5568
J. Phys. Chem. B 2001, 105, 5568-5574
Reversing-Pulse Electric Birefringence Study of Unilamellar DOPC Vesicles Viktor Peikov and Zoltan A. Schelly* Center for Colloidal and Interfacial Dynamics, Department of Chemistry and Biochemistry, UniVersity of Texas at Arlington, Arlington, Texas 76019-0065 ReceiVed: January 16, 2001; In Final Form: April 9, 2001
Unilamellar bilayer vesicles of 193 nm diameter, prepared from the zwitterionic phospholipid dioleoylphosphatidylcholine (DOPC), are investigated by the use of the reversing-pulse electric birefringence (RPEB) method with the aim of elucidating the mechanism of vesicle polarization that results in transient induced birefringence of the solution. For this purpose, characteristic parameters of the observed course of the timedependent birefringence, which ensues the sudden polarity reversal of the perturbation field, are examined as a function of the concentration of NaCl (ranging from zero to 5 × 10-4 M) placed inside and/or outside the vesicles. Effects of varying the lipid concentration and the applied field strength are also reported. On the basis of the ionic strength dependence of the extent and rate of the temporary partial loss of birefringence upon field reversal, a slow ion polarization mechanism is proposed that is consistent with the experimental results.
Introduction Perturbation of organized assemblies by a sudden change of a thermodynamic variable such as a pressure-, temperature-, or an electric field-jump provides valuable information about the structural properties, dynamic behavior, stability, and phase transitions of the particular system.1-3 Such information is vital to the numerous practical applications involving microemulsions, liquid crystals, liposomes, and biological cells. An application of particular interest is the electroporation of biological cells, where fully reversible transient pores are induced in the membrane by an externally applied electric filed.4-6 The open pores are utilized for the insertion of active chemical agents into the interior compartment of the cell. Synthetic unilamellar bilayer vesicles or liposomes have often been used as a model of the cell membrane.4-6 Electrooptic methods such as electric birefringence, electric dichroism, and electrically induced turbidity provide effective tools for studying surfactant monolayer rigidity,7 and deformation,8-10 and structural changes11,12 in vesicles. It is generally accepted that the electrooptical effects observed from originally spherical vesicles are mainly due to the field-induced polarization and deformation of the bilayer shell. Furthermore, the transients in the electrooptic response also provide information about the size of the vesicles, pore formation, and the dynamics of rotation and clustering of dipoles. Reversing-pulse electric birefringence13 (RPEB), where a sequence of two rectangular electric pulses of opposite polarity are applied to the system and the induced electric birefringence is monitored, is especially useful in determining the mechanisms of polarization and reorientation of particles.14 This method has been extensively utilized for such a purpose in various colloidal systems such as suspensions of solid particles, surfactant crystallites,15 polymers, and DNA.16-18 RPEB was successfully applied also to investigate the lateral diffusion of photopigments19 and the electric and optical anisotropy20 in photoreceptor disk membrane vesicles, and the orientation and structure of polymer-vesicle hybrids.21 In the present work, the RPEB method is used for the examination of unilamellar bilayer vesicles prepared from the * Corresponding author. E-mail:
[email protected].
zwitterionic phospholipid dioleoylphosphatidylcholine (DOPC; 1,2-dioleoyl-sn-glycero-3-phosphocholine) with the aim to elucidate the polarization mechanisms of the vesicle and to determine the effects of free ions placed in the solution. These goals were achieved through systematic variation of the concentrations of the lipid and of an added electrolyte (NaCl) inside and/or outside the vesicle. Materials and Methods Preparation and Characterization of Vesicles. The preparation and characterization of DOPC vesicles have been described in detail previously.10,22 The lipid 1,2-dioleoyl-snglycero-3-phosphocholine (DOPC, from Avanti Polar Lipids; molar mass ) 786.15 g) was used without further purification. Initially, large multilamellar vesicles (MLV; lipid concentration 4 mg/mL) were obtained by hydrating the dried lipid film in double-deionized and distilled water by vortex mixing (Vortex-2 Genie) for 5 min. The MLV suspension was repeatedly extruded (Extruder, Lipex Biomembranes) in five passes under nitrogen (overpressure of 3.4 bar) through two stacked polycarbonate filters of 0.2 µm pore size to produce large unilamellar vesicles (LUV).23 This procedure has been previously confirmed through the extent of quenching of 31P NMR signal by manganese ions to produce unilamellar DOPC vesicles of narrow size distribution.10,24 The mean hydrodynamic diameter 〈Dh〉 of freshly prepared DOPC vesicles was determined by dynamic light scattering (Brookhaven model BI-200 SM) to be 193 ( 6 nm. Vesicles of this size consist of approximately 3.2 × 105 DOPC molecules.10 Due to the zwitterionic polar headgroup of DOPC, such vesicle dispersions contain no free ions originating from the surfactant. To produce vesicles with nonzero ionic strength in their interior compartment, the dried lipid film was hydrated in aqueous solutions of NaCl of different ionic strengths (1 × 10-4 or 5 × 10-4 M), instead of using pure water as described above. The ion concentration in the outside bulk solution was adjusted by adding the requisite amount of salt during the preparation of the dilute vesicle dispersions (0.25 mg/mL of lipid) used for the majority of the electric birefringence measurements. Thus, dispersions of the following combinations of local ionic strengths
10.1021/jp010164h CCC: $20.00 © 2001 American Chemical Society Published on Web 05/19/2001
Electric Birefringence of DOPC Vesicles
J. Phys. Chem. B, Vol. 105, No. 23, 2001 5569
were prepared: ionic strength (M) inside
outside
0 0 0 1 × 10-4 1 × 10-4 5 × 10-4 5 × 10-4 5 × 10-4
0 1 × 10-4 5 × 10-4 1 × 10-4 5 × 10-4 3 × 10-5 1 × 10-4 5 × 10-4
Despite the difference in osmotic pressure ∆Π between inside and outside the vesicle, which exists in most of these cases, dynamic light scattering results showed the vesicle size to be independent of the ion concentration as well as of the difference in ion concentration between the interior and exterior of the vesicles. In the range of ion concentrations studied here, the vesicles were stable for at least 3 days, as judged from the results of dynamic light scattering and electric birefringence measurements. Instrumentation. The electric birefringence instrument has been described elsewhere.3 A 10 mW He-Ne laser (MellesGriot, wavelength λ ) 632.8 nm) was used as a light source. The optical detection system included a quarter-wave plate, with its slow axis oriented at 3π/4 relative to the direction of the applied electric field E. The signal from the photomultiplier was recorded by a digital oscilloscope (Hewlett-Packard, model 54510A) and transferred to a computer. The electric birefringence ∆n (≡ n|| - n⊥) was calculated from the experimentally observed optical retardation δ through ∆n ) δλ/2πl, where l is the optical path length in the Kerr cell (l ) 5 cm, gap between electrodes d ) 2.5 mm). The value of ∆n was corrected for the induced retardation of the cell windows (δw < 0.5°) and for stray light. The reversing-pulse electric field was produced by two pulse generators (Cober, model 605P) connected in parallel.15 The system provides up to (2 kV high-voltage reversing rectangular pulses with rise, polarity reversal, and fall times less than 100 ns. Vesicle preparation, dynamic light scattering, and electric birefringence experiments were carried out at 25 °C, well above the phase transition temperature (Tc ) -17.3 °C)25 of the DOPC bilayer. Results Reversing-Pulse Signal. Upon application of an electric field E of sufficient strength, DOPC vesicles exhibit positive induced electric birefringence that corresponds to the elongation of their time average spherical shape to prolate ellipsoid, with the major axis parallel to E. The transient birefringence and turbidity responses (i.e., initial buildup and field-off relaxations) of the system to perturbation by a single rectangular pulse have previously been analyzed in detail.10 Electrooptic responses in general reflect the induced structural anisotropy of the system. In the present paper we focus only on the transient birefringence response associated with a sudden polarity reversal of the perturbation field. To this end, when a reversing-pulse electric field is applied, three different parts of the birefringence signal can be distinguished: buildup, reverse, and decay (Figure 1). In the buildup portion of the RPEB signal, the birefringence ∆n(t) monotonically increases and reaches a steady state ∆n(∞) if the first pulse is sufficiently long. Upon reversal of the polarity of the field, the birefringence of the system initially decreases,
Figure 1. Schematic representation of the perturbation and the system response in RPEB experiments: applied reversing electric field pulse (top) and the corresponding normalized induced birefringence ∆n(t)/ ∆n(∞) signal (bottom). The duration of a pulse of each single polarity was varied from ∼1 ms (at high field strength) to ∼10 ms (at low field strength). For explanation of the characteristic parameters tm and ∆m, see the text.
passes through a minimum, and rises to a steady state ∆n(∞) equal to that reached by the end the first pulse. After the removal of the electric field, ∆n(t) monotonically decays to zero on a time scale much longer than those in the initial buildup and the reverse parts. Schematic representation of the reversing-pulse electric field and a corresponding typical RPEB signal obtained for DOPC vesicles are shown in Figure 1, where the signal is normalized as ∆n(t)/∆n(∞). The dip in the reverse part of the signal can be characterized by two parameters: the time tm at which the local minimum is reached, counted from the polarity reversal of the field, and ∆m, the normalized value of the remaining birefringence at the local minimum (Figure 1). The dip reflects a temporary, partial loss in the anisotropy of the system. Repeated application of reversing pulses leads to a slight increase of the steady-state birefringence and a deepening of the dip. These effects become more pronounced with increasing ionic strength outside the vesicle, thus signal averaging would not be applicable. All results presented here are obtained from the first reversing pulse applied. Steady-State Birefringence. The steady-state electric birefringence ∆n(∞) depends on the applied field strength E and the concentration C of the lipid. The specific steady-state electric birefringence ∆n(∞)/C as a function of E2 is plotted for several different lipid concentrations in Figure 2. In an intermediate domain of the field strength (2.4 kV/cm < E < 6.7 kV/cm), the specific steady state birefringence increases only slightly with E2. The apparent steep decline of the birefringence with decreasing values of E2 observed at extremely low field strengths (E < 2.4 kV/cm) is due to the inability of the systems to actually reach a steady state during the maximum possible pulse length (up to 10 ms) that can be produced by our pulse generators. By reason of this instrumental limitation the validity of the Kerr law could not be verified at low field strengths. Similarly, at extremely high fields (E > 6.7 kV/cm), the electric birefringence keeps increasing with the pulse duration and does not seem to approach a steady state. Because of the poor reproducibility of this effect, it will not be discussed any further. The steady-state electric birefringence is found to be linearly proportional to (i.e., the specific steady-state electric birefringence is independent of) the lipid concentration up to 0.5 mg/ mL (Figure 2, bottom curve). Above this concentration, the
5570 J. Phys. Chem. B, Vol. 105, No. 23, 2001
Figure 2. Electric field strength (E) dependence of the specific steadystate electric birefringence ∆n(∞)/C for several different DOPC concentrations C (mg/mL): (4) 0.128; ()) 0.25; (b) 0.5; (9) 2.0. Uncertainties are similar to those indicated in Figure 5.
Figure 3. Electric filed strength (E) dependence of the time tm of the local minimum in the reverse part of the RPEB signal (see Figure 1) at several different DOPC concentrations (mg/mL): (() 0.25; (b) 0.5; (9) 2.0. Typical uncertainty in tm is approximately (1.5 times the magnitude of the symbols used.
electric birefringence increases at a higher rate. The linear dependence of the birefringence on the lipid concentration (at C e 0.5 mg/mL) suggests the absence of any contribution arising from intervesicle interactions. Reverse Part of the Signal. The field strength dependence of the parameters of the local minimum in the reverse portion of the RPEB signal are shown in Figures 3 and 4 for different lipid concentrations. The time tm needed for reaching the minimum decreases with the increase of the applied electric field strength E in a very broad range (Figure 3). While at high electric field tm is of the order of only 100 µs, at low fields it reaches values of several milliseconds. In the limits of experimental error, the magnitude of tm is virtually independent of the lipid concentration in the whole concentration range studied here. The amplitude ∆m of the birefringence at the local minimum is found to be independent of the applied field strength within the experimental error (Figure 4). The value of ∆m fluctuates around 0.75 at a lipid concentration of 2 mg/mL, and decreases to about 0.6 at lower lipid concentrations. For lipid concentrations below 0.25 mg/mL, the values of tm and ∆m cannot be precisely determined because of low signal-to-noise ratio. Overall, however, the dip in the reverse part of the RPEB signal is well pronounced and its characteristics are independent of the lipid concentration up to 0.5 mg/mL. In general, the presence of a dip in the reverse part of the signals could suggest14,17,21 a possible permanent dipole moment
Peikov and Schelly
Figure 4. Electric filed strength (E) dependence of the birefringence amplitude ∆m at the local minimum in the reverse part of the normalized RPEB signal (see Figure 1) at several different DOPC concentrations (mg/mL): (() 0.25; (b) 0.5; (9) 2.0. The typical uncertainties (standard deviation) indicated apply to all curves.
Figure 5. Electric field strength (E) dependence of the specific steadystate electric birefringence at several different NaCl concentrations (M) inside (in) and outside (out) the vesicles: ()) water in and out; (() water in, 1 × 10-4 M NaCl out; (4) water in, 5 × 10-4 out; (b) 1 × 10-4 in and out; (×) 1 × 10-4 in, 5 × 10-4 out; (0) 5 × 10-4 in, 3 × 10-5 out; (9) 5 × 10-4 in, 1 × 10-4 out; (+) 5 × 10-4 in and out. C ≡ [DOPC] ) 0.25 mg/mL. The typical uncertainties (standard deviation) indicated apply to all curves.
or a contribution by a slow induced dipole moment in the deformation and/or orientation mechanism of DOPC vesicles. To clarify the origin of the dip we investigated the influence of the presence of free ions on the profile of the RPEB signals. In these experiments we kept the total lipid concentration constant at 0.25 mg/mL. At this concentration the mean distance between vesicles is estimated to be 800 nm; hence no intervesicle interactions and induced turbidity effects are expected, and the observed birefringence is due to the deformation of isolated DOPC vesicles. The ion concentration both inside and outside the vesicle was varied by the addition of NaCl, as described in the Materials and Methods. The increase of the ion concentration had two major general effects on the RPEB signals: (i) the amplitude of the steady-state electric birefringence increased, and (ii) the dip in the reverse part of the signals became shallower. In these effects, the ion concentration outside proved more important than that inside. The change of the amplitude of the steady-state electric birefringence with ionic strength is shown in Figure 5. Samples with the highest ionic strength (5 × 10-4 M) outside the vesicles exhibit by about 50% greater values of the electrically induced birefringence than samples with low or intermediate ion concentrations. Similar results have
Electric Birefringence of DOPC Vesicles
Figure 6. Change of the profile of the RPEB signals with the concentration (M) of NaCl inside (in) and outside (out) the vesicles: (a) 5 × 10-4 M NaCl in and out; (b) 5 × 10-4 in, 1 × 10-4 out; (c) 5 × 10-4 in, 3 × 10-5 out; (d) 1 × 10-4 in, 5 × 10-4 out; (e) 1 × 10-4 in and out; (f) water in, 5 × 10-4 out; (g) water in, 1 × 10-4 out; (h) water in and out. C ≡ [DOPC] ) 0.25 mg/mL; E ≈ 5.9 kV/cm.
already been reported.10 The ion concentration, however, does not affect significantly the field strength dependence of the steady-state electric birefringence (Figure 5). The change in the profile of the RPEB signals with the ion concentration is illustrated in Figure 6. The dip is the smallest (i.e., ∆m is the greatest) in the case of the highest ionic strength of 5 × 10-4 M both inside and outside the vesicles (Figure 6a). The depth of the dip increases with the decrease of the outside ionic strength at fixed inside ion concentration (compare Figure 6a with 6b, 6d with 6e, and 6f with 6g). Conversely, the depth increases only slightly with the decrease of the inside ion concentration at fixed outside ionic concentration (compare Figures 6a, d and f, and also Figures 6b, e and g). This obvious sensitivity of the depth indicates that the presence of ions plays a significant role in the polarization and deformation mechanisms of the vesicles and is in accord with a thermodynamic theory26 proposed for the electroporation of curved membranes in the presence of ionic strength gradients. A more quantitative description of the changes in the profile of RPEB signals is given in Figures 7 and 8 where the electric filed strength dependence of the position tm of the local minimum in the reverse part of the signal and the corresponding birefringence amplitude ∆m are plotted for several different ionic strengths, respectively. The position of the minimum (tm, Figure 7) remains unaffected by the ionic strength within the experimental error. However, a more than 10-fold increase of tm is observed (i.e., the local minimum is reached slower) with decreasing electric field strength. The birefringence amplitude ∆m at the signal minimum is essentially independent of the electric field strength, but it increases significantly with the ion concentration outside the vesicles (Figure 8). For instance, for samples with 5 × 10-4 M NaCl outside, the values of ∆m are greater than 0.8. Decreasing ∆m corresponds to the increasing depth of the dip in the signal. Generally, the dip is the deepest (∆m < 0.6) for samples containing a combination of water and/ or only 1 × 10-4 M NaCl outside the vesicles. Discussion Previous studies have shown that at low lipid concentrations and relatively short applied electric pulses the electric birefringence of vesicle dispersions is mainly due to electromechanical elongation of the vesicles in the direction of the field.10 However, contributions from electrostrictive thinning of the
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Figure 7. Electric filed strength (E) dependence of the time tm of the local minimum in the reverse part of the RPEB signal (see Figure 1) at several different concentrations (M) of NaCl inside (in) and outside (out) the vesicles: ()) water in and out; (() water in, 1 × 10-4 M NaCl out; (4) water in, 5 × 10-4 out; (b) 1 × 10-4 in and out; (×) 1 × 10-4 in, 5 × 10-4 out; (0) 5 × 10-4 in, 3 × 10-5 out; (9) 5 × 10-4 in, 1 × 10-4 out; (+) 5 × 10-4 in and out. C ≡ [DOPC] ) 0.25 mg/ mL. Uncertainties are similar to those in Figure 3.
Figure 8. Electric filed strength (E) dependence of the birefringence amplitude ∆m at the local minimum in the reverse part of the normalized RPEB signal (see Figure 1) at several different concentrations (M) of NaCl inside (in) and outside (out) the vesicles: ()) water in and out; (() water in, 1 × 10-4 M NaCl out; (4) water in, 5 × 10-4 out; (b) 1 × 10-4 in and out; (×) 1 × 10-4 in, 5 × 10-4 out; (0) 5 × 10-4 in, 3 × 10-5 out; (9) 5 × 10-4 in, 1 × 10-4 out; (+) 5 × 10-4 in and out. C ≡ [DOPC] ) 0.25 mg/mL. Uncertainties are similar to those in Figure 4.
membrane at the polar caps10,27 and the rotational alignment of induced10,28 and instantaneous10 dipoles cannot be fully excluded. At high vesicle concentrations and/or long electric pulses the effects due to interactions between the polarized vesicles, such as the formation of linear chains (pearling) in the field direction and the partial fusion of vesicles within the pearls, can become dominant.10 Experimental results presented in Figure 2 support these findings. With the pulse durations used in the present study, the steady-state electric birefringence normalized for different DOPC concentrations is found to be independent of the vesicle concentration up to 0.5 mg/mL. Consequently, in this concentration range, interparticle interactions do not contribute to the observed birefringence that is due to the elongation of isolated vesicles in the direction of the applied field. However, at a lipid concentration of 2 mg/mL, the excess of the specific birefringence (Figure 2) may be attributed to vesicle pearling and possibly fusion. All our RPEB signals exhibit a well-pronounced, reproducible dip upon field reversal (Figures 1 and 6), irrespective of the
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vesicle concentration used. Hence, the dip is not due to intervesicle interaction that leads to aggregation, pearling, or fusion. At lipid concentrations below 0.5 mg/mL, even the characteristic parameters of the dip (i.e., the time tm counted from the field reversal to the local minimum in the signal, and the value ∆m of the normalized birefringence at the local minimum) are independent of the vesicle concentration (Figures 3 and 4), similar to the behavior of the specific steady-state birefringence (Figure 2). Thus, we may conclude that the dip observed in the RPEB signals reflects the property of individual DOPC vesicles, and as such, represents valuable information for elucidating their polarization mechanism. For this purpose, the low lipid concentration of 0.25 mg/mL was used for all pertinent measurements because of the absence of disturbing vesicle-vesicle interactions at this concentration. The penalty for doing so is, of course, reduced signal-to-noise ratio. RPEB is often used for studying the polarization mechanisms of dispersed particles. Its main advantage compared to the application of a single electric pulse lies in the greater sensitivity of the course of the reverse part (dip in our case) of the signal to the type and magnitude of the dipole moments involved than it is the case for the buildup part of the signal. Theoretical expressions for the profile of the RPEB signals in the buildup and reverse parts are readily available for nondeformable solid particles, and polyions.14,17,18,29 The theories take into account various polarization mechanisms that may involve permanent dipole moment,14 electronic induced dipole moment,14,17,18 slow (compared to particle rotation) induced dipole17 and ionfluctuation dipole.18,29 The emergence of a dip in the signal upon field reversal is attributed to the presence of a permanent dipole moment and/or a slowly responding polarizability (such as in an ion-fluctuation dipole).14,17,18,29 A dip in the RPEB signals observed for polymer-vesicle hybrids was identified as being due to a structural permanent dipole moment.21 In contrast (also to our phospholipid vesicles), the bare unilamellar vesicles of that system, prepared from the cationic surfactant dioctadecyldimethylammonium bromide (DODAB), did not respond to field reversal.21 The DOPC vesicles studied here undergo a continuous thermal shape fluctuation and have a time average spherical shell structure in the absence of electric field, as suggested by TEM.30 The structure of the lipid bilayer is believed to be homogeneous and free of defects. The constituent DOPC molecules possess a small permanent dipole moment owing to the zwitterionic phosphatidylcholine headgroup of the surfactant. The headgroups are known to lie flat on the interior and exterior surfaces of the bilayer shell where they have a certain degree of rotational freedom around the bond that anchors them to the surface. The alignment of these permanent dipoles (especially those located in the equatorial zone of the vesicle where they can reorient without lifting the positive pole out of the surface) parallel to the field contributes to the total polarization of the vesicle. However, time domain dielectric spectroscopy measurements have revealed that their rotational relaxation time is about 1 ns10 and hence is beyond the time resolution of our transient electric birefringence instrument. For comparison, other relevant and much slower processes that are accessible to our observation are the Debye rotational relaxation time τD of the spherical vesicles and the experimentally obtained values of tm g 100 µs. The Debye relaxation time can be estimated from
τD ) 1/6 Drot ) 4πηR03/3kT ) 815 µs
(1)
where Drot is the rotational diffusion coefficient, η ) 0.89 cP is the viscosity of the solvent water, R0 ) 96.5 nm is the mean
hydrodynamic radius of the vesicles, and kT is the thermal energy. Clearly, on the time scale of our RPEB experiments, the headgroups will effectively act as fast induced dipoles that arise (align) almost instantaneously in the direction of the external field. There are neither structural nor geometrical reasons for assuming that a DOPC vesicle possesses a permanent dipole moment. Thus, the dip experimentally observed in the signals seems to be due to a slow induced ion polarization associated with ions adsorbed on or bound to the lipid/solution interfaces. The ions originate from the autodissociation of water and trace impurities (mainly dissolved CO2)sor an electrolyte added on purpose, as in our case. Each pole of a zwitterionic headgroup represents a charge-specific and site-specific binding site for ions of the opposite charge in the solution. Prior to perturbation, there are equilibrium populations of bound and free ions. When an electric field is applied, the forced migration of bound ions (hopping from one headgroup to another) over the vesicle surfaces following the direction of the field is significantly slower than the migration of any free, unbound ions because of the strong electrostatic interaction of the bound ions with the headgroups. On route to the final steady state during the first pulse, the polar cap regions both inside and outside the vesicle acquire excess charges of a sign opposite to the sign of the electrode they face. With approximately equal ionic strengths inside and outside, since the area of the exterior surface is larger than that of the interior surface, the accumulated excess charge outside a polar cap is greater than the excess charge of the same sign inside. Besides this geometric effect, the difference is also affected by the partial shielding of the applied field by the exterior charges; i.e., ions inside are exposed to a smaller force field than those outside. Action of the force field on the net excess charges in the two polar cap regions of the vesicle is a major factor in the electromechanical deformation (elongation) of the originally spherical bilayer shell to a prolate ellipsoid of revolution, with its major axis parallel to E. The arising structural anisotropy of the solution is the main source of the induced birefringence. An additional relevant event that occurs during the first pulse is the electroporation of the DOPC vesicles provided the applied field strength is in the 4-8 kV/ cm range and the pulse length exceeds ∼200 µs.8,9 The process is relevant in our case because the ion concentrations inside and outside the vesicle can equalize to a great extent through the open pores. With the much greater reservoir of free ions outside, the ion concentration inside approaches that in the bulk solution during repeated application of pulses. This explains the dominant effect of the outside ionic strength on (i) the steady-state electric birefringence and (ii) the magnitude of the dip in the signals, as mentioned before. Upon the sudden polarity reversal of the field, if vesicle rotation and the previously established ion distribution were frozen, the field now would tend to compress the prolate ellipsoidal vesicle to a symmetric oblate ellipsoid, whereby the major axis of the former would ultimately convert to the minor axis of the latter. In reality, of course, vesicle rotation is merely significantly slower (see eq 1) than the experimentally measured tm, and the ion distributions are free to adjust to the new situation. Hence, following the field reversal, the ions start migrating in the opposite direction, the ion distribution that existed in the steady state becomes less polarized, and the contribution of the bound ions to the membrane polarization and to vesicle deformation temporarily decreases. All these processes result in a partial deelongation of the vesicles and, consequently, in a partial loss (i.e., beginning of the dip) in the birefringence signal. Subsequently, the evolution of a new steady
Electric Birefringence of DOPC Vesicles state of the ion distributionsultimately equivalent to the first one but with a reversed polarityshalts the deelongation and, concomitantly, resumes the elongation. The turnaround point in the events corresponds to the local minimum (tip of the dip) in the birefringence signal. Upon the final removal of the applied electric field, the ensuing signal decay reflects a relatively slow decrease in the structural anisotropy of the solution. In the absence of added ions, the rate of this process would be determined by the rates of deelongation of the naked bilayer shells (governed by the membrane elasticity modulus κ) and the concomitant rotational randomization of the induced dipolar vesicles. In the presence of added ions, these rates are coupled to the rate of redistribution of ions bound to the surfaces of the vesicle. In the redistribution process, the ions start to migrate again to reestablish their original, field-free equilibrium distribution. In the absence of an electric field, the driving force of this process is the concentration gradient of bound ions over the vesicle surfaces. The rate of migration is governed by the rate of surface diffusion of the bound ions under the influence of existing local fields. This process is expected to be slower than the forced redistribution of the bound ions upon either the previous field reversal or the initial application of the first pulse. As a result, the rate of deelongation of the vesicles in the initial part of the decay process would be somewhat smaller than during the initial part of the field reversal process. This effect can be clearly seen from the comparison of the initial slope of the dip with that of the field-off decay of the RPEB signals in Figure 6. The electric birefringence decreases more rapidly in the initial stage of the reverse part (dip) than at the beginning of the decay part of the signal. This finding supports the slow induced ion polarization mechanism for the vesicle deformation proposed here. The proposed slow induced polarization mechanism would be very sensitive to the presence and concentration of added ions in the system, which gave the impetus for our varying the electrolyte concentration both inside and outside the vesicles. The results (Figures 5-8) confirmed that the ion concentration has a profound effect on the RPEB of the vesicle system. The steady-state electric birefringence increases and the dip in the reverse part of the signals becomes shallower with the increase of the ion concentration. Such a behavior bears both a difference from and a similarity to that observed on suspensions of solid β-FeOOH particles under analogous conditions.31 There, in contrast to our system, the steady-state birefringence ∆n(∞) decreases with increasing ionic strength, whereas the dip becomes shallower (similar to our system). The RPEB response of the β-FeOOH suspensions has been interpreted as being due to the acceleration of ion fluctuations with the increased abundance of mobile counterions in the ionic atmosphere surrounding the particles.31 A different explanation must be invoked in our case, even if a preferential adsorption of Na+ ions at the exposed PO3- sites (relative to the adsorption of Cl- ions at the sterically less accessible (CH3)3N+ sites) of the zwitterionic vesicle surface is taken into account, because the formation of no classical, unipolar counterion atmosphere can be expected. In our case, the surface density of bound ions increases with the concentration of the added salt and so does also the concentration of free counterions of both charges surrounding the vesicle. The increase of the contribution of the bound ions to the polarization and elongation of the vesicles results in the observed rise of the steady-state electric birefringence with the ion concentration. This finding is in line with the theoretical prediction26 of an increased deformation of the bilayer in the presence of large concentration gradients across
J. Phys. Chem. B, Vol. 105, No. 23, 2001 5573 the membrane and high charge densities at the membrane-water interfaces. Upon field reversal, the redistribution of bound ions speeds up with their existing surface density because of the shorter overall path on the surface over which they need and can travel. During this process, forced desorption of bound ions and forced adsorption of free ions at the respective polar cap regions of the vesicle may contribute to the accelerated charge redistribution. As a net result, the deelongation is cut short as the reversed field becomes effective sooner in resuming the elongation of the vesicles, which is reflected in a shallower dip in the signal at higher ion concentration. A decrease in tm, which is expected to be associated with the process cannot be verified by our results, most likely because of the low signal-to-noise ratio of the birefringence signals. Summary Aqueous solutions of 193 nm diameter unilamellar bilayer vesicles, prepared from the zwitterionic phospholipid DOPC, exhibit positive induced birefringence upon the application of a high-voltage electric pulse. This Kerr effect is due to the induced structural anisotropy caused by the polarization of the system which leads to the elongation of the time average spherical vesicles to prolate ellipsoids, and the partial alignment of dipoles (permanent, induced, and instantaneous), in the direction of the applied field E. If the polarity of the field is suddenly reversed after a steady state is reached, a temporary partial loss of the birefringence occurs during the repolarization of the system, which is reflected in a local minimum (dip) in the time dependent birefringence signal. The rate at which the local minimum is reached (characterized by tm) and the remaining birefringence ∆m at that point are manifestations of competing as well as parallel rate processes associated with the polarization of the system. The profile of the dip in the signal was found to be profoundly sensitive to the concentration of ions (NaCl) placed inside and/or outside the vesicles. The observed ion concentration dependence of the dip profile points to a slow induced polarization mechanism that involves the redistribution of ions of both charges adsorbed on the vesicle surfaces. Acknowledgment. This work was supported in part by the Welch Foundation and the National Science Foundation. References and Notes (1) Degiorgio, V., Corti, M., Eds. Physics of Amphiphiles: Micelles, Vesicles and Microemulsions; North-Holland: Amsterdam, 1985. (2) Schelly, Z. A. Curr. Opin. Colloid Interface Sci. 1997, 2, 37-41. (3) Tekle, E.; Ueda, M.; Schelly, Z. A. J. Phys. Chem. 1989, 93, 59665969. (4) Neumann, E., Sowers, A. E., Jordan, C. A., Eds. Electroporation and Electrofusion in Cell Biology; Plemun Press: New York, 1989. (5) Weaver, J. C.; Chizmadzhev, Yu. A. Bioelectrochem. Bioenergetics 1996, 41, 135-160. (6) Neumann, E.; Kakorin, S.; Toensing, K. In Electrochemotherapy, Electrogenetherapy, and Transdermal Drug DeliVery; Jaroszeski, M. J., Heller, R., Gilbert, R., Eds.; Methods in Molecular Medicine, Vol. 37; Humana Press: Totowa, NJ, 2000; pp 1-35. (7) Borkovec, M.; Eicke, H.-F. Chem. Phys. Lett. 1989, 157, 457461. (8) Correa, N. M.; Schelly, Z. A. J. Phys. Chem. B 1998, 102, 93199322. (9) Correa, N. M.; Schelly, Z. A. Langmuir 1998, 14, 5802-5805. (10) Asgharian, N.; Schelly, Z. A. Biochim. Biophys. Acta 1999, 1418, 295-306. (11) Kakorin, S.; Redeker, E.; Neumann, E. Eur. Biophys. J. 1998, 27, 43-53. (12) Neumann, E.; Kakorin, S.; Toensing, K. Faraday Discuss. 1998, 111, 111-125. (13) O’Konski, C. T.; Haltner, A. J. J. Am. Chem. Soc. 1957, 79, 56345649.
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