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Langmuir 1999, 15, 3430-3436
Influence of Surface Charge and Hydrocarbon Chain Length on the Sponge-Vesicle Transformation of an Ionized Phospholipid M. Koshinuma,† K. Tajima,‡ A. Nakamura,§ and N. L. Gershfeld* National Institute of Arthritis, Musculoskeletal, and Skin Diseases, National Institutes of Health, Bethesda, Maryland 20892 Received September 8, 1998. In Final Form: February 23, 1999 The influence of charge density and hydrocarbon chain length on the sponge-vesicle transformation of two bilayer-forming ionic phospholipids, the sodium salts of dilauroyl- and dimyristoylphosphatidylglycerol (NaDLPG and NaDMPG), was examined by measuring the solution properties of the lipids in water. The phase diagrams of these compounds in water indicate they undergo a transformation from a transparent, jelly-like sponge phase to unilamellar vesicles at a critical temperature T*; for NaDLPG T* ) 19 °C, and for NaDMPG T* ) 31.6 °C. At T > T* multilamellar vesicles form. The Krafft temperature for NaDLPG is ∼5 °C, just above Tm, the gel-liquid crystal transition temperature, and for NaDMPG it is ∼32 °C, slightly higher than T*. For both lipids, the charge density of the equilibrium monolayers at the air/water interface decreases dramatically as T increases and approaches T*. This effect is attributed to hydrolysis of the phosphate moiety of the lipid. Changes in solubility consistent with a decrease in bilayer charge density at T* are also observed. The transformation from the sponge phase to vesicles at T* is a structural response to the change in bilayer charge density. The results emphasize the importance of bilayer-localized chemical reactions in the transformation of the sponge state to vesicles.
I. Introduction When an ionic phospholipid, the sodium salt of dimyristoylphosphatidylglycerol (NaDMPG), is dispersed in water at temperatures below a critical value T*, a viscous, clear, jelly-like suspension forms.1a This state has been identified recently as a sponge phase (T. Heimburg, personal communication)1b and consists of an extended bilayer structure that fills the entire volume of the suspension. When heated to 32 °C (T*), the sponge phase transforms to unilamellar vesicles, which become multilamellar upon heating to higher temperatures.1 The sponge-vesicle transformation may be observed by phase contrast microscopy because the sponge phase appears structureless at T < T*, and the vesicles that form at T* are large enough to be viewed by phase contrast microscopy.1a The forces which determine the transformation conditions, and assembly of vesicles at T*, are, at present, poorly defined. The formation of the jelly-like state of the sponge phase in all likelihood represents, at least in part, a balance between the electrostatic repulsive and van der Waals attractive forces among the bilayers of this phase. Indeed, with higher homologues of these compounds (diC-16 and diC-18PG) the sponge phase does not form as the balance between the two forces shifts. In the present study we have addressed the question of the relation between these two forces in the sponge-vesicle transformation by examining the phase relations for NaDMPG and its homologue dilauroylphosphatidylglycerol (NaDLPG) in * To whom correspondence should be addressed. Address: NIH, Building 6, Room 139, Bethesda, MD 20892-2751. E-mail: ngo@ cu.nih.gov. Fax: 301-402-0009. † Present address: Teikyo University of Technology, Chiba, Japan. ‡ Kanagawa University, Yokohama, Japan. § Nagoya City University, Nagoya, Japan. (1) (a) Gershfeld, N. L.; Stevens, W. F., Jr.; Nossal, R. J. Faraday Discuss. Chem. Soc. 1986, 81, 19. (b) A schematic of a sponge phase is illustrated in Cates, M. E. Philos. Trans. R. Soc. London A 1993, 344, 339, Figure 4.
the region of T*. The phase diagrams have been obtained by studies of solutions of these compounds in water. We shall demonstrate that in each of the equilibrium condensed states that form (bilayer and the equilibrium monomolecular films at the air/water surface) the surface charge density decreases as temperatures approach T* and that the transformation from the sponge to the vesicle state is a result of this decrease in charge density. II. Experimental Section A. Materials and Preparation of Aqueous Lipid Solutions. The sodium salts of dilauroylphosphatidylglycerol (NaDLPG) and dimyristoylphosphatidylglycerol (NaDMPG) (Avanti Polar Lipids, Birmingham, AL), >99 mol %, were used without further purification. To avoid forming bilayers in the preparation of aqueous solutions, it is necessary to first dissolve the lipid in methanol and then spread this solution as a thin film over the inner surface of a 500 mL Pyrex volumetric flask. A stream of nitrogen is used to evaporate the methanol, the volume of water (triple distilled, the last two distillations from a quartz apparatus) required to form the desired solution concentration is added, and the contents of the flask are then vortexed vigorously for about 15 min. Saturated solutions of the lipid are prepared by adding water to the weighed dry lipid and vortexing the mixture in a polypropylene tube. The sample tube is flushed with nitrogen, capped, and allowed to equilibrate in a constanttemperature shaking water bath for 2-4 h. Further equilibration does not significantly influence the solubility. Generally, at least two to four times the amount of lipid required to saturate the solution is used. B. Surface Tension. The surface tensions of the lipid solutions are measured by the Wilhelmy plate method using a quartz plate suspended from a strain gauge; the output from the bridge circuit is amplified and recorded. The equilibrium surface tension is indicated by constant readings for at least 2 h. The sensitivity and reproducibility for this method are (0.2 mN/m. A constant temperature ((0.2 °C) is maintained for each isotherm. C. Solubility. Equilibrium lipid solubilities are obtained by two methods: from centrifugation studies in which the bulk lipid of saturated solutions is pelleted, freeing the supernatant for
10.1021/la981200f CCC: $18.00 © 1999 American Chemical Society Published on Web 04/22/1999
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analysis,1a,2 and from surface tension measurements. In principle, at constant temperature, equilibrium solubility requires demonstrating that the solution concentration is constant with time and is independent of the amount of bulk lipid phase. An ultracentrifuge (Beckmann Model L8-60M) is employed to separate the bulk lipid from the solution using an SW-40 rotor at 40 000 rpm; temperature control is maintained from 0 to 45 ( 0.5 °C. Since high pressures are generated in the centrifuge tube, aliquots of the supernatant are taken at different depths within the tube. The phospholipid concentration is obtained by measuring the phosphorus content of each aliquot.3 This procedure is routinely followed to establish that centrifugation is complete and to verify that the pressures generated in the centrifuge tubes do not influence the solubilities. A description of our findings with regard to these two requirements for solubility measurements is presented in the Results. The surface tension method requires that the measurement be independent of the amount of excess lipid in the dispersion. There is generally good agreement between the two methods. Since the sedimentation method requires extended centrifugation times for NaDLPG, there is a possibility that NaDLPG will degrade to the lysophospholipid and lauric acid. We therefore equilibrated suspensions of the lipid at 10, 15, and 20 °C for 48 h and analyzed for the presence of the lyso compound by thin-layer chromatography. The amount of the lyso compound formed was T*,1,2 and the present measurement confirms that conclusion. Thus, at 28 °C (11) Zana, R. J. Colloid Interface Sci. 1980, 78, 330.
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Figure 5. (A) Degree of Na ion dissociation from NaDLPG micelles R as a function of temperature. (B) cmc of NaDLPG, expressed as ln(mole fraction) versus temperature: (b) from surface tension measurements; (O) from emf measurements. Each of the curves was fitted by a second-order polynomial.
Figure 6. E, the emf of Na ions in solutions of NaDMPG as a function of concentration and temperature. The standard curve for NaCl solutions is given by the dashed line, with all curves normalized to the value of E for 10-3 M NaCl set equal to zero at each temperature. See legend to Figure 4 and Experimental Section for the description of the method: (2) 28 °C; (b) 34 °C; (0) 36 °C.
the activity of Na ions in a saturated solution of NaDMPG is identical to that of a solution of NaCl at the same concentration (no micelles form), while at 34 and 36 °C, temperatures exceeding T*, micelle formation is indicated. Na ion dissociation for these micelles is calculated to be ∼68% at 34 °C and ∼74% at 36 °C, significantly greater than that in NaDLPG micelles at these temperatures if only thermal agitation is considered (e.g., R for NaDLPG would be approximately 30% at 36 °C). For NaDMPG at
Koshinuma et al.
these temperatures the high value of Na ion dissociation is therefore primarily due to a decrease in micelle charge. As in monolayers at these temperatures above T*, hydrolysis is the likely source of the reduced micelle charge. D. Bilayer Solubility and the Phase Diagrams of NaDLPG and NaDMPG. Preliminary experiments for defining the appropriate centrifugation conditions for separating NaDLPG bilayers from solution were based on our experience with NaDMPG, where 24 h of centrifugation at 40 000 rpm was found to be sufficient. However, it quickly became apparent that these conditions are not suitable for NaDLPG. Assuming that the solubility of NaDLPG is at least an order of magnitude higher than that of NaDMPG, we prepared suspensions of 2-5 mM; the jelly-like character of these suspensions is obvious, since they are both transparent and viscous. Centrifugation at 10 °C for 24 h yielded very little sediment. Subsequent runs of 48, 65, and 120 h at 40 000 rpm found decreasing amounts of lipid in the suspension, with the two shortest centrifugation times exhibiting pronounced concentration gradients indicative of incomplete separation of solution and bilayer. For a centrifugation time of 120 h sedimentation was apparently complete because no concentration gradients were present and the lipid concentration in the supernatant was independent of the initial concentration of the dispersion. Since these are the principal requirements for bilayer-free solutions, we chose 120 h as the time necessary for ensuring complete separation of solution from the bilayer phase. With dispersion concentrations of approximately 2, 3, and 4 mM, the solubility of NaDLPG between 5 and 17 °C is independent of the initial composition and the depth within the centrifuge tube from which the samples are taken. These solubilities along with those obtained from surface tension measurements in Figure 1 are included in the phase diagram of Figure 7A. Where data overlap, the two methods show good agreement. At T g T* (19 °C), when vesicles appear, incomplete pelleting of the lipid is observed, indicating that sedimentation rates slow markedly. Since the sedimentation velocity of particles decreases as particle size decreases,12 we attribute the slow sedimentation rates to a combination of the high viscosity of the suspensions at the lipid concentrations used and the small vesicles of 1-10 µm which form from the transformation of the sponge state at T*. We do not see this affect with the higher homologue NaDMPG above its T* because the suspensions are 1/10th as concentrated and, consequently, much less viscous, and the vesicles which form are considerably larger, namely 10-50 µm. Incomplete sedimentation even after 120 h of centrifugation makes further use of centrifugation for NaDLPG solubilities at T > T* impractical. For DLPG solubilities at these temperatures we rely instead on the values obtained by the surface tension measurements in Figure 1, and they are shown in Figure 7A. Of particular significance is the fact that the solubility at T > T* is almost independent of temperature. For comparison, the phase diagram for NaDMPG is included in Figure 7B. The phase diagrams for NaDLPG and NaDMPG show the locations of the gel-liquid crystal transitions, Tm, and the temperatures where unilamellar vesicles form, T*. Both compounds form micelles when their Krafft temperatures are exceeded; for NaDLPG it is 5 °C, about 1 °C above Tm, while for NaDMPG it is 32 °C, about 8 °C above Tm and just above T*. Unlike the case for typical (12) Alexander, A. E.; Johnson, P. Colloid Science; Oxford University Press: London, 1949; Vol. 1, p 226.
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dXS/dT for NaDMPG above the Krafft temperature (T > 32 °C) is at least an order of magnitude smaller than that for a typical ionic surfactant.13 IV. Discussion
Figure 7. (A) Phase diagram for NaDLPG. Solubility XS from (4) surface tension measurements (Figure 1) and (b) centrifugation. Critical micelle concentration Xcmc from (O) emf measurements (Figures 4 and 5) and (2) surface tension measurements (Figure 1). (B) Phase diagram for NaDMPG. Solubility XS from (b) centrifugation,1a (O) emf measurements (Figure 6), and (2) surface tension (Figure 3). Critical micelle concentration Xcmc from (O) emf measurements (Figure 6) and (2) surface tension measurements (Figure 3). Table 1. Thermodynamics of Micelle Formation for NaDLPG: Data from Figure 5A kcal/mol T, °C
R, %
15 20 25
23 25 26
∆G°
a
-15.9 -16.2 -16.5
∆H° a
T∆S°
0.1 4.1 0.6
16.0 20.3 17.1
a ∆G° ) RT(2 - R) ln X 2 cmc; ∆H° ) -RT (2 - R) d ln Xcmc/dT + RT2 ln Xcmc d R/dT.
ionic surfactants, the cmc’s above their Krafft points are strongly temperature dependent, decreasing dramatically as temperatures increase. For NaDLPG, further increases in temperature find cmc behavior that is more like that for a surfactant, with an assembly process that is entropically driven (see Table 1). The equivalent temperature range for NaDMPG could not be reached because the concentrations were too low ( T*, but the close coincidence of the Krafft point with T* masks the effect, since surfactant solubility generally increases as the Krafft temperature is exceeded. Indeed, the increase in solubility
At the outset of this study we surmised that the transformation from the jelly-like sponge phase to vesicles at T* would likely reflect a decrease in electrostatic repulsive forces which arise from the electrical double layer of the bilayer. In this system, where the only counterions for the lipid structures that form are the Na and H ions, a decrease in surface charge can occur only by direct binding of the Na counterion of the lipid or by hydrolysis of the phosphate group. The distinction between the two is made by the temperature dependence of the charge density, with Na binding decreasing and hydrolysis increasing with rising temperature (see below). In virtually all of the condensed systems in this study (bilayers, monolayers, and micelles), hydrolysis of the phosphate group is the principal source of the decrease in surface charge. Two experimental results from this study provide evidence that the charge density in the bilayer has decreased. In the first, the ratio of ionized [PG-] to unionized [PGH] in condensed monolayers decreases as temperatures approach T*. Since the packing densities and internal energies of condensed films near T* are equivalent to those in the bilayer,5,14 the decrease in suface charge may be assumed to occur as well for the equilibrium bilayers at these temperatures. The second result is shown by the solubility-temperature relations of the lipids above T*. For NaDLPG the solubility becomes almost independent of temperature, while for NaDMPG the temperature dependence of the solubility is about an order of magnitude smaller than at for typical ionic surfactants.13 If the Krafft point and T* were not coincident, NaDMPG would behave similarly to NaDLPG at this temperature. Essentially, the solubilities of both lipids above T* are less than expected. A similar leveling of the solubility with temperature due to a neutralization of the charge has been observed for a cationic detergent.15 We have attributed this change in charge density to hydrolysis of the phosphate group in the phospholipid. Direct evidence for the presence of the hydrolysis reaction in these bilayers has been demonstrated by showing that the solubility of NaDMPG bilayers increases when small amounts of NaCl are added to the dispersion,2 a phenomenon that entails displacing bound H ions with Na ions.16 The micelles exhibit more complicated charge behavior with temperature than the monolayers and bilayers formed by these lipids. For NaDLPG micelles values of R and its temperature coefficient are characteristic of typical ionic surfactants, and the thermodynamics of micellization indicates that assembly is dominated by the entropy of the process (Table 1). Although above 20 °C (T*) some Na binding appears to occur, it makes only a relatively small contribution to the energetics of micellization (Table 1). The behavior of NaDMPG micelles with temperature, however, appears to resemble that of its monolayers and bilayers rather than that of typical surfactants. Although no micelles form below 31.6 °C (i.e., T*), for comparison, the charge on NaDMPG micelles at T > T* is low because Na ion dissociation is high (∼70%) and appears to increase with temperature. This high degree of Na ion dissociation (13) Alexander, A. E.; Johnson, P. Colloid Science; Oxford University Press: London, 1949; Vol. 2, pp 683. (14) Fukada, K.; Gershfeld, N. L. J. Phys. Chem. B 1997, 41, 8225. (15) Dai, Q.; Laskowski, J. S. Langmuir 1991, 7, 1361. (16) Payens, Th. A. J. Philips Res. Rep. 1955, 10, 425.
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is far greater than that expected from thermal forces alone, which would predict ∼30% dissociation according to the properties of NaDLPG (see section III.C). Thus, for NaDMPG micelles, as in the case of its monolayers and bilayers, the low charge is due to hydrolysis of its phosphate groups. Since the charge effects observed with the lipids are localized to temperatures that are near T*, it is reasonable to attribute the collapse of the jelly-like sponge phase at T* to the decrease in bilayer charge density that arises from the hydrolysis of the phosphate group. In solutions of phosphoric acid, hydrolysis of the phosphate radical is a continuously increasing function of temperature.17 As we have shown, the increase in hydrolysis of the phosphate group, and its concomitant decreased charge, with temperature occurs as well in both monolayers and bilayers of phosphatidylglycerol. However, it is puzzling that this effect in the condensed lipid states is restricted to a relatively narrow temperature range near T*, rather than being continuous over a much wider range as in solution. Since the critical temperature for NaDLPG and NaDMPG
depends strongly on hydrocarbon chain length, perhaps the change in surface charge at T* reflects an accommodation of the phosphate groups to a structural change that is inherently dominated by the hydrocarbon chainchain interactions. This seems not to be the case, because, apart from the obvious change in morphology of the sponge-vesicle transformation, there is no obvious change in bilayer state at T* despite the decrease in surface charge. At T* each of these lipids is well above the melting transition, Tm (Figure 7).. Indeed, the temperature dependence of the heat capacity for these compounds shows no latent heat but, rather, an anomaly at T* characteristic of a higher order transformation.18 We do not have an explanation for the limited temperature range in which this change in phosphate charge on the bilayers occurs. However, we have demonstrated that charge effects on condensed lipid states are highly specific. A detailed examination of the chemistry of the phosphate groups in situ is required if we are to understand their contributions to the forces which control the physical states that lipids form and the conditions leading to their transformations. Present studies are directed toward elucidating the specific chemical nature of these contributions.
(17) Nims, L. F. J. Am. Chem. Soc. 1934, 56, 1110. This report shows the pKa of the first dissociation constant of phosphoric acid increasing monotonically with increasing T; hydrolysis should therefore also increase monotonically with T.
LA981200F (18) Fukada, K.; Tajima, K.; Gershfeld, N. L. Biophys. J. 1994, 66, A289.
