Phase Behavior of Palmitic Acid/Cholesterol/Cholesterol Sulfate Mixtures and Properties of the Derived Liposomes

Phase Behavior of Palmitic Acid/Cholesterol/Cholesterol Sulfate Mixtures and Properties of the Derived Liposomes

Guillaume Bastiat and Michel Lafleur*

Department of Chemistry, UniVersite´ de Montre´al, C.P. 6128, Succursale Centre Ville, Montre´al, Que´bec, H3C 3J7, Canada

ReceiVed: February 26, 2007; In Final Form: July 9, 2007

The phase behavior of mixtures formed by palmitic acid (PA), cholesterol (Chol), and sodium cholesteryl sulfate (Schol) has been characterized by differential scanning calorimetry and infrared and ²H NMR spectroscopy. It is reported that it is possible to form, with PA/sterol mixtures, fluid lamellar phases where the sterol content is very high (a sterol mole fraction of 0.7). As a consequence of the rigidifying ability of the sterols, the PA acyl chains are very ordered. The stability of these self-assembled bilayers is found to be pH-dependent. This property can be controlled by the Chol/Schol molar ratio, and it is proposed that this parameter modulates the balance between the intermolecular interactions between the constituting species. A phase-composition diagram summarizing the behavior of these mixtures as a function of pH, at room temperature, is presented. It is also shown that it is possible to produce large unilamellar vesicles (LUVs) from these mixtures, using standard extrusion techniques. The resulting LUVs display a very limited passive release of the entrapped material. In addition, these LUVs constitute a versatile vector for pH-triggered release.

Introduction

Phospholipid-sterol mixtures have been extensively studied to understand several phenomena linked to lipid bilayers and cell membranes. The discovery of the formation of the liquid ordered (lo) phase in the presence of cholesterol (Chol)^1,2has generated a reinvestigation of the roles of Chol in membranes.

It has been shown that Chol can induce the formation of a lo phase in various phospholipid matrixes.^1,3-7The existence of this phase has been extended to matrixes formed by linear and saturated fatty acid.^8-10In this case, the resulting self-assemblies have been shown to be influenced by the pH. At pH 5.5, the palmitic acid (PA)/Chol system undergoes a transition at∼48

°C from solid and phase-separated lipids to a fluid and homogeneous lo phase. In these conditions, the system shows a eutectic behavior at a molar PA/Chol composition of 30/70.

At higher pH (g7.5), the PA/Chol mixture leads to a lo lamellar phase, stable over a wide temperature range.⁹Moreover it was recently established that it is possible to prepare large unilamellar vesicles (LUVs) from this system, using standard extrusion procedures.¹¹These LUVs were shown to have an imperme- ability drastically reduced compared to that of phospholipid LUVs, likely because of their very high Chol content. In addition, they display a pH sensitivity that can be exploited for pH-triggered release, because of the presence of the fatty acid and the different influence of its protonated and unprotonated states on the LUV stability. pH-sensitive liposomes have attracted considerable interest in recent years for their potential to release encapsulated hydrophilic molecules at specific loci in an organism where local pH is different than physiological pH.¹²

In the present study, we examined whether it is possible to modulate the phase propensities of PA/sterol systems by substituting Chol by sodium cholesteryl sulfate (Schol). This change in chemical composition provides information relative to the origin of the stability of these self-assemblies. Moreover

this change, as described below, extends the family of non- phospholipid LUVs with high sterol content and provides additional flexibility in the control of the pH-triggered release of the LUV content. Sodium cholesteryl sulfate has been shown to have an influence analogous to that of Chol on phospholipid bilayers and leads to the formation of a fluid and highly ordered phase.^13-16The rigidifying effect of Schol on lipid acyl chains was, however, found to be smaller than that of Chol.^14,16Indeed the negative charge carried by Schol is another distinctive aspect of this sterol relative to Chol. In some biological systems, the presence of Schol appears to play a key role.¹⁷For example, it was shown that the proportion of Schol modifies the fluidity of the extracellular lipidic matrix of the stratum corneum (SC), the top layer of the epidermis.^18,19The accumulation of Schol in the SC, caused by a genetic defect leading to a deficit in steroid sulfatase, is associated with a severe inhibition of the desquamation process and the formation of large scales at the surface of the skin, for the people suffering from the recessive X-linked ichthyosis.^20,21 Along the same line, the balance between Chol and Schol has been proposed to be critical for the normal fertilization by sperm.²² Schol was also found to play an essential role in blood platelets.²³

We have studied the behavior of the system based on PA, Chol, and Schol, to understand the influence of the chemical difference existing between the two sterols. First, we studied the thermotropism of the PA/Chol1-x/Scholxsystem with a PA/

total sterol molar composition of 0.3/0.7, and 0 e x e 1, substituting Chol by Schol. We established, combining dif- ferential scanning calorimetry (DSC) and infrared (IR) and²H NMR spectroscopy, a pH-composition diagram revealing the opposed behavior of the sterols as a function of pH. From this diagram, the adequate conditions to prepare nonphospholipid liposomes (NPLs) by extrusion were identified. The formation and the permeability of these liposomes were characterized as a function of temperature and pH. A straightforward correlation between the phase behavior and the stability of the liposomes was identified.

* Author to whom correspondence should be sent. Phone: (514) 343- 5936. Fax: (514) 343-7586. E-mail: michel.lafleur@umontreal.ca.

10929 J. Phys. Chem. B 2007, 111, 10929-10937

10.1021/jp0715833 CCC: $37.00 © 2007 American Chemical Society Published on Web 08/25/2007

Canada). All solvents and products were used without further purification.

Mixtures of PA, Chol, and/or Schol were prepared by dissolving weighed amounts of the solids in a mixture of benzene/methanol, from 95/5 to 75/25 (v/v) depending of the lipid solubility. The solutions were then frozen in liquid nitrogen and lyophilized for at least one night to allow complete sublimation of the organic solvent. For DSC and IR spectro- scopy, the mixtures were prepared with PA, Chol, and/or Schol, whereas PA was replaced by PA-d31for²H NMR experiments.

In this study, the mixtures had a PA/total sterol molar ratio of 0.3/0.7, except otherwise stated. The molar proportion of Chol and Schol was indicated by PA/Chol1-x/Scholxwhere x repre- sents the molar fraction of Schol relative to total sterol.

DSC, IR, and²H NMR Spectroscopy. The freeze-dried lipid mixtures were hydrated with a MES/TRIS buffer (TRIS 50 mM, MES 50 mM, NaCl 10 mM, EDTA 5 mM) providing a buffered range from pH 5 to 9. The buffer was prepared with Milli-Q water for DSC, D2O for IR spectroscopy, and deuterium- depleted water for ²H NMR spectroscopy. The final lipid concentration was 5 mg/mL for DSC, 250 mg/mL for IR spectroscopy, and 30 mg/mL for²H NMR experiments. The suspensions were subjected to five cycles of freezing-and- thawing (from liquid nitrogen temperature to 75 °C) and vortexed between successive cycles, to ensure a good hydration of the samples. The pH was measured and readjusted by the addition of HCl or NaOH (DCl and NaOD for IR experiments) diluted solutions after the hydration.

The DSC was performed with a VP-DSC microcalorimeter (MicroCal, Northampton, MA). The reference cell was filled with the buffer. The data acquisition was performed from 20 to 80°C, with a heating rate of 20°C/h.

The IR spectra were recorded on a Bio-Rad FTS-25 spec- trometer, equipped with a water-cooled globar source, a KBr beam splitter, and a DTGS detector, using an established protocol.¹⁰Briefly, an aliquot of the sample was placed between two BaF2windows separated by a 5µm thick Teflon ring. This assembly was inserted in a brass sample holder, whose tem- perature was controlled using Peltier thermopumps. Each spectrum was the result of 40 scans with a nominal 2 cm^-1 resolution and Fourier transformed using a triangular apodization function. The temperature was varied from low to high, with 2

°C steps and a 5 min incubation period prior to data acquisition.

The reported band positions correspond to the center of gravity, calculated from the top 5% of the band.²⁴

The ²H NMR spectra were recorded on a Bruker AV-600 spectrometer, using a Bruker static probe equipped with a 5 mm coil. A quadrupolar echo sequence was used with a 90°

pulse of 2.7µs and an interpulse delay of 25µs. After the second pulse, 2048 points were recorded in quadrature mode, with a dwell time of 0.5µs. The recycling time was 60 s. In absence of a slow-relaxation (solid) component, the recycling delay was

unilamellar vesicles made from PA/sterols mixtures (20 mM), loaded with calcein (80 mM), were prepared in the MES/TRIS buffer described above.

The LUVs were prepared by extrusion using a handheld Liposofast extruder (Avestin, Ottawa, Canada). The dispersions were passed 15 times through two stacked polycarbonate filters (100 nm pore size) at∼75°C. Calcein-containing LUVs were separated at room temperature from free calcein by gel permeation chromatography, using Sephadex G-50 medium (column diameter, 1.5 cm; length, 25 cm; equilibrated with a MES/TRIS buffer (MES 50 mM, TRIS 50 mM, NaCl 130 mM, EDTA 5 mM, pH 5.5 and 7.4)). The collected vesicle fraction was diluted 100 times to perform the measurements. The stock LUVs suspensions were incubated at a given temperature.

Fluorescence spectra were recorded on a SPEX Fluorolog spectrofluorimeter; the excitation and emission wavelengths were 490 and 513 nm, respectively, and the band passes were set at 1.5 nm for excitation and 0.5 nm for emission.

To determine the proportion of entrapped calcein, its fluo- rescence was measured before and after the addition of 10µL of Triton X-100 solution (10% (v/v) in the MES/TRIS buffer).

The fluorescence intensity, measured after addition of detergent, corresponded to complete calcein release and was used to normalize the leakage. To study the passive leakage at various temperatures (25, 37, and 50°C), the fluorescence intensities at 513 nm were measured from an aliquot of the stock LUVs solution prior (Ii) and after (Ii+T) the addition of Triton X-100.

These intensity values corresponded to t) 0 for the kinetic studies. After a given time, the fluorescence intensities at 513 nm were measured on another aliquot of the same stock LUVs solution before (If) and after (If+T) the addition of Triton X-100.

The percentage of encapsulated calcein remaining at that time in the LUVs was calculated as follows:

The percent of release corresponded to the balance (100 - percent of encapsulated calcein). The pH-triggered leakage of calcein was also examined. The pH was modified by adding aliquots of a diluted NaOH or HCl solution, and the calcein fluorescence intensities were measured immediately after the stabilization of pH. The percentage of released calcein was calculated with the relation presented above, except Iiand Ii+T

corresponded to the measurements at the initial pH, before and after the addition of Triton X-100, respectively. Ifand If+Twere obtained on an aliquot at a modified pH, before and after the addition of Triton X-100, respectively. The pH effect was examined either by an increase or a decrease of pH. The calcein fluorescence intensity was relatively constant over the investi- gated pH range.²⁷The hydrodynamic diameters of the LUVs were measured at 25°C using a Coulter N4 Plus quasi-elastic light-scattering apparatus coupled with a Malvern autocorrelator.

% of encapsulated calcein)

(

)

The scattering intensity was adjusted by the dilution of the dispersion with the MES/TRIS buffer. The measurements were performed∼30 min after the extrusion. In the case of the pH variations, the measurements were carried out∼2 h after the pH change.

Results

DSC Experiments. Figure 1 presents thermograms of various mixtures of PA and sterol. For PA/Chol mixtures at pH 5.5, a phase transition was observed at 50 °C, corresponding to a change from a solid to lo phase, as previously described.⁹The melting of pure hydrated PA is observed at 61 °C.^8,9 The observation of a single and relatively narrow peak is indicative of the formation of a eutectic mixture in these proportions. When a proportion of Chol was replaced by Schol (xg0.25), no phase transition was observed. The thermograms obtained from PA/

Chol0.75/Schol0.25and PA/Schol at pH 5.5, shown in Figure 1, illustrate this finding. This behavior suggested that these systems existed in the same phase over the whole temperature range and, as discussed below, it is a lo phase. Ouimet et al. have shown that for the PA/Chol system, a solid-to-lo phase transition was observed at 54°C for acidic pH, whereas the system existed in a stable lo phase between 20 and 70°C at basic pH: no transition was observed for the PA/Chol system at pH 9.⁹For

PA/Schol mixtures at pH 9, two maxima, at 43 and 55°C, were observed in the thermograms. As confirmed below, the endo- thermic process corresponded to a solid-to-lo phase transition.

The thermogram of PA/Schol mixtures at pH 9 with a molar composition of 0.2/0.8 (Figure 1e) displayed, as the 0.3/0.7 PA/

Schol mixture, peaks at 46 and 55°C. For the PA/Schol system, no eutectic composition could be observed at high pH.

IR Spectroscopy Experiments. The thermal behavior of the fatty acid in the mixtures was examined using the band associated with the symmetric C-H stretching (νCH) mode (Figures 2-4). This mode is mainly sensitive to trans-gauche isomerization along the acyl chains, and to the interchain coupling, providing a sensitive probe for transitions involving the introduction of chain conformational disorder.^29-31 The transition of pure hydrated PA, easily detected by the abrupt shift of theνCHband from 2848.5 to about 2853.5 cm^-1, was observed at about 60°C, corresponding to the melting temper- ature of the fatty acid.⁸This curve is reproduced in Figure 2 for reference purposes. The two extreme νCHpositions were representative of highly ordered and disordered chains, respec- tively.²⁹A transition was observed between 45 and 50°C, for the PA/Chol system at pH 5.5, in agreement with the thermo- grams presented above and previous results.^8,9At low temper- atures, the position of theνCHband is 2848.5 cm^-1, indicative Figure 1. Thermograms of PA/Chol (a) and PA/Chol0.75/Schol0.25(b)

at pH 5.5 and PA/Schol at pH 5.5 (c) and 9 (d). For these mixtures, the PA/total sterol molar ratio was 0.3/0.7. Thermogram e was obtained from a PA/Schol mixture, at pH 9, with a molar ratio of 0.2/0.8.

Figure 2. Thermotropism of PA in PA/sterol mixtures probed using theνCHband position. PA alone ([), PA/Chol (9), PA/Chol0.9/Schol0.1

(1), PA/Chol0.75/Schol0.25(b), PA/Chol0.5/Schol0.5(O), and PA/Schol (4) mixtures, at pH 5.5. For all these mixtures, the PA/total sterol molar ratio was 0.3/0.7.

Figure 3. Thermotropism of PA in PA/sterol mixtures probed using theνCH band position. PA/Chol (9), PA/Chol0.75/Schol0.25 (b), PA/

Chol0.5/Schol0.5 (O), PA/Chol0.25/Schol0.75 (3), and PA/Schol (4) mixtures at pH 9. For all these mixtures, the PA/total sterol molar ratio was 0.3/0.7.

Figure 4. Thermotropism of PA in PA/Schol mixtures probed using theνCHband position, at pH 5.5 (]), 7.4 (3), 8 (b), and 9 (9). For all these mixtures, the PA/Schol molar ratio was 0.3/0.7.

of PA acyl chains in solid state. At 52°C, the position of the νCHband was 2852 cm^-1, a value observed for lo phases formed by Chol-rich bilayers.^5,10,29,32When the Schol content, x, in the PA/Chol1-x/Scholxmixtures was increased, the transition slightly shifted toward lower temperatures and became less sharp compared to those observed for the PA/Chol system. Moreover, the increase in Schol content led to an increase in theνCHvalues measured at low temperatures. At 25°C, for example, theνCH

values increased from 2849 to 2851.2 cm^-1, for x varying from 0.1 to 0.5, for PA/Chol1-x/Scholx mixtures at pH 5.5. These values suggest that, on average, the acyl chains were no longer highly ordered at room temperature. Above 50 °C, the νCH

values corresponded to those observed for lo phases, indepen- dently of the sterol composition. For the PA/Schol system, any transition was observed as the νCH position remained fairly constant at∼2852 cm^-1.

Figure 3 reports the effect of temperature on theνCHposition for PA/Chol1-x/Scholx mixtures at pH 9. For x < 0.5, no

from 2849.5-2850 cm ¹at low pH, to 2851.5 cm ¹, for pH between 7.4 and 9. At pH 5.5, the amplitude of this transition was greatly reduced, a result consistent with the DSC thermo- grams. This effect was mainly associated with an increase of the νCHposition at low temperatures. These results indicates that lowering the pH had an influence mainly at low temper- atures, giving rise to a phase transition, from highly ordered acyl chains to a lo phase. At high temperatures, the PA/Schol mixtures displayed, over the investigated pH range, a similar chain order as inferred from theνCHposition, which was typical of those observed for lo-phase systems.^5,10,29,32

The CO stretching band of the protonated form (COOH) is observed between 1675 and 1725 cm^-1, whereas the CO stretching of the unprotonated form (COO^-) is found between 1525 and 1600 cm^-1.³³Figure 5 presents IR spectra of PA/

Schol mixtures in the region of the carboxylic acid/carboxylate CO stretching. At pH 5.5, only the band corresponding to protonated PA was observed, indicating complete protonation.

When the pH was increased, a decrease of the relative intensity of this band was observed and, in parallel, the relative intensity of the CO stretching band of the unprotonated form increased.

At pH 9, a broad band, with a maximum at 1560 cm^-1, was observed, indicative of the complete deprotonation of PA. This Figure 5. pH dependence of the CO stretching mode of the PA

carboxylic/carboxylate group in PA/Schol mixture (molar ratio of 0.3/

0.7) at room temperature. The spectra were normalized to provide a constant total area of the carboxylic/carboxylate bands.

Figure 6. ²H NMR spectra of PA-d31/Chol1-x/Scholxmixtures with various Schol content, x, at 25 and 70°C, at (a) pH 5.5 and (b) 9. For all these mixtures, the PA-d31/total sterol molar ratio was 0.3/0.7.

pH evolution is analogous to that previously observed for the PA/Chol system,⁹as well as for systems including ceramides and phospholipids.^34,35

2H NMR Experiments. Figure 6 presents the ²H NMR spectra for PA-d31/Chol1-x/Scholxmixtures with a molar ratio PA-d31/total sterol of 0.3/0.7, at pH 5.5 and 9, and at 25 and 70

°C. At 25 °C, the spectrum of a PA-d31/Chol mixture cor- responded to two axially symmetric superimposed powder patterns with quadrupolar splittings, measured between the maxima, of 35 and 115 kHz. This signal is consistent with solid fatty acid with all-trans immobile (on the NMR time scale) acyl chain.^8,36,37 The broader pattern was associated with the equivalent CD2 groups of “all-trans” chains, whereas the narrower powder pattern corresponded to the terminal CD3. When the temperature was increased to 70 °C, a spectrum formed by several overlapping powder patterns was obtained.

This profile was typical of a fluid lamellar phase^1,8,36,38for which a gradient of orientational order exists along the acyl chains.

The widest quadrupolar splittings, measured between the maxima, were 45 kHz, corresponding to considerably ordered acyl chains for a temperature of 70 °C. These spectra are therefore characteristic of the lo phase. These results, illustrating the solid-to-lo phase transition of the PA-d31/Chol mixtures at pH 5.5, are in agreement with previous studies performed using mixtures of Chol and PA-d31or other saturated and linear fatty acids.^8-10

When the Schol content, x, was increased in these mixtures, at pH 5.5 and 25°C, a typical spectrum of the lo phase could be observed. For 0.05exe0.25, a superimposition of spectra characteristic of solid and lo phases was obtained. The spectrum of the PA-d31/Chol0.5/Schol0.5 mixtures corresponded almost exclusively to a lo component. Figure 7 reports the proportion of solid fraction in the PA-d31/Chol1-x/Scholx mixtures as a function of the Schol content. These proportions were calculated on the basis of the relative area of each component in the spectra.

The solid-to-lo phase transition was mainly observed between x)0 and 0.5, where the solid fraction varied from 100% to 1%. The PA-d31/chol1-x/Scholxmixtures, at pH 5.5, gave rise, at 25 °C, to a spectrum typical of a lo phase when x g 0.5 (Figure 6a). At 70°C,²H NMR spectra typical of a lo phase were obtained for the PA-d31/Chol1-x/Scholxmixtures irrespec- tive of the Schol content. The expected reduction of the spectra total width, associated with the temperature-induced conforma- tional disorder, was observed. These findings are consistent with the IR experiments where the solid-to-fluid transition disap- peared in the PA-d31/Chol1-x/Scholxmixtures for Schol content

xg0.5, and that theνCHposition was about 2852 cm^-1, typical of a lo phase (Figure 2).

Cholesterol and Schol showed an opposite effect on the phase stability, at 25°C, when the pH was varied between 5.5 and 9.

Typical lo-phase²H NMR spectra were obtained from PA-d31/ Chol mixtures at pH 9 and 25°C. Therefore, a pH increase from 5.5 to 9 favored the formation of a lo phase and led to the disappearance of the solid phase. Conversely, a pH increase from 5.5 to 9 induced a lo-solid phase transition for the mixtures containing Schol as the main sterol, as shown by the

2H NMR spectra. This transition required a large Schol content as the rise of solid component (4% of the total area) was recorded for a Schol content of x)0.75. When the mixtures included exclusively Schol as sterol, the solid fraction repre- sented 87% (Figure 7). The quadrupolar splittings, measured between the maxima, for spectrum of the PA-d31/Schol mixture at pH 9 and 25°C, were 36 and 120 kHz, indicative of solid- phase PA-d31. At 70°C, as for pH 5.5, typical lo-phase spectra were obtained from the PA-d31/Chol1-x/Scholx mixtures, ir- respective of their Schol content. These findings are consistent with the IR spectroscopy results (Figure 3), indicating that, upon heating, a shift of the νCHband associated with a solid-to-lo phase transition was observed for x between 0.75 and 1.

The smoothed order profiles associated with the fluid lamellar contribution in the spectra of PA/Chol1-x/Scholxmixtures are presented in Figure 8a. These profiles were obtained at 25 or 70°C. The pH had to be different, in some cases, to obtain a fluid phase. The profiles of Schol-containing systems were obtained at pH 5.5, whereas that of the PA/Chol mixture, for which the fatty acid is solid at this pH, was obtained at pH 6.5.

These profiles were typical of fluid bilayers: in the region near Figure 7. Influence of the Schol content, x, on the proportion of solid

phase in PA/Chol1-x/Scholxmixtures, at pH 5.5 (9) and 9 (2), at room temperature. For all these mixtures, the PA-d31/total sterol molar ratio was 0.3/0.7.

Figure 8. (a) Smoothed orientational order profiles of the lamellar fraction of PA-d31/Chol mixtures at pH 6.5, for a temperature of 25 (9) and 70°C (0); PA-d31/Chol0.5/Schol0.5mixtures at pH 5.5, for a temperature of 25 °C (b); PA-d31/Schol mixture at pH 5.5 for a temperature of 25 (2) and 70°C (4). (b) Smoothed orientational order profiles obtained at 70°C, from the lamellar fraction of PA-d31/Chol mixtures, at pH 5.5 (2) and 9 (9) and PA-d31/Schol mixtures pH 5.5 (4) and 9 (0). For all these mixtures, the PA-d31/total sterol molar ratio was 0.3/0.7. The dotted lines connecting the experimental points are drawn to guide the eye only.

the head group, the orientational order did not vary considerably and this plateau was followed by a rapid decrease of SC-D. The acyl chain orientational order is high for these sterol-rich systems. At 25°C, the order parameters corresponding to the widest doublet were 0.46 and 0.42 for PA-d31/Chol and PA- d31/Schol systems, respectively. These values are close to the maximum value, i.e., 0.5, corresponding to an all-trans chain with fast rotation along the long axis of the molecule. This system seems to be more ordered than phospholipid/Chol ones.

For example, order parameters slightly below 0.4 have been typically measured for phospholipid bilayers containing 45 mol

% Chol.^5,38For a given temperature, the order parameters were decreased when Schol content in the mixture was increased.

Typically there was a decrease by about 9% when Chol was completely substituted by Schol. The PA-d31chain orientational order was indeed sensitive to temperature. A decrease of the order parameter along the deuterated acyl chain was observed upon increasing temperature, for PA-d31/Chol and PA-d31/Schol (presented in Figure 8a) as well as for the other investigated PA/Chol1-x/Scholxmixtures (data not shown). This behavior, expected because of the thermal excitation of gauche conformers along the chains, is analogous to that of phospholipid/Chol matrixes.^5,38,39

The influence of Schol on the smoothed order profiles can be also highlighted by the comparison of the profiles for PA/

Chol and Schol at 70°C, at pH 5.5 and 9, where the four systems are in lo phase (see Figure 6). For both pH values, a decrease of the order parameters was observed when Schol replaced Chol in the mixture PA/sterol (Figure 8b); this decrease, calculated for the plateau region (3ene8), was 16% and 6% at pH 9 and 5.5, respectively.

Phase-Composition Diagram of the PA/Chol1-x/Scholx

Ternary System. In order to summarize the phase behavior revealed by the present spectroscopic study, a phase-composi- tion diagram of the PA/Chol1-x/Scholxsystem (with a PA/sterol molar ratio of 0.3/0.7), at 25°C, is presented in Figure 9. Three areas can be defined on the diagram. First, at low pH and low Schol content, there is a coexistence of the solid and lo phases, and the proportion of lo phase increases when x increases. Upon heating, in these conditions, there is a phase transition, where the solid fraction is transformed into a lo phase, for Tg55°C.

Second, an analogous area is observed at high pH and high Schol content where there is the coexistence of solid and lo phases.

In this region, the proportion of lo phase increases when x decreases. As for the other region, the solid phase is transformed into a lo phase upon heating above 50°C. Third, between these two areas of the diagram, there is a region where the lo phase

dominates. Indeed, in these conditions, no transition is observed upon heating and the lo phase appears to be stable up to 70°C.

PA/Chol1-x/ScholxLUVs. It has been shown that, despite the very high Chol content, it is possible to extrude the PA/

Chol mixtures at pHg7.5, to form LUVs.¹¹We have therefore examined the possibility to prepare LUVs from the PA/Chol1-x/ Scholx mixtures, using the phase diagram presented above to identify the conditions leading to the formation of stable lamellar structures. These systems were expected to present two valuable characteristics. First, they should allow a convenient modulation of the surface charge density by selecting the Chol/Schol ratio.

Second, these formulations could show a potential for pH- triggered release. These systems should show distinct behavior because the pH variation causing the release should be in opposite directions for the Chol- and Schol-containing formula- tions, on the basis of the presented phase-composition diagram.

Various PA/Chol1-x/Scholxmixtures were examined at pH 5.5 and room temperature. For xg0.1, the mixtures included a prevailing fraction of lo phase and it was possible to extrude these preparations to form stable LUVs. Table 1 presents some characteristics of the resulting LUVs. Their hydrodynamic diameter corresponded to the diameter of the polycarbonate filter pores (Table 1). The trapping capacity of these LUVs was assessed by fluorescence; the initial self-quenching of calcein entrapped in the LUVs was between 0.87 and 0.95. These values are those expected for a 80 mM calcein solution,⁴⁰demonstrating the efficient encapsulation of the fluorophore.

The passive release from calcein-loaded vesicles was deter- mined, at pH 5.5 (Figure 10). For PA/Schol mixtures, calcein was progressively released from the LUVs, reaching a 42%

release after 50 days. When a fraction of Schol was replaced by Chol, the passive release of entrapped calcein was reduced.

liquid ordered phases (lo s), and (9) corresponds to only liquid ordered phase (lo). The phase identification was carried out by the²H

NMR experiments. The PA/total sterol molar ratio was 0.3/0.7. Figure 10. Passive leakage from PA/Chol LUVs at pH 5.5 (0) and 7.4 (as external pH) (9), PA/Chol0.9/Schol0.1(O), PA/Chol0.75/Schol0.25

LUVs (4), PA/Chol0.50/Schol0.50LUVs (b), and PA/Schol LUVs (1) at pH 5.5. The LUVs were prepared at pH 5.5. The incubation was performed at room temperature. The PA/total sterol molar ratio was 0.3/0.7.

TABLE 1: Characteristics of Extruded LUVs for Various PA/Chol1-x/ScholxCompositions^a

LUV composition pH

dLUVs

(nm)

initial self-quenching

PA/Chol 7.4 117(22 0.87(0.01

PA/Chol 5.5 impossible to extrude

PA/Chol0.9/Schol0.1 5.5 99(10 0.88(0.09 PA/Chol0.75/Schol0.25 5.5 118(15 0.93(0.02 PA/Chol0.50/Schol0.50 5.5 101(16 0.95(0.01

PA/Schol 5.5 130(20 0.91(0.05

aThe PA/total sterol molar ratio was 0.3/0.7.

After 50 days, 82%, 89%, and 93% of the encapsulated calcein remained inside the LUVs formed with PA/Chol1-x/Scholx

mixtures with x ) 0.1, 0.25, and 0.5, respectively. These performances are considerably better than those obtained with phospholipid-based vesicles that are typically leakier. As a control, the passive release from PA/Chol LUVs, at pH 7.4, was measured. This pH was used because, in agreement with the phase-composition diagram, it was impossible to extrude the PA/Chol mixture at pH 5.5 (Table 1). The leakage from PA/Chol LUVs was very limited, as previously shown;¹¹even after 60 days, no significant release could be detected. The extent of passive release appeared to be consistent with the effects of the sterols on the acyl chain orientational order (Figure 8) as it was shown that Schol was less efficient than Chol to order the PA acyl chains.

The effect of temperature on the passive leakage was observed (data not shown). As expected, the passive leakage was systematically faster at higher temperatures. After a 15 day incubation at 37°C, only from 10% to 20% of calcein remained encapsulated in PA/Chol0.9/Schol0.1 and PA/Schol LUVs, re- spectively. If the incubation was carried out at 50 °C, the entrapped calcein was released considerably faster. The time for which half the encapsulated calcein was released (t1/2) corresponded to 3 and 2 days for PA/Chol0.9/Schol0.1 LUVs, and 5 and 3 days for PA/Schol LUVs, at 37 and 50 °C, respectively. This enhanced passive leakage is a phenomenon also observed for phospholipid LUVs^41,42and is consistent with the diminution of the order parameters (Figure 8).

The release of the entrapped content of these LUVs can be triggered by a pH variation. Because of the phase stability described above, the pH-controlled release could be predicted in a rational manner. It has been shown that a decrease of pH can induce the release of the LUV content for PA/Chol systems.¹¹ This pH-triggered release is also illustrated in the present study by the very rapid liberation of calcein when the PA/Chol LUVs were incubated at pH 5.5, at room temperature (Figure 10). Figure 11 presents the pH-triggered release of calcein for the PA/Schol LUVs, measured within 2 min after the pH modification. Calcein release could be caused, with this formulation, by an increase of the external pH. At pH 5.5, these LUVs were stable, whereas the calcein release became signifi-

cant at an external pH ∼8. At pH g 8.5, about 80% of the content or more was abruptly released. The light-scattering measurements performed subsequently on these systems re- vealed a drastic size increase of the particles in these conditions;

these morphological changes appeared to occur in a concomitant manner with the content release. At pH g 7.5, it was found that only a small proportion of particles corresponded to 100 nm LUVs (<15%), whereas the aggregated structures, with an average hydrodynamic diameter of 400 nm, represented more than 85% of the particle distribution. The pH-triggered release and morphological changes can be rationalized (and actually beforehand predicted) by the phase-composition diagram: a lo-to-solid phase transition was identified to start at about pH 6.5 for PA/Schol mixtures. The formation of solid phase within the vesicle bilayer is likely the triggering event leading to the aggregation of the liposomes.

Discussion

This work demonstrates that it is possible to form stable lo lamellar phases with PA/Schol mixtures under certain condi- tions, namely, at acidic pH. These compounds do not form individually fluid lamellar phases. These self-assemblies formed from these mixtures are analogous to the PA/Chol lo phases recently reported.^8-10 They are characterized by high sterol content, a high orientational order of the PA acyl chains, and a rotational diffusion sufficiently fast to lead to axially symmetric powder pattern in²H NMR spectroscopy. At a molecular level, it has been shown that the hydrophobic matching between the chain of the acid and Chol is essential to form lo phases.¹⁰Schol is proposed to be positioned vertically inside phospholipid bilayers, similar to Chol.^16,43If one supposes that Schol mixed with PA adopts a similar molecular arrangement to that with Chol, it is expected that PA ensures also a proper hydrophobic match with Schol. The order parameters of the PA/sterol systems are remarkably high. For example, SC-Dat the plateau is 0.46 for PA/Chol bilayers, at pH 6.5 and 25°C. This is a value close to the maximum of 0.5 corresponding to an all-trans chain experiencing fast rotational mobility along the bilayer normal.

Ouimet et al. have obtained SC-Dat the plateau of 0.45 for PA- d31/Chol at pH 8.8, at 60°C.⁹Similarly, mixtures rich in Chol prepared with myristic and stearic acid lead to SC-D at the plateau of 0.41, at pH 5.5, and 66°C.¹⁰In the present systems, since the fluid lamellar self-assemblies are stable at low temperatures, even greater order could be obtained when the temperature was decreased. Schol is slightly less efficient for rigidifying the PA acyl chains. At 70°C, the SC-Dvalues, at the plateau, for PA/Chol bilayers, at pH 5.5 or 9, are typically from 5% to 20% higher than those obtained from PA/Schol bilayers in the same conditions. A similar difference (10%) is observed at 25°C, from the spectra obtained at pH 6.5 for PA/

Chol bilayers and 5.5 for PA/Scholsthis pH difference is necessary to form stable lo phases. It was shown in phospholipid bilayers that both sterols could lead to the formation of a lo lamellar phase, but the chain ordering effect of Schol was found slightly smaller than that of Chol.^15,16,44This difference, also observed for the PA/sterol bilayers, was explained by the increase of the interfacial area of Schol compared to Chol, due to the electrostatic repulsion, the increased hydration, and the bulkiness of the sulfate groups.^15,16,44The ordering effect of the sterol appears to be robust as the thermal expansion coefficient of the bilayers, estimated from the variation of the orientational order as a function of temperature,^10,45seems to be unusually high. If one considers only the PA molecules in the mixtures, this coefficient, calculated over the temperature range between Figure 11. pH-triggered release of calcein from PA/Schol LUVs (9).

The variations of the hydrodynamic diameters of the LUVs (measured

∼2 h after the pH variation) as a function of pH are also reported (0).

For pHg7.5, populations with two size distributions were observed.

In these conditions, the liposomes with a∼100 nm diameter represented

<15% of the detected particles. The PA/Schol molar ratio was 0.3/

0.7, and the measurements were carried out at room temperature.

respectively.^16,48,49 These peculiar predicted values suggest distinct micromechanical properties for these LUVs, an aspect that should be investigated in detail by more direct approaches.

It should be noted that, relative to the PA/Chol system, at pH 5.5,⁸the phase behavior of the PA/Schol system appears to be more complex at basic pH. Conditions leading to a sharp solid- to-lo phase transition could be not found, suggesting the absence of a eutectic point in the phase diagram. This behavior could be reminiscent of that observed in the absence of water because, by opposition to the PA/Chol system, the PA/Schol system did not present a eutectic behavior in the dry and solid phase.⁵⁰

The ability of a limited amount of fatty acid to solubilize a considerable amount of sterol is indeed related to the intermo- lecular interactions existing between the different components.

In PA/sterol systems, it is expected that van der Waals interactions, between the fatty acid acyl chain and the hydro- phobic part of the sterol, are a main contribution, by analogy to phospholipid/sterol interactions.⁵¹This contribution should be similar for Chol- and Schol-containing systems because both sterols display a similar condensing effect, and, consequently, a similar distance between the lipid acyl chain and the sterol hydrophobic part. This hypothesis is supported by the compa- rable results obtained for dipalmitoylphosphatidylcholine mem- branes with Schol and Chol.¹⁶The most important changes in the energetics must be associated with the substitution of the hydroxyl group in Chol by the sulfate group in Schol. Com- bining the results presented here and those describing the stability of PA/Chol systems,^8,9 the conditions leading to the formation of fluid lamellar phases by fatty acid/sterol systems can be rationalized on the basis of intermolecular interactions.

The PA/Chol system at pHg 7.4, and PA/Schol at pHe7, form lo lamellar phases. In the first system, a significant proportion of PA should be unprotonated, leading to electrostatic inter-PA repulsive interactions. In the latter system, electrostatic repulsive interactions exist between the sterol molecules. In both cases, these repulsions reduce the like-like interactions relative to the like-unlike ones. When the PA/Chol system is brought at pH<7.4, the protonation of the carboxylic groups leads to a reduction of the inter-PA repulsion. This change increases the like-like interactions, favoring a phase separation; this leads to the formation of solid PA and solid Chol phases.⁸ In an opposite way, the increase of pH above 7 for the PA/Schol system leads to the deprotonation of PA molecules, and this leads to electrostatic repulsion between both negatively charged PA and Schol. This change decreases the like-unlike interac- tions favoring also the phase separation of the two components.

This qualitative rationale suggests that a fine balance of electrostatic interactions is at the origin of the lamellar phase stability. In the seminal work of Hargreaves and Deamer⁵²on lamellar phases prepared with monoacylated lipids, it was

fluid lamellar phases in fatty acid/sterol systems.

Despite the very high Chol content, it is possible to prepare LUVs, at room temperature, using a standard extrusion method, from PA/Schol dispersions. These NPLs show a very low permeability and the capacity of pH-triggered release of its content. The PA/Schol LUVs are slightly more permeable than those prepared with PA/Chol mixtures, a phenomenon which is consistent with the slightly more ordered acyl chains observed in the former composition. As expected, the LUVs become more permeable upon heating, a variation concomitant with the acyl chain disordering. Lamellar lo phases could also be prepared from PA and a mixture of the sterols. These mixtures display behaviors that are intermediate of those observed for PA/Chol and PA/Schol mixtures. PA/Chol1-x/ScholxLUVs with x>0.1 display a predominant lo-phase proportion (>60%), and the extruded LUVs remain stable between pH 5.5 and 8.5, in a manner consistent with the pH-composition diagram (Figure 9).

Concluding Remarks. The present results extend the com- positions of the NPLs that can be formed with very high sterol content. These vesicles display Chol contents significantly higher than the Chol solubility generally observed in phospholipid bilayers.^41,53Moreover, they display a very high chain order and low passive permeability, two distinct features that are likely associated with their unique composition. The substitution of Chol by Schol in fatty acid/sterol LUVs offers the valuable advantage of tuning the liposome stability as a function of pH.

Furthermore, the balance of Chol/Schol proportions provides a simple approach to tune the pH-triggered released from these liposomes. When Chol is the main sterol, the LUVs are stable at neutral pH and become leaky at acidic pH, whereas the formulation containing mainly Schol releases its content at basic pH. One can therefore plan to tailor these sterol-rich NPLs for pH-controlled released in a very flexible way.

In addition to leading to interesting self-assembled structures, these findings may also have an impact in the understanding of the phase behavior of biological systems. Normal stratum corneum (SC) includes free fatty acids and Chol as main components. Its lipids are proposed to be mainly organized in small crystalline domains embedded in a fluid lipid matrix, and this organization could be essentially responsible for the remarkable skin impermeability.^54-56On the basis of the present results, it is possible that the lipid distribution in the crystalline and fluid phase is sensitive to the Chol/Schol balance in the SC. This balance has been actually demonstrated to be crucial in the desquamation process of the skin.^20,21,57In normal skin, Schol is a minor component representing 2% (w/w) of the lipids, and the Chol/Schol ratio is about 7.^57,58 This proportion is considerably increased in the case of people suffering from X-linked recessive ichthyosis. Schol represents in these cases

about 12% (w/w) of the total lipid of the SC, and the Chol/

Schol ratio drops to 1.^20,57The persons suffering from this type of ichthyosis have a severe inhibition of the desquamation, and as a consequence, large scales, associated with an accumulation of SC, cover their bodies. On the basis of our results, it is possible that the increased Schol content in the SC favors the formation of fluid phase. Moreover, this feature would be promoted by the acidic pH of the skin that is reported to be around 5.5.^59,60The presence of an abnormally large proportion of fluid phase may lead a more cohesive SC and prevent the crumbling of material at the surface. Actually it has been already reported on mixtures mimicking the SC lipids that an increased proportion of Schol (from 2 to 10 mol %) induces the disappearance of crystalline Chol as a consequence of an increase of its solubility in the SC matrix.⁶¹The interactions between the free fatty acids and the sterol, that dictates the final structures of the simple binary systems discussed here, may play an essential role in the complex phase behavior of SC lipids, and this aspect should clearly be investigated in detail.

Acknowledgment. The authors thank the Natural Sciences and Engineering Research Council of Canada for the financial support. They also thank Ce´dric Malveau, Department of Chemistry, Universite´ de Montre´al, for his technical support on the NMR experiments. This work was also funded by Fonds Que´be´cois de la Recherche sur la Nature et les Technologies through its financial support to the Center for Self-Assembled Chemical Systems (CSACS).

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