally the samples were sonicated in a water bath above the lipid transition temperature for 30 min, followed by the heating and vortexing cycle mentioned above. Samples were stored at 4 °C for 12-16 h. Hydration of samples was always carried out a day before collecting the DSC scans. A Thermal Analysis 2920 differential scanning instrument was used for the DSC studies. The hydrated samples were weighed into DSC aluminum pans. The DSC cell was purged with 60 mL/min and the refrigerated cooling system with 120 mL/min dry nitrogen, respectively. Samples were cooled to 4 °C at a heating rate of 10 °C/min and then heated from 4 to 80 °C with a heating rate of 5 °C/min.24,25 All samples were subjected to two subsequent heating cycles. All experiments were carried out in triplicate. Onset, maximum, and offset temperatures as well as peak width of the pretransition and the main phase transition were determined for the second run using the Universal Analysis NT software.22
Results The present study investigates the mixing behavior of 11-(perfluorohexyl)-undecanoic acid with five diacyl phosphatidylcholines in insoluble monolayers at the air-water interface and in completely hydrated mixtures using DSC. The chain length of the acyl group of the phosphatidylcholines under investigation ranged from 14 to 18 carbon atoms. It was not possible to study dinonadecanoylphosphatidylcholine (D19PC) because of solubility problems. Interaction at the Air-Water Interface. The concentration dependence of (i) the breakpoint of a phase transition and (ii) the collapse pressure are criteria for the miscibility of binary mixtures at the air-water interface.17,19,26,27 In short, a concentration-dependent (24) Koynova, R. D.; Boyanov, A. I.; Tenchov, B. G. Biochim. Biophys. Acta 1987, 903, 186. (25) Eliasz, A. W.; Chapman, D.; Ewing, D. F. Biochim. Biophys. Acta 1976, 448, 220. (26) Do¨rfler, H.-D. Adv. Colloid Interface Sci. 1990, 31, 1. (27) Motomura, K. J. Colloid Interface Sci. 1974, 48, 307.
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Figure 1. Compression isotherms of mixtures of 11-(perfluorohexyl)-undecanoic acid with (a) D14PC (0, 0.20, 0.40, 0.60, 0.79, 1), (b) D15PC (0, 0.21, 0.42, 0.60, 0.81, 1), (c) D16PC (0, 0.20, 0.40, 0.61, 0.79, 1), (d) D17PC (0, 0.18, 0.38, 0.56, 0.78, 1), and (e) D18PC (0, 0.20, 0.40, 0.58, 0.79, 1) at 37 ( 2 °C on hydrochloric acid, pH ) 1.9-2.1. Numbers in parentheses are the mole fractions of the respective phospholipids (from left to right).
phase transition and/or collapse pressure points to the complete miscibility of the components, whereas concentration independence is a sign of immiscibility of the components.26 The concentration dependence of the average molecular area at constant film pressure is another criterion to assess the miscibility of two components at the air-water interface. In ideal behavior, the average area per molecule of any mixture is the sum of the areas occupied by each species at the surface:
A(π) ) XAcidAAcid + XPCAPC
(1)
(X is the mole fraction of each component present at the air-water interface, A is the average area per molecule of the pure component, Acid is 11-(perfluorohexyl)undecanoic acid, and PC is any of the phosphatidylcholines under investigation.) This “additivity rule” gives the expected area per molecule, A(π), for ideal mixing or complete phase separation. (Partial) miscibility can be observed as a result of interaction between the two components. An attractive interaction will lead to a negative deviation, and a repulsive interaction will lead to a positive deviation from ideal behavior. To investigate the mixing behavior, the compression isotherms of mixtures of 11-(perfluorohexyl)-undecanoic acid with the respective phosphatidylcholine were recorded at various compositions at 37 ( 2 °C (Figure 1). The breakpoint of the phase transition and the collapse pressure for all π-A isotherms were determined where applicable and are summarized in Figure 2. The A-XPC
diagrams (eq 1) of all carboxylic acid-phosphatidylcholine mixtures are shown at several surface pressures in Figure 3. 11-(Perfluorohexyl)-undecanoic Acid and D14PC. The compression isotherm of D14PC (neat) is of the expanded type and shows no phase transition. The limiting molecular area of D14PC of 70.2 ( 0.9 Å2/molecule and the collapse pressure of 47.4 ( 0.9 mN/m are in agreement with previously reported values. As shown in Figure 2a, the phase transition characteristic of pure 11-(perfluorohexyl)-undecanoic acid22 cannot be detected in mixtures at a mole fraction of X(D14PC) g 0.2. The collapse pressure is concentration dependent over the entire concentration range studied. The A-XD14PC diagram of this mixture shows a negative deviation from additivity at 5 mN/m (Figure 3a). This negative deviation is much less pronounced at higher surface pressures and lies within the experimental error. The (concentration-dependent) disappearance of the phase transition of 11-(perfluorohexyl)undecanoic acid and the negative deviation from additivity at 5 mN/m both suggest that this system is at least partially miscible at lower surface pressures, while the concentration-dependent collapse pressure suggests miscibility at higher surface pressures. 11-(Perfluorohexyl)-undecanoic Acid and D15PC. The compression isotherm of D15PC exhibits a phase transition from a liquid-expanded to a liquid-condensed phase and, overall, resembles the isotherm of D16PC. The limiting molecular area of D15PC is 51.1 ( 0.3 Å2/molecule, and the phase transition occurs at 37.8 ( 0.5 mN/m, which is higher compared to the value for D16PC (20.3 ( 0.1 mN/m). The phase transition characteristic of pure 11-
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Figure 2. Mole fraction dependence of the phase transition (2) and collapse pressure (() of mixtures of 11-(perfluorohexyl)undecanoic acid with (a) D14PC, (b) D15PC, (c) D16PC, (d) D17PC, and (e) D18PC (37 ( 2 °C, hydrochloric acid, pH ) 1.9-2.1). All data points are averages of at least three experiments ( one standard deviation.
(perfluorohexyl)-undecanoic acid cannot be detected in mixtures at a mole fraction of X(D15PC) g 0.2 (Figure 2b). The phase transition pressure of D15PC decreases from pure D15PC to X(D15PC) ) 0.8 and remains constant between X(D15PC) ) 0.6 and 0.8. No phase transition is observed for lower concentrations of D15PC. The collapse pressure is concentration dependent at high acid concentrations. Because of the experimental setup, we were not able to determine the collapse pressure for mole fractions of D15PC between 0.4 and 1. The A-XD15PC diagram of this mixture shows a very slight negative deviation from additivity at 5 mN/m and ideal behavior at higher surface pressures. Considering the nearindependence on concentration of the liquid-expanded to liquid-condensed phase transition, we suggest that the two components of this mixture show very limited miscibility or are phase separated at low surface pressures. An unambiguous interpretation of the miscibility at high surface pressures is not possible because we were unable to determine the collapse pressure over a wide concentration range. Considering the near-ideal behavior at high surface pressures, the binary monolayer can either be miscible or not miscible. 11-(Perfluorohexyl)-undecanoic Acid and D16PC. The compression isotherm of D16PC is in good agreement with previous reports.17 As shown in Figure 2c, the liquidexpanded to liquid-condensed phase transition at high mole fractions of D16PC is concentration dependent and decreases steadily down to a mole fraction of X(D16PC) ∼ 0.6. Only an expanded phase appears to be present between mole fractions of e0.2 and g0.4. Although we
were unable to determine the collapse pressure at high mole fractions of D16PC, the collapse pressure appears to be concentration dependent. The A-XD16PC diagram exhibits a negative deviation from ideality over the entire surface pressure range studied with maxima at X(D16PC) between 0.6 and 0.8. This binary system is therefore (partially) miscible at the air-water interface at low and high surface pressures. 11-(Perfluorohexyl)-undecanoic Acid and D17PC. The compression isotherm of D17PC exhibits a transition from a liquid-expanded to a liquid-condensed phase at 6.6 ( 0.2 mN/m. The limiting molecular area of D17PC is 49.2 ( 0.8 Å2/molecule, and the collapse pressure is 71.7 ( 0.7 mN/m. With the exception of low concentrations of D17PC (X(D17PC) ∼ 0.2), a concentration-independent phase transition can be observed over the entire concentration range (Figure 2d). The collapse pressure appears to be concentration independent between a mole fraction of D17PC of 0.4 and 0.8, whereas it is concentration dependent at high and low concentrations of the acid. The A-XD17PC diagram in Figure 3d shows a complex behavior at 5 and 10 mN/m, which is due to the existence of a phase transition in this pressure range. At higher pressures (30 mN/m), ideal behavior is observed. Considering the nearindependence on concentration of the liquid-expanded to liquid-condensed phase transition, we suggest that the two components of this mixture are phase separated over large concentration ranges. This interpretation is supported by the concentration independence of the collapse pressure over a relatively broad concentration range. Thus,
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Figure 3. A-XPC diagrams of mixtures of 11-(perfluorohexyl)-undecanoic acid with (a) D14PC, (b) D15PC, (c) D16PC, (d) D17PC, and (e) D18PC (37 ( 2 °C, hydrochloric acid, pH ) 1.9-2.1). All data points are averages of at least three experiments ( one standard deviation.
the mixing behavior of this system is similar to that of the 11-(perfluorohexyl)-undecanoic acid-D15PC mixture. 11-(Perfluorohexyl)-undecanoic Acid and D18PC. In agreement with previous reports,33 the compression isotherm of D18PC is of the condensed type and exhibits no phase transition. The limiting molecular area of D18PC is 51.4 ( 0.5 Å2/molecule, and the collapse pressure is 71.5 ( 1.2 mN/m. As shown in Figure 2e, a concentrationdependent phase transition can be observed in mixtures rich in 11-(perfluorohexyl)-undecanoic acid (X(D18PC) e 0.4). The collapse pressure is mostly concentration dependent. However, between mole fractions of D18PC of 0.6 and 0.8, the collapse pressure is concentration independent. As a result of the phase transition in the acid-rich region of the phase diagram (Figure 2e), the A-XD18PC diagram in Figure 3e is complex at low surface pressures (5 and 10 mN/m). A slight negative deviation from ideal behavior can be observed at 30 mN/m. Overall, these observations suggest (partial) miscibility of this binary mixture. Interactions of 11-(Perfluorohexyl)-undecanoic Acid and Phosphatidylcholines in an Excess of Water Studied by DSC. DSC is a straightforward tool to study the phase behavior of binary mixtures. Since our ultimate interest is biological systems, we limited our DSC study to the biologically more relevant phosphatidylcholine-rich part of the phase diagram. The procedure for the preparation of the acid-phospholipid mixtures in excess water and the temperature program of 5 °C/min for the DSC studies was similar to that of previous studies,24,25 thus allowing us a direct comparison with the results from these investigations.
11-(Perfluorohexyl)-undecanoic Acid and D14PC. Thermograms recorded with acid-D14PC dispersions at different acid contents are shown in Figure 4a. For pure hydrated D14PC, the maximum of the pretransition (14.8 ( 0.1 °C) and main transition (Tm ) 24.1 ( 0.2 °C) are in agreement with literature data.28 Upon addition of 11(perfluorohexyl)-undecanoic acid to pure D14PC, the peak of the pretransition widens and occurs at higher temperatures. The pretransition merges with the main transition at X(D14PC) e 0.90. In this concentration range, the peak of the main phase transition broadens slightly with increasing content of the acid (Figure 4a). The onset of the main phase transition remains constant up to X(D14PC) ) 0.79. Significant broadening of the peak and a shift of the peak maximum to higher temperatures are observed between mole fractions of 0.83 and 0.79 D14PC. This increase continues down to a mole fraction of 0.70 D14PC, where again a sharp peak is observed. A decrease of the onset and maximum peak temperature as well as peak broadening can be observed at a further decreasing mole fraction of D14PC. According to the partial phase diagram (Figure 5a), gel separation takes place at mole fractions below X(D14PC) ) 0.79. At least three separate phase transitions are present at mole fractions from 0.83 to 0.79. The fluidus line indicates some miscibility in the fluid phase. A homogeneous phase is observed at a mole fraction of 0.7, indicating that a single phase is present at a molar ratio of phosphatidylcholine/acid of 2:1. However, the phase (28) Huang, C.-h.; Li, S. Biochim. Biophys. Acta 1999, 1422, 273.
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Figure 4. Calorimetric scans for mixtures of 11-(perfluorohexyl)-undecanoic acid with even-numbered phosphatidylcholines, i.e., (a) D14PC, (b) D16PC, and (c) D18PC, in excess water. The mole fraction of the phosphatidylcholine is indicated beside each scan. The heating rate was 5 °C/min from 4 to 80 °C (only the part of the curve with a phase transition is shown).
Figure 5. Partial phase diagram of mixtures of 11-(perfluorohexyl)-undecanoic acid with (a) D14PC, (b) D15PC, (c) D16PC, (d) D17PC, and (e) D18PC in excess water. (9) Onset temperature of pretransition, (×) offset temperature of pretransition, (() onset temperature of main transition, and (2) offset temperature of main transition.
transition of this complex is only marginally increased compared to the transition of pure dipalmitoyl-phosphatidylcholine.
11-(Perfluorohexyl)-undecanoic Acid and D15PC. Thermograms of this mixture at different mole fractions are shown in Figure 6a. The pure phospholipid in excess
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Figure 6. Calorimetric scans for mixtures of 11-(perfluorohexyl)-undecanoic acid with odd-numbered phosphatidylcholines, i.e., (a) D15PC and (b) D17PC, in excess water. The mole fraction of the phosphatidylcholine is indicated beside each scan. The heating rate was 5 °C/min from 4 to 80 °C (only the part of the curve with a phase transition is shown).
water shows a pretransition at 23.4 ( 0.1 °C and a main transition at 33.0 ( 0.2 °C, which is in good agreement with literature data.28 The pretransition shifts to higher temperatures with increasing acid content and merges with the main transition at X(D15PC) e 0.95. Up to a mole fraction of 0.84, the onset of the main phase transition remains constant while the peak slightly broadens. At 0.84, the peak broadens significantly and exhibits a shoulder at approximately 34 °C. This shoulder appears to be also present at a mole fraction of 0.81. The peak shape suggests that in this mole fraction range two solidto-liquid phase transitions occur. Below this mole fraction of D15PC, the peak shifts to higher temperatures and the peak width increases as well. Similar to the 11-(perfluorohexyl)-undecanoic acid and D14PC system, the partial phase diagram (Figure 5b) indicates that separation occurs in the gel phase at mole fractions above X(D15PC) ) 0.84 and in the fluid phase above 0.95. The increase in the fluidus line below 0.95 indicates some miscibility in the fluid phase. Above a mole fraction of 0.84, both the gel and fluid phase show miscibility. 11-(Perfluorohexyl)-undecanoic Acid and D16PC. The pretransition and main transition of fully hydrated D16PC occur at 36.8 ( 0.1 and 41.8 ( 0.1 °C, respectively (for literature values, see ref 28). As seen in Figure 4b, the pretransition of D16PC bilayers with small mole fractions of the fluorinated acid behaves similarly to the one observed for bilayers of either D14PC or D15PC. The temperature of the pretransition shifts to higher temperatures with increasing acid content. At the same time, the width of the peak increases. The pretransition merges
with the main transition at X(D16PC) ∼ 0.95. The main transition occurs at a constant temperature of ∼42 °C up to X(D16PC) g 0.95, indicating immiscibilities in both the gel and fluid phase. Between 0.95 and 0.90, the onset temperature remains constant and at least two phase transitions can be observed (shoulder at 42 °C). In addition, the peak width increases, all of which indicates immiscibilities in the gel phase up to a mole fraction of approximately 0.90. At a mole fraction of 0.90, the maximum of the phase transition begins to shift to higher temperature. A sharp phase transition can be observed at a mole fraction of 0.80, which indicates the formation of a 4:1 phospholipid-acid complex. The onset of the main phase transition further increases below mole fractions of 0.8, and the width of the peak begins to increase as well. 11-(Perfluorohexyl)-undecanoic Acid and D17PC. D17PC in excess water shows a pretransition at 43.8 ( 0.4 °C and a main transition at 49.0 ( 0.1 °C, which is in good agreement with previous reports (Figure 6b).28 The pretransition shifts to higher temperatures with increasing acid content and merges with the main transition at X(D17PC) ≈ 0.95. The maximum of the main transition increases slightly with increasing acid content. Similar to the acid-D15PC and acid-D16PC systems, the onset of the main transition remains constant at low acid content (until X(D17PC) ) 0.87), indicating immiscibilities in the gel phase. At this mole fraction, the main phase transition shows a shoulder, that is, several phase transitions, at 48.9 ( 0.3 °C and is broader compared to the peaks observed at smaller and larger mole fractions. A sharp phase transition can be observed at a mole fraction of
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0.80, which indicates the formation of a 4:1 phospholipidacid complex. At X(D17PC) > 0.87, the peak of the main phase transition begins to broaden, resulting in a constant maximum and decreasing onset temperature. 11-(Perfluorohexyl)-undecanoic Acid and D18PC. The thermograms of the acid-D18PC system in excess water are shown in Figure 4c. D18PC in excess water has a pretransition a 53.1 ( 0.2 °C and a main transition at 55.2 ( 0.2 °C.28 The pretransition cannot be observed at a mole fraction of D18PC of 0.94. Above this mole fraction, the transition shows a very slight increase in onset and maximum temperature (X(D18PC) ) 0.85) followed by a decrease in both temperatures. This decrease in temperature is accompanied by an increased peak width starting at X(D18PC) g 0.70. In contrast to the binary phase diagrams discussed previously, this mixture does not show a clearly defined area of immiscibility at low concentrations of acid. However, the changes in the onset of the phase transition are only small, which makes it difficult to interpret this phase diagram. Discussion Monolayer Studies. To the best of our knowledge, this is the first study that reports the mixing behavior of a partially fluorinated carboxylic acid with a series of diacyl phosphatidylcholines with acyl chains ranging from C14 to C18 at the air-water interface. In earlier studies, negative deviations from ideality were observed for mixtures of various hydrocarbon29-33 and fluorocarbon acids17,18 with D16PC and/or D18PC. In the present study, we observe a negative deviation from ideality for mixtures of phosphatidylcholines with even-numbered acyl chains (Figure 2). The negative deviation and, hence, the interaction between the two components decrease in the following order: D16PC > D14PC > D18PC. The behavior of the acid-D18PC system resembles that of the octadecanoic acid-D18PC system.33 In this perhydrocarbon system, the two components of the monolayer show a weak interaction which is not strong enough to cause miscibility. The two odd-numbered phospholipids, D15PC and D17PC, show a weak negative deviation at a surface pressure of 5 mN/m but nearly ideal behavior above 10 mN/m (D15PC) and at 30 mN/m (D17PC). The phase transitions of both phospholipids are only slightly dependent on concentration. Thus, the miscibility of the partially fluorinated acid in monolayers composed of odd-numbered-chain phosphatidylcholines appears to be reduced compared to that of even-numbered-chain phosphatidylcholines. In all five mixtures, an increase in surface pressure results in (more) ideal behavior. In addition, the collapse pressures are in general concentration dependent, with D17PC and D18PC being an exception. These two observations suggest that the miscibility of the acid in the respective phosphatidylcholine increases with increasing surface pressure. However, further studies are necessary to confirm this interpretation of our data. Our results suggest that the fluorinated acid interacts with all five phospholipids at the air-water interface, thus resulting in (partial) miscibility. This macroscopic (29) Lee, K. Y. C.; Gopal, A.; von Nahmen, A.; Zasadzinski, J. A.; Majewski, J.; Smith, G. S.; Howes, P. B.; Kjaer, K. J. Chem. Phys. 2002, 116, 774. (30) Matuo, H.; Motomura, K.; Matuura, R. Chem. Phys. Lipids 1981, 28, 385. (31) Matuo, H.; Motomura, K.; Matuura, R. Chem. Phys. Lipids 1981, 28, 281. (32) Matuo, H.; Motomura, K.; Matuura, R. Chem. Phys. Lipids 1982, 30, 351. (33) Matuo, H.; Motomura, K.; Matuura, R. Chem. Phys. Lipids 1982, 31, 353.
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behavior, that is, attractive interactions, appears to be typical for all binary mixtures of carboxylic acids and phosphatidylcholines investigated to date at the air-water interface.17,18,29-33 Because only the chain length of the acyl groups in the phospholipid has changed, these attractive interactions are most likely interactions between the headgroups. The formation of hydrogen bonds between the carboxylic acid group and the phosphate diester function in the monolayer seems to be a plausible explanation. Similar interactions have been postulated in acid- and alcohol-phospholipid bilayers and result in the formation of 1:2 up to 1:4 complexes.34 Formation of a 1:1 complex has also been postulated for monolayers of hexadecanoic acid and D16PC.29 Differential Scanning Calorimetry Studies. At no mole fraction investigated for any of the five systems (i.e., mole fraction of the phosphatidylcholine between 1 and 0.6) was a peak corresponding to excess acid (melting point ) 62.5 °C22) observed. This observation suggests that all of the added acid is associated with the lipid. Similar to simple linear alcohols and carboxylic acids,34 the partially fluorinated acid is most likely incorporated into the lipid bilayer. The carboxylic acid group will be located near the phosphatidylcholine headgroup, where it could form hydrogen bonds with water as well as the headgroup of the phospholipids. The partially fluorinated tail is assumed to be aligned with the alkyl chains of the respective phospholipid. Most phosphatidylcholines show a pretransition from a tilted to a vertical configuration, that is, a Lβ′ (tilted gel) to Pβ′ (ripple gel) phase transition.28 Mixtures of alkanes, alcohols, and carboxylic acids with a chain length of g12 carbon atoms as well as numerous small molecules and proteins are known to reduce or eliminate the pretransition at low concentrations.34 Similarly, 11-(perfluorohexyl)undecanoic acid eliminates the pretransition of all five phosphatidylcholines at mole fractions ranging from X(phospholipid) e 0.94 to 0.90. In perhydrocarbon systems, the tilted arrangement of the hydrophobic chains below the pretransition temperature results from packaging constraints caused by the mismatch of the area of the tails (40 Å2 for both alkyl chains35) and the headgroup (46 Å2 36,37). A more optimal, that is, vertical, alignment of the tails is possible in the presence of small quantities of carboxylic acids and other small molecules, thus resulting in an elimination of the tilted gel phase and, hence, the pretransition. The elimination of the pretransition after addition of the fluorinated acids suggests a change in tilt angle. In addition to eliminating the pretransition, longer chain perhydrocarbon carboxylic acids (12-18 carbon atoms) are known to cause an increase and broadening of the onset temperature of the main phase transition of D16PC and other phosphatidylcholines. The increase of the transition temperature is caused by the formation of a 2:1 compound (i.e., an azeotropic behavior of the mixture).21 Although some peak broadening was observed in all five mixtures (Figure 7), 11-(perfluorohexyl)-undecanoic acid hardly increases the temperature of the phase transition of all five phosphatidylcholines. The partial phase diagrams shown in Figure 7a,c,d suggest the putative formation of 4:1 or 3:1 compounds resulting in slightly to (34) Lohner, K. Chem. Phys. Lipids 1991, 57, 341. (35) Gaines, G. L. Insoluble monolayers at liquid-gas interfaces; Interscience: New York, 1966. (36) Helm, C. A.; Mohwald, H.; Kjaer, K.; Als-Nielsen, J. Biophys. J. 1987, 52, 381. (37) Albrecht, O.; Gruler, H.; Sachmann, E. J. Phys. (France) 1978, 39, 301.
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diluted in a given biological system, and thus, changes in the thermodynamic properties of lipid assemblies may be, on average, too small to have any adverse effect. The partial phase diagrams of the three biologically relevant phospholipids shown in Figure 5 indicate immiscibilities at physiological temperature in D16PC (fluid phase) and, probably, D18PC (gel phase), whereas D14PC is miscible in the phase present under physiological conditions (i.e., the fluid phase). Such immiscibilities could give rise to the formation of domains within a bilayer where the concentration of the partially fluorinated acid is particularly high. Furthermore, the partial miscibility observed in monolayers of the fluorinated acid and phosphatidylcholines suggests that domains of different composition may be present at the air-water interface. Despite the overall low concentration of the fluorinated acid, such domains could locally cause an enrichment of the acid and, thus, alter the functions of biological phospholipid mono- or bilayers and cause nonspecific toxic effects, that is, after pulmonary administration. Conclusions
Figure 7. Changes in the half-width of the main phase transition of (a) even-numbered and (b) odd-numbered phosphatidylcholines. The ratio of the half-width is defined as the ratio between the half-width of the main transition of the mixed system and that of pure phospholipid. All data points are averages of at least three experiments ( one standard deviation.
marginally higher melting points (∆T ) 7.0, 2.5, and 1.8 °C, respectively) compared to that of the pure phosphatidylcholine. In comparison, the melting point of the hexadecanoic acid-D16PC 2:1 compound is more than 20 °C above that of pure D16PC.21 This drastic increase in the temperature of the phase transition in the perhydrocarbon system has been explained by the more optimal packing of the alkyl chains in the 2:1 mixture and, hence, an increase in chain-chain interactions. Obviously, the partially fluorinated tail of 11-(perfluorohexyl)-undecanoic acid would not be expected to favor improved packing of the bilayer. These packing problems can be explained with the structure of the perfluorinated tail. The perfluorinated terminus of 11-(perfluorohexyl)-undecanoic acid is lipophobic, significantly larger compared to an alkyl chain. In addition, the -CF2-CH2- linkage is highly polar. We, and others, have shown that such a structure results in packing problems and, thus, weaker chain-chain interactions in monolayers of partially fluorinated amphiphiles.17,19,22,23 Therefore, none of the 11-(perfluorohexyl)undecanoic acid-phosphatidylcholine mixtures under investigation showed the drastic increase in temperature typical for similar hydrocarbon systems. Ultimately, the observed changes in the thermodynamic properties of both biological mono- and bilayers may (adversely) influence their biological function as well. As a xenobiotic, the partially fluorinated acid will be highly
On the basis of our monolayer and DSC studies, the partially fluorinated acid is able to incorporate into phosphatidylcholine mono- and bilayers, aligning itself with the acyl chain of the phospholipids where it affects the packing of the lipids and, ultimately, their thermodynamic properties. Our monolayer results show attractive interactions between the fluorinated acid and the respective phospholipids. There is some evidence in the DSC data for the formation of 4:1 and 3:1 complexes in several mixtures, which suggest the presence of such interactions in the bilayers as well. In comparison to binary perhydrocarbon systems, the partially fluorinated acid does not drastically alter the macroscopic characteristics of the phase behavior of the composed monolayers (e.g., concentration dependence of the phase transition and collapse point). This is different from our bilayer studies, where the partially fluorinated acid shows little effect on the main phase transition whereas perhydrocarbon acids of similar chain length cause a drastic increase and broadening of the main phase transition. The difference in the fluorocarbon- versus perhydrocarbon-phosphatidylcholine system is probably the result of packing constraints in the bilayer which result from the necessity to accommodate the strong dipole moment of the -CF2-CH2linkage. The partial phase diagrams of both monolayers and bilayers show immiscibilities which could give rise to the formation of domains with high concentrations of the partially fluorinated acid. Unfortunately, the impact of domain formation, that is, the microscopic phase behavior, on the biological function of biological mono- and bilayers is hardly understood, even in typical hydrocarbon systems. Considering the great potential of partially fluorinated materials for a variety of biomedical applications, a further understanding of the microscopic phase behavior of partially fluorinated surfactants with biological surfactants on a molecular level is warranted. Acknowledgment. This work was in part supported by a grant from the American Lung Association (RG-024N) and a grant from the National Science Foundation (NIRT 0210517). LA034734B
