Study on the Correlation between Lateral Diffusion Effect and Effective

pubs.acs.org/Langmuir © 2009 American Chemical Society

Study on the Correlation between Lateral Diffusion Effect and Effective Charge in Neutral Liposomes ,§
Elisa Galera-Cort
es,† Juan de Dios Solier,† Joan Estelrich,‡ and Roque Hidalgo-Alvarez* †

Departamento de Fı´sica Aplicada, Universidad de Extremadura, Avda. de Elvas s/n
, 06071 Badajoz, Spain, ‡ Departament de Fisicoquı´mica, Facultat de Farm acia, Universitat de Barcelona, Joan XXIII s/n
, 08028 Barcelona, Spain, and §Departamento de Fı´sica Aplicada, Universidad de Granada, Campus Fuentenueva, 18071 Granada, Spain Received August 7, 2009. Revised Manuscript Received October 16, 2009

An experimental investigation is described on the variables that affect the lateral diffusion coefficient (Dlat) of dimyristoylphosphatidylcholine, a zwitterionic phospholipid, and the effective charge (Zef) on liposomes. The lateral diffusion coefficient was obtained from the dielectric relaxation time of the zwitterionic phospholipids in the bilayer, and the effective charge on the external monolayer was estimated from microelectrophoretic mobility measurements by means of the Henry and Coulomb equations. The measurements were performed at different pH values and salt (KBr) concentrations as well as in two physical states of the phospholipid: the liquid-crystalline phase and gel phase. The Zef and Dlat values in the gel phase are always lower than those in the fluid phase. A very small change of pH (∼0.5 pH units) caused a pronounced variation of the effective charge and the lateral diffusion coefficient. Both variations are correlated, which demonstrates that the adsorption of the ions that determine the electrokinetic potential also controls the lateral diffusion of dipolar phospholipids in the bilayer and the effective charge on the external surface of the liposomes.

1. Introduction Liposomes are lipid structures used as a model of biological membranes. They can encapsulate biological molecules, such as proteins, enzymes, or drugs. Lipids used in the preparation of liposomes are predominantly phospholipids (PLs), the same components also found in biological membranes. Depending on the processing conditions, liposomes are formed with one concentric bilayer (the so-called unilamellar liposomes) or with several concentric bilayers (multilamellar liposomes). The principal barrier to permeation in biological membranes is the lipid bilayer, and the lateral diffusion inside the bilayer is a process of importance for the diffusion of their components in the biological membranes, the lateral diffusion coefficient being one of the parameters that inform us about the dynamic state of the membrane.1 Therefore, it is of great interest to know which factors affect the diffusion of phospholipids in their bilayer. This diffusion has fundamental implications in functional coupling between membrane components through collisional mechanisms as, for example, in (a) visual transduction, the process by which light initiates a nerve impulse;2 (b) receptor-mediated endocytosis, a process by which molecules are internalized into a cell (endocytosis) by the inward budding of plasma membrane vesicles containing proteins with receptor sites specific to the molecules being internalized;3 and (c) intercellular adhesion molecules, which promote adhesion among cells, for example, the adhesion of most white blood cells, related to their immunological response to wound or bacterial infection.4 *To whom correspondence should be addressed. E-mail: [email protected]. (1) Tocanne, J. F.; Dupou-C
ezanne, L.; Lopez, A. Prog. Lipid Res. 1994, 33, 203. (2) Lamb, T. D. Biophys. J. 1994, 67, 1439. (3) Schlessinger, J. Biopolymers 1993, 22, 47. (4) Leckband, D. E.; Israelachvili, J. N.; Schmitt, F. J.; Knoll., W. Science 1992, 255, 1419–1421.

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Lateral diffusion of phospholipids in membranes has been studied experimentally over the years by a variety of methods: fluorescence recovery after photobleaching (FRAP),5-7 electron spin resonance (ESR),8 nuclear magnetic resonance (NMR),9-11 and quasielastic neutron scattering (QENS).12,13 Lateral diffusion is also determined by dielectric spectroscopy.14,15 This determination is possible due to the correlation existing between the translational diffusion process and the rotational relaxation of the phospholipids in the bilayer.14 In a previous study the dielectric spectrum of charged liposomes was analyzed.15 The liposomes were prepared with an anionic phospholipid (phosphatidylserine) and a zwitterionic phospholipid (phosphatidylcholine) at a molar ratio of 99:1, respectively. In that study, it was observed that the lateral diffusion in the bilayer decreased with the ionic strength. In the present work, we have studied the electric response of liposomes composed exclusively of a zwitterionic phospholipid, the dimyristoylphosphatidylcholine (DMPC), which possesses the common chemical structure of a phospholipid: a phosphate group and a choline group as the headgroup. DMPC presents a transition temperature of 23.5
C, which implies that, as a function of (5) Vaz, W. L. C.; Clegg, R. M.; Hallmann, D. Biochemistry 1985, 24, 78. (6) Rubenstein, J. L. R.; Smith, B. A.; McConnel, H. M. Proc. Natl. Acad. Sci. U.S.A. 1979, 76, 15. (7) Merkel, R.; Sackmann, E.; Evans, E. J. Phys. France 1989, 50, 1535. (8) Devaux, P. F.; McConnel, H. M. J. Am. Chem. Soc. 1972, 94, 4475. (9) Bloom, M.; Burnell, E. E.; Mackay, A. L.; Nicol, C. P.; Valic, M. I.; Weeks, G. Biochemistry 1978, 17, 5750. (10) Lindblomm, G.; Johansson, L. B. A.; Arvidson, G. Biochemistry 1981, 20, 2204. (11) Kuo, A. L.; Wade, C. G. Biochemistry 1979, 18, 2300. (12) Tabony, J.; Perly, B. Biochim. Biophys. Acta 1990, 1063, 67. (13) K€onig, S.; Pfeiffer, W.; Bayerl, T. M.; Richter, D.; Sackmann, E. J. Phys. II 1992, 2, 1598. (14) Haibel, A.; Nimtz, G.; Pelster, R.; Jaggi, R. Phys. Rev. E 1998, 57, 4838. (15) Solier, J. D.; Galera-Cort
es, E.; Sabat
e, R.; Estelrich, J. Colloids Surf., A 2005, 270-271, 88.

Published on Web 11/03/2009

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temperature, we can have liposomes in a fluid phase (the crystalliquid state) or in a rigid phase (the gel state). In these liposomes, we have analyzed the effect of the pH and the concentration of counterions in the bulk on the lateral diffusion of DMPC in the bilayer, when the phospholipid was in each one of the physical states mentioned above. The aim of this work was to investigate a possible relationship between the effective charge on the external surface of the liposomes and the lateral diffusion of the zwitterionic phospholipids, using DMPC as a model. Moreover, the effect of the rigidity of the bilayer was also studied by performing experiments well below and above the crystal-liquid-gel transition.

2. Materials and Methods 2.1. Materials. Dimyristoylphosphatidylcholine (DMPC) was purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.) and used without further purification. The cation- and anionexchange resin Amberlite IRN-150 was from Supelco (Bellefonte, PA, U.S.A.). Organic solvents of ACS grade (methanol and chloroform) were obtained from Merck (Darmstadt, Germany) and used without further purification. All inorganic reagents were of analytical grade and aqueous solutions were prepared with doubly distilled water. 2.2. Methods. 2.2.1. Liposome Preparation. A total of 60 μmol of DMPC was dissolved in CHCl3/CH3OH (2:1, v/v), placed in a round-bottom flask, and dried in a rotary evaporator under reduced pressure at 40
C to form a thin film on the inner surface of the flask. This film was hydrated with 2 mL of a 1 mM KBr solution to give a lipid concentration of 30 mM. Multilamellar vesicles (MLVs) were initially formed by constant vortexing for 4 min on a vortex mixer, followed by sonication in a Ultrasonic Digitals bath sonifier (Elma, Germany) for 10 min. MLVs were downsized to form oligolamellar vesicles by extrusion at 40
C in a extruder device (Lipex Biomembranes, Canada) through polycarbonate membrane filters of variable pore size under nitrogen pressures up to 55 105 Pa.16 Briefly, liposomes were extruded in three steps: first, three consecutive extrusions through a 0.8 μm pore diameter filter and three other consecutive extrusions through 0.4 μm membranes. The resulting lipid suspension was then extruded three consecutive times through 0.1 μm filters. The extrusion method has been demonstrated to produce unilamellar vesicles,17 and extruded DMPC liposomes are commonly used as a model of unilamellar vesicles for several biophysical studies.18 By means of freeze-fracture electronic microscopy, these vesicles were shown to be spherical (see the Supporting Information, Figure SI1). As the headgroup of the DMPC is zwitterionic, at nonextreme pH values, the obtained liposomes have no net charge on their surface. 2.2.2. Characterization of the Liposomes. Particle size distribution was determined at 25
C by photon correlation spectroscopy with a commercial light-scattering setup (Zetasizer Nano ZS90, Malvern, U.K.) using a 5 mW He-Ne laser. For viscosity and the refractive index, water values were used. To measure the particle size distribution of the dispersion, a polydispersity index, ranging from 0.0 for an entirely monodisperse sample up to 1.0 for a polydispersity sample, was used. It is generally accepted that a liposome sample with a polydispersity index smaller than 0.2 can be considered practically a monodisperse sample. (16) Bally, M. B.; Hope, M. J.; Van Echteld, C. J. A.; Cullis, P. R. Biochim. Biophys. Acta 1985, 812, 55–65. (17) Armengol, X.; Estelrich, J. J. Microencapsulation 1995, 12, 525–535. (18) Kucerka, N.; Kiselev, M. A.; Balgav
y. Eur. Biophys. J. 2004, 33, 328–334.

2666 DOI: 10.1021/la902916y

Galera-Cort es et al. Table 1. Physical Characteristics of Extruded DMPC Liposomes DMPC liposome 126 ( 1 63 58.5 0.084 23.5 0.11 ( 0.01

mean size (nm) outside radius (nm) inside radius (nm) volume fraction (φ) fluid-gel transition (
C) mean polydispersity index

The volume fraction of the samples was determined from the mass fraction using the equation proposed by Haro-P
erez et al.19 x φ ¼ 4πða3 -R3 Þ 1

3

4πa3 F0 3

ð1Þ

where a is the outer liposome radius, R1 = a - Δ is the inner radius, Δ is the thickness of the phospholipid shell (4.5 nm), x is the phospholipid weight fraction used in each synthesis, and F0 is the density of the phospholipid shell on the colloidal particle (1.015 g/cm3). The liposomes were dispersed in a 1 mM KBr solution and were kept over a bed of the Amberlite IRN-150 (0.1 mg of resin by mL of liposomal suspension) for different times. The particle size distribution of the liposomes was determined before (0 h) and after mixing with the resins (24, 96, and 168 h). It was observed that the contact of the liposomes with the resins did not affect either the size or the polydispersity index of the vesicles, and therefore, the vesicles can be considered stable in the colloidal sense. The content of potassium in the vesicles incubated for different times in resins was determined using an UNICAM PU 939 flame absorption spectrometer equipped with an acetylene/air (1:1) burner and a selenium hollow cathode lamp (Photron) that was operated at 776.5 and 0.5 nm band pass. A reference potassium solution (0.5 μg/mL) was prepared by dilution of a 1000 μg/mL solution (VWR, Titrisol, Darmstadt, Germany) with doubly distilled water containing 1% (v/v) of nitric acid (VWR, Titrisol, Darmstadt, Germany). Over the concentration range of 0.1-0.8 μg/mL, the measurement of potassium was linear (R2 > 0.999). The reproducibility of the assay was determined by repeated measurements of the reference solution as part of a run or from run to run. Each value is the average of three replicates. The experiments of atomic absorption of potassium performed with the DMPC liposomes showed that the concentration of this cation on the liposome surface remains unchanged and very close to zero, after 20 h on the resins. Table 1 shows a summary of the properties of the liposomes used in the dielectric and electrophoresis measurements. 2.2.3. Impedance Measurements. To determine the complex dielectric permittivity, impedance measurements were carried out by means of an automated procedure covering the frequency range from 20 Hz to1 GHz. Three different Hewlett-Packard impedance analyzers, HP4284A (20 Hz to 1 MHz), HP4285A (75 kHz to 30 MHz), and HP4191A (1 MHz to 1 GHz) were used. A low ac voltage with an amplitude of 22 mV was applied in all measurements; this voltage corresponds to a field strength of ∼10 nV/nm. The data were obtained using a cell designed in such a shape and size that they allowed for its insertion in a coaxial transmission line of very short length,and for the whole setup to be connected directly (without wire) to the impedance analyzers. (19) Haro-P
erez, C.; Quesada-P
erez, M.; Callejas-Fern
andez, J.; Casals, E.;
Estelrich, J.; Hidalgo-Alvarez, R. J. Chem. Phys. 2003, 119, 628.

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With this setup, the contribution of parasite impedance at the highest frequencies range (1 MHz to 1 GHz) is avoided and, after some ad-hoc calibration procedure, the resolution of the measurements is improved considerably. The experimental procedure employed in the dielectric measurements and the cell features have been described elsewhere.20 The entire system (impedance analyzers, cell, and samples) was kept at the desired temperature inside a thermally insulating cabinet connected at all times to a circulation ultrathermostat (Selecta Frigiterm-30, Spain), which allows making quick measurements. The influence of salt concentration and pH on the dielectrical response and on the lateral diffusion of the PLs was assessed 48 h after the liposome samples had been produced. Subsequent measurements were performed after having the liposome samples on the matrix of the ion-exchange resin for 43, 50, 72, and 113 h. It is important, however, to clarify that the dielectric and microelectrophoretic mobility measurements were carried out in the absence of ion-exchange resins. 2.2.4. pH Measurements. The pH measurements were carried out at 19 and 35
C with a GLP22 pH meter (Crison, Spain). This instrument is equipped with a glass electrode and automatic temperature compensation. It presents an accuracy of ( 0.01 units and was calibrated with two buffer solutions of pH 7.02 and 4.00. 2.2.5. Electrophoretic Mobility Measurements. The microelectrophoretic mobility (μe) measurements were made with a Zetasizer 4 device (Malvern, U.K.) based on the laser-Doppler shift. Results were the average of four measurements. The standard deviation of the microelectrophoretic mobility values was lower than 5% of the average value. Throughout these measurements, liposomes were diluted with 10-3 M KBr in order to obtain a suspension of 10-3 M lipid concentration. All experiments in this study were performed in triplicate. Similar to the dielectric measurements, the influence of salt concentration and pH on the electrophoretic response of the liposomes was assessed 48 h after the liposome samples had been produced. Subsequent measurements were performed after having the liposome samples on the matrix of the ion-exchange resin for 63, 74, and 113 h. The measurements were carried out at 35
C (liquid-crystalline phase) and at 19
C (gel phase). The values of the ζ-potential were obtained by means of the H€uckel-Onsager equation and considering the Henry’s correction factor ζ ¼

3ημe 1 2ε0 εr f ðKaÞ

3. Results and Discussion Figure 1 shows the variation of pH and ionic strength of a liposome sample in the fluid phase (T > 23.5
C) as a function of the ion-exchanging time. Evidently, the salt concentration decreases with increasing time. The pH, however, decreases

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monotonously in the first 72 h, and subsequently, it increases slightly. The time dependence of the pH will be commented on below. The experimental dielectric permittivity spectra are obtained from impedance spectra once the electrode effect has been removed. The procedure used to remove the electrode effect has been described elsewhere.15,20 The experimental complex dielectric permittivity (ε*) is then
 σdc 00 ε ðωÞ ¼ ε ðωÞ - j ε ω /

0

ð3Þ

where ε0 and ε00 are the real and imaginary part of the complex dielectric permittivity (CDP), σdc is the dc conductivity, and ω is the angular frequency. The real part is related to the polarization phenomena of the particle (electric double layer, polarization of dipolar headgroup), and the imaginary part in eq 3 is associated with the dissipation of energy owing to the reorientation of those dipoles and the electric conduction. In the frequency range analyzed, the dissipation term is dominated by the dc ionic conduction (σdc/ω . ε00 ) so that the relaxation peaks are not observed. To remove the dc-conductivity contribution to the imaginary part of the CDP, the Kramer-Kronig integral transform for ε*(ω) can be used.15,20,21

ð2Þ

where εr =78.5, ε0 = 8.85418 3 10-12 F 3 m-1, and η = 8.9 3 10-3 Pa 3 s. For the liposome sample at 10-3 M KBr, a Henry’s factor of 1.5 was used, whereas for the deionized samples, a Henry’s factor of 1 was used. Once the ζ-potential values were obtained, they were converted into effective charge (Zef) using the Coulomb equation. All the experiments were carried out under atmospheric conditions and at constant temperature.

(20) Rold
an-Toro, R.; Solier, J. D. J. Colloid Interface Sci. 2004, 274, 76.

Figure 1. Variation of the ionic strength (b) and pH (f) with the conditioning time of liposome samples in contact with ion-exchange resins.

ε0 ðωÞ -ε0 ð¥Þ ¼


Z ¥ 2 xε00 ðωÞ dx π x2 - ω2 0


Z ¥ 2 xε0 ðxÞ dx ε ðωÞ ¼ π x 2 - ω2 0 00

ð4Þ

ð5Þ

Figure 2 shows the ε0 and ε00 spectra (ε00 is obtained from ε0 using eq 5) of the measurements for two DMPC samples, both in fluid phase: one without contact with the ion-exchange resin (0 h) and the other after 72 h of ion exchanging (72 h). Two relaxations are observed, one just below 105 rad/s and the other around 108 rad/s. The relaxation corresponding to the low frequency is associated with the diffusion of the counterions condensed into a relatively thin layer around the vesicle electric double layer. Because the liposome in our case has a bilayer formed predominantly of (21) B€ottcher, C. J. F.; Bordewijk, P. Theory of Electric Polarization; Elsevier Scientific: Amsterdam, 1978; Vol. II.

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Figure 3. Lateral diffusion coefficient (Dlat, b) and pH (f) as a function of ionic strength for DMPC liposomes in the fluid phase.

Debye-type behavior with a single relaxation time (τH). In a fluid lattice of a liposome, the Debye relaxation time gives an estimate of the 2D lateral self-diffusion coefficient, assuming the phospholipid molecules can be modeled as continuous cylinders with two degrees of freedom, one of rotation and the other of translation. In that case14 rm 2 Dlat e pffiffiffi 2τ H

Figure 2. ε0 (a) and ε00 (b) spectra for DMPC samples in the fluid phase, without contact with the ion-exchange resin (0 h) and after 72 h subjected to the effect of the ion-exchange resin in the liquid phase (72 h). Solid lines correspond to the fitting obtained by Havriliak-Negami equations.

zwitterionic phospholipids, the relaxation corresponding to the high frequency is associated with the lateral diffusion of zwitterionic molecules by rotation around the normal to the bilayer. Such rotation gives rise to the lateral movement14,15 mentioned above. In the low-frequency range, the dielectric relaxation strength decreases due to the decrease of the number of ions condensed on the external surface of liposome as a consequence of the deionization process caused by the ion-exchange resins. The dielectric spectra of all samples are described by the superposition of two Havriliak-Negami (HN) equations:21,22 εðωÞ ¼

ΔεL RL βL

ð1þðjωτL Þ Þ

þ

ΔεH ð1þðjωτH ÞRH ÞβH

þ ε¥

ð6Þ

where Δε is the relaxation strength or intensity, R and β are numbers between 0 and 1, and τ is the characteristic time associated with the peaks of the imaginary part (L and H indicate low and high frequencies, respectively). Parameters R and β are measures of the peak broadening due to the superposition of the dielectric response of different dipoles in the local structures. For a Debye-type process with a single relaxation time, one has R = β = 1, and the frequency at which ε00 is maximum is a good measure of the average relaxation time.23 In this work, in all cases, R ≈ β ≈ 1 for the peak at high frequencies that corresponds to (22) Havriliak, S.; Negami, S. Polymer 1961, 8, 161. (23) Filippov, A.; Or€add, G.; Lindblom, G. Biophys. J. 2003, 84, 3079.

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ð7Þ

where rm is the molecule’s mean headgroup radius. This expression is obtained by assuming that the fluid phase of the membrane has vacancy sites and also considering each degree of freedom takes up the same thermal energy. Then, taking14 rm = 0.4 nm and the fitted relaxation times (τH) from each spectrum, the corresponding average value of the lateral self-diffusion coefficient Dlat can be obtained. 3.1. DMPC in the Fluid Phase. Figure 3 shows the lateral self-diffusion coefficient (Dlat) and pH values as a function of ionic strength. As can be seen in that figure, a close inverse correlation exists between the values of Dlat and the solution pH. It seems that, under these experimental conditions, the pH controls the lateral self-diffusion of DMPC in the bilayer of liposomes in fluid phase. The values of Dlat obtained are of the order of 10-12-10-11 m2/s, and they are of a similar order to those determined by pulsed field gradient NMR23 and a fluorescence technique.24 In 100 nm diameter liposomes, the diffusion of the liposome itself should not be neglected. Considering that the liposome is a sphere with such diameter, the hydrodynamic diffusion model predicts that the 100 nm liposome itself is moving with a diffusion rate of ∼5  10-12 m2/s at 30
C. However, from a dielectric point of view, the corresponding characteristic or typical relaxation time for this particle is of the order of ∼1 ms and the relaxation times of the phospholipid molecules are approximately ∼10 ns. Obviously, these relaxation times correspond to ranges of frequencies clearly separated and easy to differentiate. Therefore, the Dlat determined by dielectric spectroscopy in the MHz range of frequencies has only the contribution of the rotational relaxation of the phospholipid molecules. The dielectric spectroscopy allows us to discriminate between both diffusive processes. Regarding the dramatic change in Dlat of approximately 1 order of magnitude with a relatively small change in pH, it (24) Almeida, P. F. F.; Vaz, W. L. C.; Thompson, T. E. Biochemistry 1992, 31, 6739.

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Figure 4. Effective charge (Zef, b) and pH (f) as a function of the ionic strength of the DMPC liposomes in the fluid phase.

should be noted that changes of the same order have been observed in the fluid phase of DMPC liposomes when the lateral diffusion coefficient was measured as a function of the temperature.14,23 These changes in Dlat with temperature were even larger than the change associated with the gel-to-liquid-crystalline phase transition.14 Makino et al.25 showed experimentally some years ago that neutral liposomes composed of DMPC phospholipids exhibit a nonzero effective charge (Zef) when they are dispersed in aqueous solutions at approximately neutral pH. Obviously, this effective charge is due to the selective adsorption of certain ions from the bulk solution. We have estimated the effective charge on the external surface of DMPC liposomes measuring their microelectrophoretic mobility at different pH and ionic strength values. First, we converted the experimental values of mobility into ζpotential using the classical theoretical treatment (Henry’s equation). Second, we converted the ζ-potential into effective charge using the Coulomb equation. As can be seen in Figure 4, the effective charge (Zef) of the DMPC liposomes is negative and this indicates that anions are preferably adsorbed on these neutral liposomes. The Zef depends on both the solution pH and its ionic strength. There is an important change of Zef when the ionic strength varies 3 orders of magnitude approximately. A close inverse correlation can be seen again between the values of both Zef and pH as in the case of Dlat (see Figure 3). Also, these results indicate that the effective charge on the DMPC liposomes is a very sensitive function of the solution pH. A small change in pH (∼0.5) led to significant variations in the effective charge on the liposomes’ surface (see Figure 4). This, however, is not a new situation because similar changes of ζ-potential (equivalent to Zef) related to a similar range of variation of pH values have been observed by other authors.26 Hence, pH controls the negative effective charge of DMPC liposomes, and this means that OH- is the ion that determines the potential. Therefore, the pH of the solution controls the lateral diffusion in the bilayer and the effective charge on the external surface of these liposomes. Both results can be explained assuming a selective adsorption of OH-. As a consequence of this anion adsorption, the dipolar headgroup of phospholipids varies its orientation with respect to the direction normal to the surface of (25) Makino, K.; Yamada, T.; Kimura, M.; Oka, T.; Ohshima, M.; Kondo, T. Biophys. Chem. 1991, 41, 175. (26) Fatouros, D. G.; Klepetsanis, P.; Ioannou, P. V.; Antimisiaris, S. G. Int. J. Pharm. 2005, 288, 151.

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Figure 5. Lateral diffusion coefficient (Dlat, b) and the effective

charge (Zef, f) as a function of pH of the DMPC liposomes in the fluid phase.

Figure 6. Lateral diffusion coefficient (Dlat, b) and the effective

charge (Zef, f) as a function of pH of the DMPC liposomes in the gel phase.

the liposome; this means that dipoles are more or less perpendicular to the surface, depending on the OH- concentration close to the surface, which is given by the pH changes. Depending on the orientation of the dipolar headgroup of the phospholipids with respect to the external surface, the lateral diffusion of phospholipids is more or less difficult. The correlation between Zef and Dlat is shown in Figure 5 for different pH values. There is a good agreement between both magnitudes for each value of pH. Accordingly, the effective charge of the liposomes is a macroscopic effect of the dynamic or local structure of the DMPC phospholipids in the bilayer.27 It is quite evident that the pH plays a crucial role in the effective charge and the lateral diffusion of the PLs in DMPC liposomes. Now we can give a plausible explanation to the time dependence of the pH (see Figure 1). Comparing Figures 1 and 4, we can see that the pH of the solution is clearly determined by the effective charge on the liposomes’ surface and that the presence in the bulk solution of the potential-determining ions (OH- or Hþ) depends on their specific adsorption on the external surface of the liposome. Some authors have indicated that the decrease in the pH of the solutions is probably due to the diffusion toward the solution (27) Schrader, W.; Kaatze, U. J. Phys. Chem. B 2001, 105, 6266.

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Figure 7. Effective charge (Zef) of the DMPC liposomes in the gel (f) and in the fluid phases (b) versus pH.

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the pH, although the values of Zef and Dlat are smaller for the gel phase than for the liquid phase. Hence, the local structure and the “lashing” of the phospholipids to each other in the bilayer determine the effective charge acquired by OH- adsorption/ desorption as well as the lateral diffusion. Neither the friction process and the dipolar orientation of the headgroup nor their effects are simple, easy, and linear phenomena. The effective charge in both phases has a similar behavior in relation to pH, but the values in the gel phase are always smaller than the corresponding values in the fluid phase (see Figure 7).14 The changes with pH are also smaller in the gel phase. This abrupt change of Zef is a consequence of higher stiffness of the bilayer in the gel phase in comparison with the fluid phase. Furthermore, the different number of bonds changes the friction between phospholipids during their rotation and explains their behavior. The stiffness effect in the gel bilayer is more evident when looking at the lateral diffusion versus pH (see Figure 8). The changes in the lateral diffusion in the fluid phase are more meaningful than in the gel phase, although, in both cases, the pH effect is quite similar. The control of lateral diffusion by effective charge is meaningfully higher in the fluid than in the gel phase. This is due to a greater difficulty for the rotation/translation of the phospholipids in the gel phase.

4. Conclusion

Figure 8. Lateral diffusion coefficient (Dlat) of the DMPC liposomes in the gel (f) and in the fluid phases (b) versus pH.

of the atmospheric CO2.28,29 In our case, this effect is probably negligible because all the ionic impurities of this atmospheric contamination would be removed from the solution by the ionexchange resins. 3.2. Comparison of Gel/Fluid Phases. We changed the temperature from 25 to 19
C to cause a phase transition from the fluid to the gel phase of the zwitterionic phospholipids in the bilayer. By doing this, we were able to study the dielectric response of a liposome with a gel structure in the bilayer. Figure 6 shows the data of Zef and Dlat obtained with a gel structure in the bilayer as a function of pH. As can be seen in this figure, when Zef increases in absolute value, Dlat also increases and we observe basically the same behavior for both properties with the pH. In this case, a clear inverse correlation also appears with (28) Carrique, F.; Ruiz-Reina, E. J. Phys. Chem. B 2009, 113, 8613. (29) Ruiz-Reina, E.; Carrique, F. J. Phys. Chem. B 2008, 112, 11960.

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Broad-band dielectric spectroscopy has been shown to be an effective tool to study the dynamic and even the local structure of phospholipids in the bilayer. Measurements of the dipolar relaxation time provided the average diffusion coefficient of these molecules for the fluid as well as for the gel phases of the liposomes. In the range of salt concentration analyzed (