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Interaction of Cetylpyridinium Chloride with Giant Lipid Vesicles Vesna Arrigler,*,† Ksenija Kogej,‡ Janja Majhenc,† and Sasˇa Svetina† Institute of Biophysics, Faculty of Medicine, University of Ljubljana, Lipicˇ eva 2, SI-1000 Ljubljana, Slovenia, and Department of Physical Chemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Asˇ kercˇ eva 5, SI-1001 Ljubljana, Slovenia Received January 5, 2005. In Final Form: April 26, 2005 The interaction of cationic surfactant cetylpyridinium chloride, CPC, with giant lipid vesicles prepared from 1-palmitoyl-2-oleoylphosphatidylcholine, POPC, was examined at various concentrations of the lipid component. The lipid concentration was determined by a spectrophotometric method. The potentiometric method based on surfactant-selective electrode was used for the determination of surfactant concentration in the external water solution. From these results, moles of surfactant incorporated in the membrane per mole of lipid (parameter β) and two kinds of partition coefficients were calculated. Their values were found to be considerably larger than the available literature data. A three stage process of surfactant-induced solubilization of lipid vesicles was observed. First, stable mixed bilayers form, which become saturated with CPC at a value βsat larger than 0.8, which then gradually disintegrate. Just prior to the breakdown of the vesicular structure, formation of ellipsoidal vesicles was observed by optical microscopy. This phenomenon was attributed to the cooperative incorporation of surfactant into the bilayer. Fluorescence measurements have shown that the second stage in the solubilization process of POPC by the C16 chainlength surfactant does not involve mixed micelles. These are formed only in the third stage, which is the complete solubilization of POPC bilayers. The corresponding critical micellization concentration decreases with increasing concentration of the lipid component.
1. Introduction Single-component phospholipid vesicles are extensively used as model systems for biological membranes.1,2 Their properties like fluidity, headgroup dipole density, membrane permeability, and packing geometry can be affected by the presence of other compounds.2-5 Because many biological compounds are amphiphilic in nature, it is of considerable interest to study interactions between surfactants and lipid vesicles. It has been shown that the addition of surfactant to the suspension of lipid vesicles can cause disintegrationssolubilizationsof their membranes.4-7 Several stages in this process have been identified, which depend on the amount of surfactant added.4-8 At low surfactant-to-lipid ratios, surfactant is distributed between the water phase and the vesicles without causing any major change in the general structure of the bilayer (stage 1). At a certain critical surfactantto-lipid ratio, which is characteristic for each system, the bilayers become saturated with surfactant and progressive * Corresponding author. Telephone: ++386-1-543-76-04. Fax: ++386-1-431-51-27. E-mail: vesna.arrigler@biofiz.mf.uni-lj.si. † Institute of Biophysics, Faculty of Medicine, University of Ljubljana. ‡ Department of Physical Chemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana. (1) Lasicˇ, D. D. Liposomes from Physics to Application; Elsevier: Amsterdam, 1993. (2) Cevc, G.; Marsh, D. Phospholipid Bilayers, Physical Principles and Models, John Wiley & Sons: New York, 1997. (3) Hiemenz, P. C.; Rajagopalan, R. Principles of Colloid and Surface Chemistry; Marcel Dekker: New York, 1997. (4) Helenius, A.; Simons, K. Biochim. Biophys. Acta 1975, 415, 2979. (5) Lichtenberg, D.; Robson, R. J.; Dennis, E. A., Biochim. Biophys. Acta 1983, 737, 285-304. (6) Lichtenberg, D. Biochim. Biophys. Acta 1985, 821, 470-478. (7) Lantzsch, G.; Binder, H.; Heerklotz, H.; Wendling, M.; Klose, G. Biophys. Chem. 1996, 58, 289-302. (8) Kragh-Hansen, U.; Le Maire, M.; Moeller, J. Biophys. J. 1998, 75, 2932-2946.
disintegration of the vesicle starts (the beginning of stage 2). At even higher surfactant concentrations (stage 3), the lipid component is completely solubilized by the surfactant and mixed lipid/surfactant micelles are formed. The type and the structure of the aggregates formed in the transition region between stable mixed vesicles and mixed micelles (stage 2) were found to vary considerably with the type of the surfactant, in particular with its chain length. Often, coexistence of mixed micelles and a lamellar phase saturated with surfactant is assumed.4-9 The majority of this type of studies focused on interactions between different nonionic surfactants and small lipid vesicles.9-18 Among ionic surfactants, anionic alkyl sulfates were used in the study of the mechanism of surfactant solubilization of liposomes19-22 and protein(9) Lasch, J. Biochim. Biophys. Acta 1995, 1241, 269-292. (10) Schubert, R.; Beyer, K.; Wolburg, H.; Schmidt, K. H. Biochemistry 1986, 25, 5263-5268. (11) Lasch, J.; Schubert, R. The Interaction of Detergents with Liposomal Membranes. In Liposome Technology; Gregoriadis, G., Ed.; Entrapment of Drugs and Other Materials; CRC Press: Boca Raton, FL, 1993; Vol. 2. (12) Heerklotz, H.; Binder, H.; Lantzsch, G.; Klose, G. Biochim. Biophys. Acta 1994, 1196, 114-122. (13) Heerklotz, H.; Binder, H.; Lantzsch, G.; Klose, G.; Blume, A. J. Phys. Chem. B 1997, 101, 639-645. (14) Keller, A.; Kerth, A.; Blume, A. Biochim. Biophys. Acta 1997, 1326, 178-192. (15) Levy, D.; Gulik, A.; Signeuret, M.; Rigaud, J. L. Biochemistry 1990, 29, 9480-9488. (16) Lasch, J.; Hoffman, J.; Omalyanenko, W. G.; Klibanov, A. A.; Torchilin, V. P.; Binder, H.; Gawrisch, K. Biochim. Biophys. Acta 1990, 1022, 171-180. (17) Paternostre, M.; Meyer, O.; Grabielle-Madelmont, C.; Lesieur, S.; Ghanam, M.; Ollivon, M. Biophys. J. 1995, 69, 2476-2488. (18) Wenk, M. R.; Seelig, J. Biophys. J. 1997, 73, 2565-2574. (19) Silvander, M.; Karlsson, G.; Edwards, K. J. Colloid Interface Sci. 1996, 179, 104-113. (20) Lopez, O.; Cocera, M.; Wehrli, E.; Parra, J. L.; de la Maza, A. J. Colloid Interface Sci. 1990, 367, 153-160. (21) Cocera, M.; Lopez, O.; Estelrich, J.; Parra, J. L.; de la Maza, A. Chem. Phys. Lipids 2001, 110, 19-26.
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containing membranes.8 In an extensive review, Almgren23 has discussed solubilization of lipid bilayers also by cationic surfactants of the alkyltrimethylammonium family, for example by cetyltrimethylammonium bromide, C16TAB. Therein,23 emphasis is given to the phase behavior and structures resulting from the solubilization process, whereas a quantitative treatment of binding was not undertaken. Recently, Kadi et al.24 have determined the isotherms of binding for four alkyltrimethylammonium chlorides, CnTAC, to vesicles made of a nonionic lipid glycerol monooleate, GMO. The normal route of vesicle solubilization predicted by the three stage model was observed only for surfactants with 12 (C12) and 14 (C14) C atoms in the hydrocarbon chain, whereas a different behavior was found for the C16 one. The purpose of our work is to fill the gap in the field of vesicle-surfactant interactions by providing a study involving another cationic surfactant with a long hydrocarbon chain (C16), cetylpyridinium chloride, CPC, and giant lipid vesicles. CPC was chosen for the study because it is one of the surfactants that show extremely strong interaction with other species, for example with anionic polyelectrolytes.25-30 The purpose of using giant unilamellar vesicles (GUVs) is that they can be easily observed under the optical microscope. It has to be especially emphasized that the use of GUVs presents an important advantage in comparison with most of other studies in this field by offering the possibility of following the morphological changes that are caused by the addition of surfactant on unperturbed (“living”) vesicles. In previous reports, morphologies of small unilamellar vesicles (SUVs) and large unilamellar vesicles (LUVs) were usually followed by cryo-transmission microscopy (cryo-TEM), which requires vitrified samples.19,23,24 This procedure may cause some irreversible changes that would not take place at room temperature. A similar approach to the present one was recently undertaken by Tanaka et al.31 They have prepared giant vesicles made of cholesterol and dipalmitoylphosphatidylcholine, and studied vesicle fission induced by a nonionic surfactant, lysophosphatidylcholine. In contrast to our study, which includes also the quantitative treatment of binding (see below), this was not undertaken by Tanaka. As a further step in the characterization of the interaction between surfactants and phospholipid membranes, a quantitative analysis on the partitioning of surfactant between the water and the lipid phase is necessary. In former studies on interaction of surfactants with lipid vesicles, equilibrium dialysis8 was used to obtain this information, or the amount of the membrane-bound surfactant was deduced from calorimetric22 or spectroscopic12 measurements. In this work, we use the surfactant (22) Tan, A.; Ziegler, A.; Steinbauer, B.; Seelig, J. Biophys. J. 2002, 83, 1547-155. (23) Almgren, M. Biochim. Biophys. Acta 2000, 1508, 146-163. (24) Kadi, M.; Hansson, P.; Almgren, M. J. Phys. Chem. B 2004, 108, 7344-7351. (25) Kogej, K.; Sˇ kerjanc, J.: Surfactant Binding to Polyelectrolytes. In Physical Chemistry of Polyelectrolytes, Radeva, T., Ed.; Surfactant Science Series 99; Marcel Dekker: New York 2001; Chapter 21, pp 793-827. (26) Satake, I.; Yang, J. T. Biopolymers 1976, 15, 2263-2275. (27) Hayakawa, K.; Santerre, J. P.; Kwak, J. C. T. Macromolecules 1983, 16, 1642-1645. (28) Kwak, J. C. T., Ed. Polymer-Surfactant Systems; Surfactant Science Series 77; Marcel Dekker: New York, 1998. (29) Goddard, E. D.; Ananthapadmanabahn K. P.; Eds. Interactions of Surfactants with Polymers and Proteins; CRC Press: Boca Raton, FL, 1993. (30) Sˇ kerjanc, J.; Kogej, K.; Vesnaver, G. J. Phys. Chem. 1988, 92, 6382-6385. (31) Tanaka, T.; Sano, R.; Yamashita, Y.; Yamazaki, M. Langmuir 2004, 20, 9526-9534.
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ion-selective electrode for this purpose, which allows an easy and straightforward determination of the amount of binding of surfactant by other compounds, e.g. by polyelectrolytes.26-30 The advantages of using surfactantselective electrodes include small required sample volume, electrode tolerance to large excess of additives like simple electrolytes and nonelectrolytes (e.g., simple sugars), and excellent sensitivity and reproducibility,29 which are normally far superior to results obtained from equilibrium dialysis experiments.8 The latter experiments rely on analytical determination of the amount of bound surfactant and require extremely long times for the attainment of the equilibrium.8 Clearly, the potentiometric method is applicable only in the case of ionic surfactants such as CPC. The only case of the determination of binding isotherms by surfactant-selective electrodes in vesiclesurfactant suspensions, that the authors are aware of, is for the GMO/CnTAC/NaCl system,24 where small vesicles were involved. To summarize, in the present study we have combined potentiometric determination of the amount of membraneincorporated C16-type surfactant with observation of the induced changes in GUV shape by the optical microscope. Diameters of GUVs in this study were up to 100 µm. This is in variance with previous works, where mostly SUVs (diameters up to 50 nm) and LUVs (diameters up to 500 nm) were investigated.1,2,19,23,24 Furthermore, the solubilization process was followed also by a fluorescence technique using pyrene as a fluorescence probe. As far as we could ascertain, pyrene was not used in such studies until now. It is expected that this method will give us information on the solubilization structures (presence or absence of mixed micelles) in the transition region. 2. Treatment of Surfactant Incorporation into Lipid Membrane In the treatment of interactions of surfactants with lipid vesicles it is convenient to define the partition coefficient as the ratio of surfactant concentrations in the membrane and in the water phase. The electrochemical potential, µ˜ , of surfactant ions (S) is the sum of the chemical potential, µ, and the electrical work zFφ needed to bring 1 mol of z-valent ions from infinity to a place with a potential φ, i.e., µ˜ ) µ + zFφ. In equilibrium, the electrochemical potentials of surfactant ions (S) incorporated in the membrane, (µ˜ S)L, and of the ones remaining in the external aqueous solution, (µ˜ S)W, are equal:
(µ˜ S)W ) (µ˜ S)L
(1)
Indices W and L refer to the water and the lipid phase, respectively. If we express chemical potentials with standard chemical potentials, µ°, as µ ) µ° + RT ln c, we obtain the partition coefficient Kc as the ratio of surfactant concentrations in the lipid and the water phase:
Kc )
(nS)L/VL (nS)W/VW
)
(cS)LVW (cS)WVL
(
exp -
)
) ( )
(µS0)L - (µS0)W zF ∆φ (2) exp RT RT
Here, (nS)L and (nS)W are the number of moles of surfactant in the lipid and in the water phase, respectively, VL and VW are the corresponding volumes, (cS)L is the concentration of surfactant incorporated into the membrane, and (cS)W is the concentration of surfactant monomer in water, where these concentrations are given in moles per total
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b)
(cS)T - (cS)W (cS)T
(4)
The value of b close to 1 implies that almost all surfactant ions are aggregated or incorporated into the membrane. From the binding isotherm, one identifies several stages in the interaction of surfactants with lipid vesicles (see the Introduction) that are displayed by two or more linear regions. The slopes of these regions can be characterized by effective partition coefficients KS with dimensions of liters per mole. 9-11 3. Experimental Section
Figure 1. Response of the CP+ electrode (E) to changes in total CPC concentration (cS)T in aqueous solutions at 25 °C; calibration curve in solution without added POPC: cL ) 0 (0), and potentiometric curves in the presence of POPC: cL ) 2.4 × 10-6 (2), 4.9 × 10-6 (b), and 7.3 × 10-6 mol/L (9). The evaluation of the surfactant concentration in the water phase (cS)W, at a given (cS)T, is indicated by the arrows.
volume of the sample V0 () VL + VW). Note that VL, required for the calculation of Kc, can be obtained from the volume of one lipid molecule incorporated in the membrane,2 vL,1, by multiplying it with the number of lipid molecules in the sample. In eq 2, ∆φ is the potential difference between the lipid and the water phase, T is the absolute temperature, F is the Farraday constant, and R is the gas constant. Equation 2 is valid in dilute solutions where activities can be replaced by concentrations. We have assumed that this condition is satisfied in the present study since the concentration of surfactant is practically always below 1 × 10-3 M. The potentiometric method using surfactant-selective electrode is based on the determination of free surfactantion concentration in the water phase. Following the approach adopted from studies of polyelectrolyte-surfactant interactions,25-27 we define parameter β that gives the number of moles of surfactant ions incorporated into the membrane (or other aggregates) per mole of lipid as
β)
(cS)L (cS)T - (cS)W ) cL cL
(3)
where cL is the concentration of lipid molecules in the sample. (cS)L can be calculated as the difference between the total surfactant concentration, (cS)T, and the concentration of surfactant in the water phase, (cS)W. These values are obtained directly from potentiometric curves measured in the absence and in the presence of lipid vesicles as demonstrated in Figure 1. In contrast to Kc (eq 2), no knowledge of the volumes of lipid and water phases is necessary for the evaluation of β. Various designations and expressions can be found in the literature for β, for example the effective detergent/ phospholipid molar ratio5,6,12,14 Re or simply binding8 νj in the field of surfactant interactions with lipid vesicles or the degree of binding β in the field of surfactant binding by polyelectrolytes.25-30 The data are usually represented in the form of binding isotherms, i.e., plots of β (or Re or νj) vs (cS)W (monomer surfactant concentration in the water phase or simply free surfactant concentration25). It is convenient to know the fraction of surfactant ions that are incorporated into the lipid phase and/or in other aggregates. This is given by parameter b in the following way:25
3.1. Materials. 1-Palmitoyl-2-oleoylphosphatidylcholine (POPC) was purchased from Avanti Polar Lipids Inc., and was used as received. Sucrose (C12H22O11) and glucose (C6H12O6) were from Fluka BioChemica, Switzerland. N-Cetylpyridinium chloride monohydrate (CPC‚H2O, Kemika, Croatia) and hexadecyltrimethylammonium bromide (C16TAB, Fluka, Switzerland) were thoroughly purified by repeated recrystallization from hot acetone and dried under vacuum at 50 °C. The fluorescent probe pyrene (Aldrich, Germany) was used as received. A 70% perchloric acid (HClO4), ammoniumheptamolybdate tetrahydrate ((NH4)6Mo7O24‚ 4H2O), potassium phosphate, monobasic (KH2PO4), and Malachite Green (all from Fluka, Switzerland), 36.5% hydrochloric acid (Kemika, Croatia), and sodium dodecyl sulfate (SDS, Sigma) were used for the determination of the total amount of POPC in vesicle samples. All solutions were prepared in triple distilled water. 3.2. Preparation of Giant Lipid Vesicles with Electroformation. Unilamellar giant lipid vesicles were prepared from a 1 mg/mL solution of POPC in a 2:1 chloroform/methanol mixture by using the modified method of Angelova:32 25 µL of lipid solution was spread on a pair of platinum wire electrodes and vacuumdried for 2 h. The electrodes were then placed 4 mm apart in an electroformation chamber. The chamber was filled with water or with 0.2 mol/L sucrose or glucose solution, and an ac current (10 Hz, 10 V) was applied. After 2 h the frequency and the voltage were reduced in several steps to final values of 1 Hz and 1 V, and the chamber was flushed with water or with 0.2 mol/L glucose solution. This preparation usually results in more or less spherical and unilamellar vesicles with diameters up to 100 µm.33 Vesicles containing sucrose (or glucose) solution were used for observations by inverted optical microscope and for fluorimetric measurements, whereas those in water without added sugars were used for potentiometric measurements. For fluorimetric measurements, sugar solutions were prepared with pyrene-saturated water. 3.3. Determination of Lipid Concentration. The concentration of POPC in lipid vesicle samples was determined as the concentration of inorganic phosphate using optical density measurements. The method was developed previously for the determination of lipoprotein phospholipids in serum34 and was in our study adapted for the determination of lipid concentration in lipid vesicle samples. The concentration region of inorganic phosphate in serum34 was from 0.063 to 4 mmol/L, whereas concentrations in our samples were much lower. Consequently, special care was taken in all procedures in order to prevent any contamination of samples by external phosphorus. This includes soaking of all glassware prior to use in triple distilled water overnight. The following protocol was carried out: 1 mL of lipid solutions was transferred to the test tubes and thoroughly dried on a sand bath at around 300 °C for about 1 h. Then 0.225 mL of perchloric acid (0.5 mol/L) was added to each tube, and the samples were reheated and then heated until the dry residue was obtained. The test tubes were cooled and 1.5 mL of distilled water and 3 mL of color reagent were added and stirred well. Free inorganic phosphate (that was liberated after the destruc(32) Angelova, M. I.; Soleau, S.; Meleard, Ph.; Faucon, J. F.; Bothorel, P. Prog. Colloid Polym. Sci. 1992, 89, 127-131. (33) Akashi, K.; Miyata, H.; Itoh, H.; Kinoshita, K. Biophys. J. 1996, 71, 3242-3250. (34) Jurman-Gros, T. Period. Biol. 1992, 94, 9-12.
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tion) formed a stable complex with color reagent. The absorbance was measured 20 min after the destruction in a 10 mm cuvette at a wavelength 620 nm using a spectrometer Beckmann DU 64. This wavelength corresponds to the absorption maximum of the dye that was determined from measured spectra and differs somewhat from the one reported in the literature.34 With every sample, a blank (triple distilled water) and a standard were measured. Calibration curve was determined by using a series of standard inorganic phosphate solutions with concentrations ranging from 2 × 10-6 to 1 × 10-5 mol/L. The precision of the method was evaluated by the coefficient of variation (C.V.). The obtained value of the C.V. for 10 aliquots of the same sample of lipid vesicles was 11%, and for 10 aliquots of standard inorganic phosphate solution with a concentration 3 × 10-6 mol/L, it was 10%. The described procedure for the determination of lipids was successful only in the case of aqueous solutions without added simple sugars. The destruction of lipid vesicles in sugar solutions with perchloric acid resulted in an inorganic carbon precipitate, and the samples were therefore unsuitable for spectrometric determination. Inorganic phosphate standard solution with a concentration 1 mmol/L was prepared in triple distilled water. The concentrations of ammoniumheptamolybdate and hydrochloric acid solutions were 34 in 5 mmol/L, respectively, whereas those of Malachite Green and SDS were 0.45 and 2 g/L, respectively. Color reagent was prepared by mixing one aliquot of ammoniumheptamolybdate solution with three aliquots of Malachite Green solution. After 30 min, the mixture was filtered and 20 µL of SDS solution was added per 1 mL of the mixture. The reagent was stored in the refrigerator. 3.4. Potentiometric Measurements. The equilibrium concentration of free CP+ ions in aqueous suspensions of lipid vesicles was determined potentiometrically with a CP+-selective membrane electrode. Electrode preparation was as described before,28,29 with the carrier complex prepared by reacting CPC with purified SDS. As a reference, the saturated calomel electrode was used and the voltage E was measured with an ISKRA MA 5740 pH meter. A titration technique28,29 was employed to determine both, E vs the logarithm of total surfactant concentration in pure water (calibration curve) and the corresponding curve in vesicle suspensions. The voltage reached a constant value a few minutes after the change in surfactant concentration. Within the experimental error in the determination of E, the measured slope of the calibration curve did not change. To carry out the measurements, 2 mL of water or lipid vesicle samples was transferred into a temperature controlled measuring cell, the electrodes were immersed into the suspension, and a CPC solution with known concentration was successively added under continuous stirring. All measurements were performed at 25 °C. Calibration curve was scanned every time before and after the measurements in lipid vesicle samples. The response of the electrode (E) in pure CPC solutions had in a wide concentration range the slope 62 ( 2 mV, which is close to its theoretical value (59.2 mV at 25 °C). The incorporation of CPC into POPC vesicles was studied at various concentrations of the lipid component. For this purpose, about 15 mL of vesicle suspension obtained by electroformation was transferred into the evaporation flask and the volume was reduced to about one-quarter of the initial value by vacuum distillation of water at 30 °C, resulting in a higher lipid concentration. The amount of the lipid in vesicle samples was determined before and after the evaporation. For potentiometric measurements both, the concentrated suspensions prepared at various POPC concentrations (concentrated samples), and the original dilute solutions (unconcentrated samples) were used. Furthermore, one of the concentrated vesicle suspensions was diluted to two-thirds and one-third of its concentration and also to the initial concentration value before solvent evaporation. 3.5. Observation of Vesicles. The CPC partitioning into lipid membranes was followed also by observations under the inverted optical microscope (Opton IM 35 or Nikon Diaphot 200) based on a phase contrast technique. The images were recorded with Panasonic VCR AG-7350 video recorder. When Nikon microscope was used, the images were stored directly on a hard disk. With lipid vesicles prepared in 0.2 mol/L sucrose solution (the inside solution) and suspended in a mixture of 0.2 mol/L glucose and
Arrigler et al. Table 1. Partition Coefficients KS1 and KS2, βsat, and Cmc Values Determined in POPC Vesicle Solutions with Added CPC sample no. 1 2 3c 4 5 6d,e 7d,e 8e
cL, 10-6 mol/L
KS1, 105 L/mol
KS2, 105 L/mol
βsat
cmc, 10-4 mol/L
0.79a 0.79a 0.62a 0.62b 3.3b 1.4a 2.4b 4.9b 7.3b
6.0 5.2 7.1 7.7 9.2 6.1 0.7 1.2 1.4
0.27 0.22 0.53 0.51 0.47 0.15 0.16 0.48 0.97
1.6 1.5 1.5 1.5 1.6 0.9 0.8 0.8 0.8
4.5 4.5 4.0 5.0 5.5 6.0 4.2 3.5 1.8
a Unconcentrated samples. b Concentrated samples. c Sample 3 was prepared by dilution of sample 4. d Samples 6 and 7 were prepared by dilution of sample 8. e The binding plots for these samples are given in Figure 2.
0.2 mol/L sucrose solutions (the outside solution) two different experiments were performed. In the first type of experiment, 2 mL of vesicle suspension was poured into a Petri dish and a small amount of concentrated CPC solution was added so that a desired concentration of surfactant in the sample was obtained. After a spherical vesicle without protrusions was found, it was observed for about 10 min before the next addition of CPC solution was made. In the second type of experiment, a small amount (5 µL) of vesicle suspension was transferred into 2 mL of CPC solution with a desired concentration and a suitable vesicle was traced. The first type of experiment was done also with vesicles containing 0.2 mol/L glucose solution on both sides of the membrane in order to avoid osmotic flow between the inner and the outer solution. 3.6. Fluorescence Measurements. Pyrene fluorescence spectra were recorded on a Perkin-Elmer model LS-50 luminescence spectrometer with a water-thermostated cell holder at 25 °C. A 10 mm quartz cuvette was used. The instrument was controlled with a personal computer using Perkin-Elmer Fluorescence Data Manager software. The titration technique was used to vary the concentration of surfactant. The emission spectra of pyrene were recorded from 350 to 500 nm after excitation at 330 nm. The excitation and emission spectral widths were 2.5 nm and the scan rate was 240 nm per minute. From the spectra, the ratio of intensities of the first and the third vibrational peaks, I1/I3, was calculated. These two peaks appeared at approximately 373 and 384 nm.
4. Results 4.1. Results of Potentiometric Measurements. Typical response of surfactant selective electrode to the change in CPC concentration is shown in Figure 1. The calibration curve shows a linear dependency of E on log(cS)T in a wide concentration range from approximately 2 × 10-6 mol/L to the critical micelle concentration, cmc. The value of cmc for pure CPC is indicated by the break in this curve and is equal to 6 × 10-4 mol/L, in good agreement with literature data.30 The E vs log(cS)T curves in the presence of POPC deviate from the calibration curve, implying that CPC is taken up by the vesicles. The shift of the curves to the right increases with increasing lipid concentration. In all cases, the incorporation of CPC into lipid membranes starts at extremely low surfactant concentrations, clearly below 5 × 10-7 mol/L. At higher (cS)T, a similar break is observed in these curves as seen previously for pure CPC solutions (see the calibration curve), indicating that micelles are formed also in the presence of POPC. From this break, the cmc in mixed lipid/surfactant solutions was determined, and it is reported in the last column of Table 1. The obtained values depend on the concentration of the lipid component and are lower than the cmc of pure surfactant.
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Figure 3. Dependence of the partition coefficient Kc on total surfactant concentration (cS)T: cL ) 7.9 × 10-7 (0), 2.4 × 10-6 (2), 3.3 × 10-6 ([), 4.9 × 10-6 (b), and 7.3 × 10-6 (9) mol/L.
Figure 2. (a) Typical binding plots (dependence of β on surfactant concentration in the water phase (cS)W) for samples with different lipid concentration: cL ) 2.4 × 10-6 (2), 4.9 × 10-6 (b), and 7.3 × 10-6 mol/L (9). (b) Enlarged region at low concentrations (cf. frame in Figure 2a) for the sample with cL ) 4.9 × 10-6 mol/L. The error bars are given for β values at the lowest three concentration points. The lines in Figure 2b are the result of a linear regression analysis of the data points in regions A and B, respectively.
From the data in Figure 1, parameter β was calculated (eq 3), and typical binding plots are shown in Figure 2a. The calculation was carried out in the following way: for known value of (cS)T in the presence of POPC vesicles, the voltage E was obtained. The CPC concentration in the water phase (cS)W was then determined from the calibration curve at the same voltage E as indicated in Figure 1. The values of (cS)T and (cS)W involved in this procedure are for the given example indicated by arrows. The uncertainty in the resulting β values is rather low (approximately 3%) at surfactant concentrations higher than 2 × 10-6 mol/L. The part of the calibration curve below (cS)T ) 5 × 10-7 mol/L (no data points) was determined from an anticipated course by taking into account the value of E (around -4 mV) measured in solution without added CPC (in pure water; not shown in the logarithmic plot). Therefore, the uncertainty in β is considerably higher in this concentration region; it may reach 45% at the lowest concentration studied. It has to be stressed that this large uncertainty quickly diminishes with increasing surfactant concentration and has a noticeable, but not large, influence only on the first two β values in the binding plot. This is indicated with the error bars in Figure 2b. In Figure 2a, the steep increase of β at high (cS)W (this threshold CPC concentration depends on the lipid concentration) is clearly seen. We note that total surfactant concentration (cS)T at this point corresponds to cmc values reported in Table 1. Such behavior of β is in strong analogy
with micelle formation35 (see for example Figure 11.2 in ref 35). In the region (cS)T > cmc, total solubilization of mixed lipid/CPC membranes and subsequent formation of mixed micelles occurs.4-12 More interesting is the region with β values below 5. A characteristic binding plot in this region (see enlargement in Figure 2b for one POPC concentration) shows two distinctive linear regions (region A and region B) that differ in slopes. The alteration in slope indicates a change in the binding regime. From the slopes of the curves in regions A and B, the partition coefficients KS1 and KS2, respectively, were calculated.9-11 They are summarized in Table 1 for all POPC concentrations studied. It will be demonstrated further on that the region of surfactant concentrations where the slope of β vs (cS)W curves decreases corresponds to the saturation of the membrane by the surfactant and to the onset of membrane solubilization. The values of β that were obtained from the intercept of the lines in regions A and B are therefore referred to as the “saturation” β values, βsat, and are also reported in Table 1. In some cases, the transition from region A to region B was more gradual than the one shown in Figure 2b. When this occurred, βsat was taken at the point where the deviation of the data points from the linearity in region A was observed. In addition to KS1 and KS2, the partition coefficient Kc (eq 2) was calculated. The dependence of Kc on total surfactant concentration, (cS)T, for different lipid concentrations is shown in Figure 3. All plotted curves display a similar trend in Kc. At low total surfactant concentrations Kc is high (larger than 1 × 106) and decreases with increasing (cS)T. After that, it is almost constant in a wide concentration range from somewhat below 1 × 10-5 mol/L to nearly 1 × 10-3 mol/L, but it increases steeply at higher surfactant concentrations due to mixed micelle formation. Parameter b (eq 4) is plotted in Figure 4 as a function of (cS)T. This plot shows that at low total surfactant concentrations the fraction of surfactant incorporated into the membranes is high. In samples with POPC concentrations 4.9 × 10-6 and 7.3 × 10-6 mol/L it is between 0.9 and 1, implying that more than 90% of CPC is included in the lipid vesicles and less than 10% remains free in the external aqueous phase. With increasing surfactant concentration, the parameter b starts to decrease indicating that additional CPC remains in the monomer form. This region coincides with the region of almost constant Kc values reported in Figure 3. As it will be demonstrated (35) Jo¨nsson, B.; Lindman, B.; Holmberg, K.; Kronberg, B. Surfactants and Polymers in Aqueous Solution; John Wiley & Sons: New York, 1999; Chapter 11.
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Figure 4. Dependence of the fraction of surfactant ions incorporated in aggregates (parameter b; eq 4) on the total surfactant concentration (cS)T: the symbols are the same as in Figure 3.
below by the observations of samples by the inverted microscope, the attainment of constant value of Kc agrees with the region where vesicles start to rupture. At higher surfactant concentration (close to the cmc), b increases again due to micellization. 4.2. Results of Observations by the Inverted Optical Microscope. We shall first describe the observations when sugar solutions inside and outside of the vesicle were different (see Experimental Section, part 3.5) At CPC concentrations below 3 × 10-6 mol/L, there was no observable effect of the added surfactant on the shape of the vesicle, either in the experiment of the first type (addition of CPC solution to the vesicle suspension) or in the one of the second type (addition of vesicle suspension to the CPC solution). Vesicles were stable (time interval of observations was 10 min) and appeared similar to the picture on the first slide in Figure 5, parts a and b. Slow rupture of lipid vesicles was noticed in a narrow concentration range from approximately 3 to 5 × 10-6 mol/L of CPC, which matches with the previously reported region at low surfactant concentrations, where Kc reached a more or less constant value (see Figure 3 for comparison). For about 5 min, no changes could be seen in vesicle shape, but then in a time interval of approximately 1 min vesicle became ellipsoidal and large pores were formed at its poles. This behavior was accompanied by the loss in contrast between the inner and the outer solution. The progress of this change in vesicle shape is shown in Figure 5a for the experiment of the first type. When the concentration of CPC is higher than 5 × 10-6 mol/L, the vesicles disintegrate immediately. Consequently, no pictures could be taken at higher concentrations than this. The same behavior was noticed upon transferring the vesicle suspension into the CPC solution. This is shown in Figure 5b. Observations of vesicles that contained 0.2 mol/L glucose solution both inside and outside (see Experimental section) are presented in Figure 5c. The first frame shows a giant vesicle 10 min after the addition of surfactant solution so that CPC concentration was (cS)T ) 4 × 10-6 mol/L. The second and the third frame present the vesicle 5 and 10 min, respectively, after the further increase of CPC concentration to (cS)T ) 5 × 10-6 mol/L. The last frame corresponds to (cS)T ) 8 × 10-6 mol/L and was recorded 30 min after the last increase of CPC concentration. It can be seen that in this case (no osmotic pressure shock) the vesicle survives an even higher surfactant concentration. 4.3. Results of Fluorescence Measurements. The experimental curves obtained by measuring the fluores-
Arrigler et al.
cence of pyrene are shown in Figure 6 as plots of the intensity ratio I1/I3 vs the logarithm of total C16TAB concentration. C16TAB was used instead of CPC in these measurements because CPC acts as a quencher for the fluorescence of pyrene. These measurements were performed with a suspension of lipid vesicles in sugar solution in order to be comparable with observations under optical microscope. Because the quantitative determination of the lipid component was not carried out in this case (see Experimental Section), it was assumed that the concentration of lipid in these samples was not higher than approximately 1.4 × 10-6 mol/L (this is the largest concentration for the “unconcentrated” samples prepared in water; see for example sample 2 in Table 1). It is known that the change in I1/I3 reflects the formation of micelles or other similar aggregates.25 The value of I1/I3 is high in polar environment like water (around 1.7) and lower in less polar solvents (e.g. in methanol it is around 1.2).36 In the present case of a suspension of lipid vesicles in sugar solutions, I1/I3 is larger than 1.8 indicating a rather polar environment sensed by pyrene. When pure or mixed micelles are formed, pyrene solubilizes in the less polar interior of aggregates and the ratio I1/I3 decreases to 1.2. The critical micelle concentration of C16TAB in sugar solution and in vesicle suspension can be determined from the sharp decrease of the I1/I3 value above the cmc. The cmc of pure C16TAB in 0.2 mol/L glucose solution at 25 °C is around 1.8 × 10-3 mol/L, which is somewhat higher than the value in water (9.1 × 10-4 mol/L).25 In the unconcentrated POPC sugar suspension, the cmc for mixed micelle formation is essentially the same as the one in the absence of the lipid. This can be attributed to the low POPC concentration. 5. Discussion Before discussing the results, we would like to make a comment on the samples of lipid vesicles. The lipid concentration in our GUV samples was very low. Although the use of electroformation for the preparation of giant vesicles results in the highest concentration of the lipid component in comparison with other procedures,38 still the concentration of POPC in our samples was not higher than approximately 1 × 10-6 mol/L (i.e., around 6 × 10-4 w/w %). This fact hampers the quantitative determination of the amount of surfactant that partitions in the membranous phase per mole of lipid. Two steps were undertaken in order to circumvent this difficulty: first, suspensions were carefully concentrated by vacuum evaporation of the solvent (water) and second, very sensitive methods were used for the determination of lipid concentration and free surfactant concentration in the aqueous phase. In similar previous measurements using surfactant selective electrodes24 the concentration of the lipid component was much higher, i.e., around 1 × 10-3 mol/L, and was obtained simply from the weighted amount of the sample. Millimolar lipid concentrations were used also in equilibrium dialysis studies.8 It seems that the micromolar concentration region was investigated only in the present paper. It is important to note that values of partition coefficients for the concentrated samples and for the ones used as prepared do not suggest any influence of sample treatment. This can be concluded by comparing the values of KS,1 and KS,2 for sample 3 in Table 1, which was diluted to the initial concentration from a concentrated (36) Karpovich, S. D.; Blanchard, G. J. J. Phys. Chem. 1995, 99, 3951-3958. (37) Stryer, L. Biochemistry; W. H. Freeman & Co.: New York, 1998. (38) Reeves, J. P.; Dowben, R. M. J. Cell Physiol. 1969, 73, 49-60.
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Figure 5. (a) Rupture of a giant vesicle caused by the addition of surfactant solution to the vesicle suspension (inner solution 0.2 mol/L sucrose; outer solution a mixture of sucrose and glucose): (cS)T ) 5 × 10-6 mol/L, phase contrast objective 40×. The elapsed time between the first and the last slide was around 1 min. Bar ) 10 µm. (b) Rupture of a giant vesicle that was observed upon transferring vesicle suspension into surfactant solution (inner solution 0.2 mol/L sucrose sucrose; outer solution a mixture of sucrose and glucose): (cS)T ) 3 × 10-6 mol/L, phase contrast objective 40×. The elapsed time between the first and the last slide was around 1 min. Bar ) 10 µm. (c) Giant vesicle (inner and outer solution 0.2 mol/L glucose) after the consecutive additions of surfactant solution: (cS)T ) 4 × 10-6 (first frame), 5 × 10-6 (second and third frame) and 8 × 10-6 mol/L (last frame), phase contrast objective 60×. The elapsed time between the first and last frame was about 1 h. Bar ) 10 µm.
Figure 6. Intensity ratio of the fluorescence of pyrene (I1/I3) in solutions of C16TAB in 0.2 mol/L glucose without (2) and with POPC vesicles (9).
suspension, with the ones for samples 1 and 2 (unconcentrated). This likewise holds for the cmc values. High values of the partition coefficients show a strong tendency of CPC to incorporate into the POPC bilayers. In the region where vesicles are stable and the solubilization has not started yet (i.e., at surfactant concentrations below 1 × 10-5 mol/L), the values of Kc are several times 106, up to 1.2 × 107, and they increase with increasing lipid concentration (Figure 3). The affinity of CPC for the membranes decreases rapidly in the progress of incorporation into vesicles and Kc falls to less than 1 × 104 at the point of saturation, characterized by βsat. This pro-
nounced decrease of Kc can be accounted for by electrostatics. The insertion of CP+ ion into an electrically neutral lipid vesicle produces a positively charged surface that repels other surfactant cations. The incorporation of new CP+ ions becomes increasingly more difficult as the amount of bound CP+ and concurrently the charge on the vesicle increases. To the best of our knowledge, there are no literature data on Kc values for incorporation of a cationic surfactant into giant phospholipid vesicles; therefore, no direct comparison can be made with our results on CPC. However, the available literature data9 on nonionic (e.g., octaethylene glycol monododecyl ether, C12EO8; Kc around 1 × 104) and anionic surfactants (e.g., SDS; Kc in the range 400-800) reveal that our highest values of Kc are much higher than usually reported until now.7,9,12-22 Other relevant data, including LUVs and SUVs, can be found in a review by Lasch9 and are also lower than ours. Contrary to the dependence of Kc on cL in the region of stable vesicles (where β < βsat) βsat values display nearly no dependence on lipid concentration. They are around 0.8 (samples 5-8) or around 1.5 (samples 1-4; a new delivery of POPC), suggesting that somewhere between 1 and 2 CPC molecules can be incorporated into the membrane per one POPC molecule before causing vesicle disintegration. In the previously mentioned GMO/C16TAC/ NaCl system24 (the vesicles used were SUVs) the maximum incorporation of C16TAC was found at the surfactantto-lipid ratio equal to 0.57. The higher value for CPC is in agreement with the larger tendency of CPC for
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Figure 8. Different regions in the Kc vs (cS)T plot and the observed changes under the inverted optical microscope.
Figure 7. Model representations of CPC and of POPC molecule.
37
aggregate formation in comparison with C16TAC3,25 (for example, the cmc of CPC is lower than the one of C16TAC25). The effective partition coefficients KS are also higher than those found in the literature. For C12EO8 partitioning into EPC vesicles, Levy et al.15 reported only one KS (which can be compared with our KS,1) equal to 1600 L/mol. For the interaction of sodium cholate with EPC vesicles, three partition coefficients of the KS family were determined by Lasch and Schubert:11 values equal to 290, 52, and 730 L/mol were obtained for KS,1, KS,2, and KS,3, respectively. The third partition coefficient KS,3 is found at increasing amounts of surfactant in solution11 and was not determined inhere. By our opinion, possible reasons for high KS and Kc values obtained in our investigation should be sought in the geometric parameters of the CPC and POPC molecules. Figure 7 shows model representations of both the POPC molecule and the CP+ ion. The shape of the aggregates that are likely to be formed in associating systems is restricted by basic packing considerations, which are governed by the volume-to-surface area ratio (v/a) of an aggregate, and are expressed by the packing parameter v/al, where l is the hydrocarbon chain length.1,3 An important threshold value of v/al is 0.5: when v/al exceeds 0.5 a transition from cylindrical micelles to bilayers is expected.3 The packing shape of POPC is a truncated cone with a packing parameter1,3 larger than 0.5 and the favored shape of the aggregates in water is a bilayer (vesicle).1,3 The length of the hydrocarbon chain in CPC is 16 C atoms, comparable with the lengths of tails in POPC molecule, which are 15 and 17 C atoms. The shape of CPC is also conical (packing parameter1,3 is close to 0.5) with the pyridinium headgroup having a rather small cross-sectional area. These geometrical characteristics actually favor the formation of aggregates with lower curvature in CPC solutions3 and could lead to a tighter interaction of the cetyl chain with the phospholipid fatty acyl chains in GUVs. In comparison with other surfactants, especially nonionic ones, which typically have a bulky headgroup7-18 and a much shorter hydrocarbon chain, the incorporation tendency of CPC into membranes is thus considerably increased. In agreement with this, Silvander et al.19 have found that less alkyl sulfate surfactant with a shorter hydrocarbon chain is needed for the solubilization of lipid bilayers. Surfactant chain length has influence also on the type of structures formed in the transition region (stage 2).19,24 For example, Kadi et al.24 report
different structures in stage 2 of vesicle solubilization when C16TAC is used in place of shorter chain analogues C12TAC or C14TAC. It is of interest to correlate well-defined regions in the concentration dependence of Kc with observations of vesicles by inverted phase microscope (see Figure 8). The region of stable lipid-surfactant mixed vesicles (stage 1), where no ruptures were observed and the shape of the vesicle was not changed, is limited to the first part of this plot at (cS)T below 3 × 10-6 mol/L and characterized by Kc values larger than 1 × 106. At these surfactant-to-lipid ratios, the membrane is impermeable for sugar molecules. In a narrow range from 3 to 5 × 10-6 mol/L, slow ruptures were observed (the beginning of stage 2; see Figure 5, parts a and b). The pores remained opened at the poles and their size increased slowly in time. This enabled the exchange of inner and outer sugar solutions. Because the diffusion constant of glucose is larger than the one of sucrose39 the volume of the vesicle increased owing to a more extensive penetration of glucose into the vesicle until the membrane tension reached a critical value and vesicle disintegrated. The whole process was accompanied by the change of vesicle shape from spherical to ellipsoidal and by the loss of contrast between the inner vesicle compartment and the outer solution (see Figure 5, parts a and b). At higher surfactant concentrations (in the broad region of almost constant Kc), the vesicle disintegrated immediately; therefore, we presume that a larger number of small pores were formed. We could not identify these small pores, not even by using fluorescently labeled POPC vesicles.40 It was concluded, based on the resolution of the optical microscope, that they must be smaller than 0.5 µm. In agreement with our observations, perforated vesicles of almost spherical shape were observed (GMO/ C16TAC/NaCl system) by cryo-TEM.24 To identify possible intermediate forms in the transition region (stage 2), experiments with vesicles suspended in the same medium as included inside the vesicles were performed (see Experimental Section). In this way, the problem of osmotic pressure difference across the membrane was avoided. The experiments have shown that under such conditions vesicles survived even at CPC concentrations around 8 × 10-5 mol/L (this concentration lies well in the region of constant Kc; see Figure 8) and their shape gradually changed from spherical to distorted spherical one (see the first three frames in Figure 5c) and ultimately they formed myelin-like bilayer structures (the (39) Mally, M.; Majhenc, J.; Svetina, S.; Zˇ eksˇ, B. Biophys. J. 2002, 83, 944-953. (40) Gomisˇcˇek, G.; Arrigler, V.; Gros, M.; Zupancˇicˇ, M.; Svetina, S. Pflu¨ gers. Arch.sEur. J. Physiol. 2000, 440, R51-R52.
Interaction of CPC with Giant Lipid Vesicles
last frame in Figure 5c). Similar behavior, where vesicles opened and turned partly into irregular and curved bilayer flakes, has been observed in various other systems.19,23,24 The last region (stage 3) of recurrent increase in Kc was not possible to characterize with the optical microscope, due to the extremely rapid process of vesicle disintegration. Also, the size of mixed micelles is below the resolution of the optical microscope. Their existence was evidenced by fluorescence measurements (see discussion below). It can be concluded that the overall picture for the interaction of CPC with giant POPC vesicles agrees with the three stage model; however, it includes different solubilization structures in the transition region (stage 2) from the ones usually reported in the literature.4-6,9 In the first stage, only intact/stable vesicles are found. Then, at βsat the bilayers become saturated with surfactant and vesicles start to rupture. The formation of pores is a consequence of the cooperativity phenomenon8 that drives CP+ ions to join one with another and may in this way make some parts of the bilayer enriched in surfactant.24 The curvature of mixed vesicles increases in those parts, where it is plausible to expect that more surfactant is incorporated into the membrane, that is in vesicle regions
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where membrane curvature is larger. This could be due to the difference in packing parameters of surfactant and lipid molecule. The described behavior seems to indicate the beginning of the second stage that extends over a broad region of surfactant concentrations, from approximately 1 × 10-5 to somewhat above 4 × 10-4 mol/L. It is usually believed that mixed vesicles coexist with mixed micelles in this region. However, the high and almost constant I1/I3 fluorescence ratio of pyrene (around 1.8, see Figure 6) denied the existence of mixed C16TAB/POPC micelles at C16TAB concentrations below 1 × 10-3 mol/L. Inspection of cmc values in Table 1 suggests the same conclusion for CPC/POPC system. Therefore, we can conclude that in the case of long chain cationic surfactants like C16TAB or CPC, the aggregate structures in stage 2 do not include mixed micelles as usually proposed for the majority of cases.4-9 Instead of a mixed vesicle-mixed micelle coexistence region, vesicles gradually transform into less regular bilayer structures, while the formation of mixed micelles is limited to the third stage of the solubilization process. LA050028U