Cholesterol Sulfate

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

10929

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)1,2 has 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.1,3-7 The existence of this phase has been extended to matrixes formed by linear and saturated fatty acid.8-10 In 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.9 Moreover it was recently established that it is possible to prepare large unilamellar vesicles (LUVs) from this system, using standard extrusion procedures.11 These LUVs were shown to have an impermeability 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 * Author to whom correspondence should be sent. Phone: (514) 3435936. Fax: (514) 343-7586. E-mail: [email protected].

this change, as described below, extends the family of nonphospholipid 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.13-16 The rigidifying effect of Schol on lipid acyl chains was, however, found to be smaller than that of Chol.14,16 Indeed 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.17 For 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.18,19 The 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/Scholx system 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 differential scanning calorimetry (DSC) and infrared (IR) and 2H 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.

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

10930 J. Phys. Chem. B, Vol. 111, No. 37, 2007 Experimental Methods Cholesterol (>99%), Schol, PA (99%), tris(hydroxymethyl)aminomethane (TRIS) (99%), 2-[N-morpholino]ethansulfonic acid (MES) (>99%), ethylenediaminetetraacetic acid (EDTA) (99%), NaCl (>99%), Triton X-100 (99%), deuterium oxide (D2O) (>99.9%), and deuterium-depleted water (>99.99%) were purchased from Sigma Chemical Co. (St. Louis, MO). Deuterated palmitic acid (PA-d31) (98.9%) was supplied from CDN isotopes (Pointe-Claire, QC, Canada). Calcein (high purity) has been obtained from Molecular Probes (Eugene, OR). Sephadex G-50 medium was purchased from Pharmacia (Uppsala, Sweden). Methanol (spectrograde) has been obtained from American Chemicals Ltd. (Montreal, QC, Canada). Benzene (high purity) was purchased from BDH Inc. (Toronto, ON, 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 spectroscopy, the mixtures were prepared with PA, Chol, and/or Schol, whereas PA was replaced by PA-d31 for 2H 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/Scholx where x represents the molar fraction of Schol relative to total sterol. DSC, IR, and 2H 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 deuteriumdepleted 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 for 2H NMR experiments. The suspensions were subjected to five cycles of freezing-andthawing (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 spectrometer, equipped with a water-cooled globar source, a KBr beam splitter, and a DTGS detector, using an established protocol.10 Briefly, an aliquot of the sample was placed between two BaF2 windows separated by a 5 µm thick Teflon ring. This assembly was inserted in a brass sample holder, whose temperature 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 2.7 µs and an interpulse delay of 25 µs. After the second pulse, 2048 points were recorded in quadrature mode, with a dwell time of 0.5 µs. The recycling time was 60 s. In absence of a slow-relaxation (solid) component, the recycling delay was

Bastiat and Lafleur reduced to 0.3 s. Typically 2000 FID were coadded. The temperature was regulated using a Bruker VT-3000 controller. The characteristic fluid-phase spectra were dePaked,25 and the smoothed order profiles were extracted from the dePaked spectra using an established procedure.26 This approach assumes a monotonic decrease of the orientational order along the alkyl chain, from the head group toward its end. The quadrupolar splitting of the terminal CD3 was, however, measured directly on the dePaked spectra as it is always well resolved. The orientational order parameters, SC-D, were calculated from these splittings. Permeability Measurements. The permeability of the LUVs was measured using a standard procedure based on the selfquenching property of calcein at high concentration.27,28 Large 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 fluorescence was measured before and after the addition of 10 µL 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:

% of encapsulated calcein )

(

)

(If+T - If)/If+T (Ii+T - Ii)/Ii+T

× 100

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 Ii and Ii+T corresponded to the measurements at the initial pH, before and after the addition of Triton X-100, respectively. If and If+T were 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 investigated pH range.27 The 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.

Phase Behavior of Palmitic Acid/Sterol Mixtures

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 the νCH band 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.

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.9 The 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 (x g 0.25), no phase transition was observed. The thermograms obtained from PA/ Chol0.75/Schol0.25 and 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 9.9 For

J. Phys. Chem. B, Vol. 111, No. 37, 2007 10931

Figure 3. Thermotropism of PA in PA/sterol mixtures probed using the νCH 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 the νCH band 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.

PA/Schol mixtures at pH 9, two maxima, at 43 and 55 °C, were observed in the thermograms. As confirmed below, the endothermic 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 the νCH band from 2848.5 to about 2853.5 cm-1, was observed at about 60 °C, corresponding to the melting temperature of the fatty acid.8 This curve is reproduced in Figure 2 for reference purposes. The two extreme νCH positions were representative of highly ordered and disordered chains, respectively.29 A transition was observed between 45 and 50 °C, for the PA/Chol system at pH 5.5, in agreement with the thermograms presented above and previous results.8,9 At low temperatures, the position of the νCH band is 2848.5 cm-1, indicative

10932 J. Phys. Chem. B, Vol. 111, No. 37, 2007

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.

of PA acyl chains in solid state. At 52 °C, the position of the νCH band was 2852 cm-1, a value observed for lo phases formed by Chol-rich bilayers.5,10,29,32 When the Schol content, x, in the PA/Chol1-x/Scholx mixtures 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 the νCH values measured at low temperatures. At 25 °C, for example, the νCH 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, independently 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 the νCH position for PA/Chol1-x/Scholx mixtures at pH 9. For x < 0.5, no

Bastiat and Lafleur transition was observed as the position of the νCH band was practically constant at ∼2851 cm-1, a value compatible with lo phases. For PA/Schol mixtures at pH 9, a transition was observed at about 40 °C, on the basis of the shift of the νCH band from 2850 to 2851.5 cm-1. This is in agreement with the DSC measurements presented above where an endothermic peak appeared at 43 °C (Figure 1d). For PA/Chol0.5/Schol0.5 and PA/ Chol0.25/Schol0.75 mixtures, a broad shift of νCH, from 2850.6 to 2851.5 cm-1, was observed. By contrast to PA/Chol1-x/Scholx mixtures at pH 5.5, the acyl chains became more ordered at room temperature, when x Schol content increased at pH 9. Figure 4 presents the thermotropism of PA/Schol mixtures, as probed by the νCH position, for pH varying from 5.5 to 9. A shift of the νCH position was observed below 40 °C, increasing from 2849.5-2850 cm-1 at 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 thermograms. This effect was mainly associated with an increase of the νCH position at low temperatures. These results indicates that lowering the pH had an influence mainly at low temperatures, 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 the νCH position, 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-1.33 Figure 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 6. 2H NMR spectra of PA-d31/Chol1-x/Scholx mixtures 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.

Phase Behavior of Palmitic Acid/Sterol Mixtures

J. Phys. Chem. B, Vol. 111, No. 37, 2007 10933

Figure 7. Influence of the Schol content, x, on the proportion of solid phase in PA/Chol1-x/Scholx mixtures, 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.

pH evolution is analogous to that previously observed for the PA/Chol system,9 as 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/Scholx mixtures 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 corresponded 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 phase1,8,36,38 for 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-d31 or 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 0.05 e x e 0.25, 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/Scholx mixtures, 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/Scholx mixtures irrespective of the Schol content. The expected reduction of the spectra total width, associated with the temperature-induced conformational disorder, was observed. These findings are consistent with the IR experiments where the solid-to-fluid transition disappeared in the PA-d31/Chol1-x/Scholx mixtures for Schol content

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/Schol0.5 mixtures 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.

x g 0.5, and that the νCH position 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-phase 2H 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 represented 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 solidphase PA-d31. At 70 °C, as for pH 5.5, typical lo-phase spectra were obtained from the PA-d31/Chol1-x/Scholx mixtures, irrespective of their Schol content. These findings are consistent with the IR spectroscopy results (Figure 3), indicating that, upon heating, a shift of the νCH band 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/Scholx mixtures 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

10934 J. Phys. Chem. B, Vol. 111, No. 37, 2007

Figure 9. pH-composition diagram of the PA/Chol1-x/Scholx system, at room temperature. (2) corresponds to the coexistence of solid and liquid ordered phases (lo + s), and (9) corresponds to only liquid ordered phase (lo). The phase identification was carried out by the 2H NMR experiments. The PA/total sterol molar ratio was 0.3/0.7.

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 PAd31/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.5,38 For 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-d31 chain 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/Scholx mixtures (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 (3 e n e 8), 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-composition diagram of the PA/Chol1-x/Scholx system (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 T g 55 °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

Bastiat and Lafleur

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/Schol0.50 LUVs (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/Scholx Compositionsa LUV composition

pH

PA/Chol PA/Chol PA/Chol0.9/Schol0.1 PA/Chol0.75/Schol0.25 PA/Chol0.50/Schol0.50 PA/Schol

7.4 5.5 5.5 5.5 5.5 5.5

a

dLUVs (nm)

initial self-quenching

117 ( 22 0.87 ( 0.01 impossible to extrude 99 ( 10 0.88 ( 0.09 118 ( 15 0.93 ( 0.02 101 ( 16 0.95 ( 0.01 130 ( 20 0.91 ( 0.05

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

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/Scholx LUVs. It has been shown that, despite the very high Chol content, it is possible to extrude the PA/ Chol mixtures at pH g 7.5, to form LUVs.11 We 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 pHtriggered 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 formulations, on the basis of the presented phase-composition diagram. Various PA/Chol1-x/Scholx mixtures were examined at pH 5.5 and room temperature. For x g 0.1, 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,40 demonstrating the efficient encapsulation of the fluorophore. The passive release from calcein-loaded vesicles was determined, 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.

Phase Behavior of Palmitic Acid/Sterol Mixtures

J. Phys. Chem. B, Vol. 111, No. 37, 2007 10935 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 revealed 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 (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 compositions 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.41,53 Moreover, 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.54-56 On 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.20,21,57 In 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

Phase Behavior of Palmitic Acid/Sterol Mixtures about 12% (w/w) of the total lipid of the SC, and the Chol/ Schol ratio drops to 1.20,57 The 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 5.5.59,60 The 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.61 The 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). References and Notes (1) Vist, M. R.; Davis, J. H. Biochemistry 1990, 29, 451-464. (2) Ipsen, J. H.; Mouritsen, O. G.; Bloom, M. Biophys. J. 1990, 57, 405-412. (3) Thewalt, J. L.; Bloom, M. Biophys. J. 1992, 63, 1176-1181. (4) Linseisen, F. M.; Thewalt, J. L.; Bloom, M.; Bayerl, T. M. Chem. Phys. Lipids 1993, 65, 141-149. (5) Pare´, C.; Lafleur, M. Biophys. J. 1998, 74, 899-909. (6) Filippov, A.; Ora¨dd, G.; Lindblom, G. Biophys. J. 2006, 90, 20862092. (7) Chachaty, C.; Rainteau, D.; Tessier, C.; Quinn, P. J.; Wolf, C. Biophys. J. 2005, 88, 4032-4044. (8) Pare´, C.; Lafleur, M. Langmuir 2001, 17, 5587-5594. (9) Ouimet, J.; Croft, S.; Pare´, C.; Katsaras, J.; Lafleur, M. Langmuir 2003, 19, 1089-1097. (10) Ouimet, J.; Lafleur, M. Langmuir 2004, 20, 7474-7481. (11) Bastiat, G.; Oliger, P.; Karlsson, G.; Edwards, K.; Lafleur, M. Langmuir 2007, 23, 7695-7699. (12) Drummond, D. C.; Zignani, M.; Leroux, J.-C. Prog. Lipid Res. 2000, 39, 409-460. (13) Le Grimellec, C.; Daigneault, A.; Bleau, G.; Roberts, K. D. Lipids 1984, 19, 474-477. (14) Xu, X.; London, E. Biochemistry 2000, 39, 843-849. (15) Kitson, N.; Monck, M.; Wong, K.; Thewalt, J.; Cullis, P. R. Biochim. Biophys. Acta 1992, 1111, 127-133. (16) Smondyrev, A. M.; Berkowitz, M. L. Biophys. J. 2000, 78, 16721680. (17) Strott, C. A.; Higashi, Y. J. Lipid Res. 2003, 44, 1268-1278. (18) Elias, P. M. J. InVest. Dermatol. 1983, 80, 44s-49s. (19) Epstein, E. H.; Williams, M. L.; Elias, P. M. Arch. Dermatol. 1981, 117, 761-763. (20) Williams, M. L.; Grayson, S.; Bonifas, J. N.; Epstein, E. H., Jr.; Elias, P. M. Stratum Corneum; Marks, R., Plewing, G., Eds.; SpringerVerlag: Heidelberg, 1983; pp 79-84. (21) Yardley, H. J.; Summerly, R. Pharmacol. Ther. 1981, 13, 357383.

J. Phys. Chem. B, Vol. 111, No. 37, 2007 10937 (22) Roberts, K. D. J. Steroid Biochem. 1987, 27, 337-341. (23) Blache, D.; Becchi, M.; Davignon, J. Biochim. Biophys. Acta 1995, 1259, 291-296. (24) Cameron, D. G.; Kauppinen, J. K.; Moffatt, D. J.; Mantsch, H. H. Appl. Spectrosc. 1982, 36, 245-250. (25) Sternin, E.; Bloom, M.; MacKay, A. L. J. Magn. Reson. 1983, 55, 274-282. (26) Lafleur, M.; Fine, B.; Sternin, E.; Cullis, P. R.; Bloom, M. Biophys. J. 1989, 56, 1037-1041. (27) Allen, A. T. Liposome Technology; CRC Press: Boca Raton, FL, 1984; pp 177-182. (28) Benachir, T.; Lafleur, M. Biochim. Biophys. Acta 1995, 1235, 452460. (29) Mantsch, H. H.; McElhaney, R. N. Chem. Phys. Lipids 1991, 57, 213-226. (30) Kodati, R. V.; Lafleur, M. Biophys. J. 1993, 64, 163-170. (31) Kodati, V. R.; El-Jastimi, R.; Lafleur, M. J. Phys. Chem. 1994, 98, 12191-12197. (32) Chen, H.-C.; Mendelsohn, R.; Rerek, M. E.; Moore, D. J. Biochim. Biophys. Acta 2001, 1512, 345-356. (33) Go´mez-Ferna´ndez, J. C.; Villalaı´n, J. Chem. Phys. Lipids 1998, 96, 41-52. (34) Lieckfeldt, R.; Villalaı´n, J.; Go´mez-Ferna´ndez, J.-C.; Lee, G. Colloids Surf., A 1994, 90, 225-234. (35) Lieckfeldt, R.; Villalaı´n, J.; Go´mez-Ferna´ndez, J.-C.; Lee, G. Pharm. Res. 1995, 12, 1614-1617. (36) Davis, J. H. Biophys. J. 1979, 27, 339-358. (37) Kitson, N.; Thewalt, J.; Lafleur, M.; Bloom, M. Biochemistry 1994, 33, 6707-6715. (38) Lafleur, M.; Cullis, P. R.; Bloom, M. Eur. Biophys. J. 1990, 19, 55-62. (39) Seelig, J. Q. ReV. Biophys. 1977, 10, 353-418. (40) El Jastimi, R.; Lafleur, M. Biospectroscopy 1999, 5, 133-140. (41) Mayer, L. D.; Bally, M. B.; Cullis, P. R. J. Liposome Res. 1990, 1, 463-480. (42) Mayer, L. D.; Bally, M. B.; Hope, M. J.; Cullis, P. R. Biochim. Biophys. Acta 1985, 816, 294-302. (43) Faure, C.; Dufourc, E. J. Biochim. Biophys. Acta 1997, 1330, 248252. (44) Faure, C.; Tranchant, J.-F.; Dufourc, E. J. Biophys. J. 1996, 70, 1380-1390. (45) Bloom, M.; Evans, E.; Mouritsen, O. G. Q. ReV. Biophys. 1991, 24, 293-397. (46) Needham, D.; McIntosh, T. J.; Evans, E. Biochemistry 1988, 27, 4668-4673. (47) Takahashi, H.; Sinoda, K.; Hatta, I. Biochim. Biophys. Acta 1996, 1289, 209-216. (48) Chen, Z.; Rand, R. P. Biophys. J. 1997, 73, 267-276. (49) Ipsen, J. H.; Karlstro¨m, G.; Mouritsen, O. G.; Wennerstro¨m, H.; Zuckermann, M. J. Biochim. Biophys. Acta 1987, 905, 162-172. (50) Rehfeld, S. J.; Williams, M. L.; Elias, P. M. Arch. Dermatol. Res. 1986, 278, 259-263. (51) Vanderkooi, G. Biophys. J. 1994, 66, 1457. (52) Hargreaves, W. R.; Deamer, D. W. Biochemistry 1978, 17, 37593768. (53) Bach, D.; Wachtel, E. Biochim. Biophys. Acta 2003, 1610, 187197. (54) Forslind, B. Acta Derm.sVenereol. 1994, 74, 1-6. (55) Pilgram, G. S. K.; Engelsma-van Pelt, M. A.; Bouwstra, J. A.; Koerten, H. K. J. InVest. Dermatol. 1999, 113, 403-409. (56) Kitson, N.; Thewalt, J. Acta Derm.sVenereol. Suppl. 2000, 208, 12-15. (57) Elias, P. M.; Crumrine, D.; Rassner, U.; Hachem, J.-P.; Menon, G. K.; Man, W.; Choy, M. H. W.; Leypoldt, L.; Feingold, K. R.; Williams, M. L. J. InVest. Dermatol. 2004, 122, 314-319. (58) Weerheim, A.; Ponec, M. Arch. Dermatol. Res. 2001, 293, 191199. (59) O ¨ hman, H.; Vahlquist, A. J. InVest. Dermatol. 1998, 111, 674677. (60) O ¨ hman, H.; Vahlquist, A. Acta Derm.sVenereol. 1994, 74, 375379. (61) Bouwstra, J. A.; Gooris, G. S.; Dubbelaar, F. E. R.; Ponec, M. J. Lipid Res. 1999, 40, 2303-2312.