Critical Conditions for Adsorption of Calcium Ions onto Dipolar Lipid

Article pubs.acs.org/JPCA

Critical Conditions for Adsorption of Calcium Ions onto Dipolar Lipid Membranes Oren Lotan,† Lea Fink, Asaf Shemesh, Carmen Tamburu, and Uri Raviv* Institute of Chemistry, the Hebrew University of Jerusalem, Jerusalem 91904, Israel ABSTRACT: Dipolar lipid membranes may adsorb multivalent ions. The binding constant depends on the type of lipid and ions. In this paper, we focus on the adsorption of calcium ions onto 1,2dilauroylphosphatidylcholine (DLPC) membrane. Using small-angleX-ray scattering we found that at ambient room temperature ca. 0.6 mM CaCl2 is a critical concentration at which calcium ions adsorbed to 30 mg/mL (ca. 48 mM) DLPC membrane. We then determined the structure of the lamellar phases formed at CaCl2 concentrations below and above the critical concentration and characterized the effect of temperature and incubation time on the adsorption process. Our findings suggest that calcium adsorption to DLPC membranes requires an initial nucleation phase.

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INTRODUCTION Lipid bilayers are the focus of extensive research owing to their important role in cell membranes.1−13 Lipid molecules have two hydrophobic tails and a hydrophilic headgroup (Figures 1

Figure 2. 1,2-Dilauroylphosphatidylcholine (DLPC) lipid. The positively charged N(CH3)3+ group is closer to the water layer, whereas the negatively charged phosphate group is deeper in the bilayer headgroup.

membranes. The electrostatic repulsion between the membranes is stronger than other forces that determine the lamellar repeat distance (vdW attraction, hydration repulsion, and undulation repulsion).15−18,32−37 At low concentrations of CaCl2, Ca2+ ion adsorption to the membranes is incomplete, and a coexistence of the two phases is observed: one is rich in lipid molecules that adsorb calcium ions and another that is rich in lipid molecules that do not adsorb calcium ions.22 The lamellar repeat distance does not grow gradually with the concentration of added CaCl2. Rather, a sudden increase in the lamellar repeat distance appears when ca. 1 mM Ca2+ ion concentration is added to the lipid solution.22,38−42 To better understand the adsorption mechanism of calcium cations onto lipid bilayers, we followed the transition from the adsorbed to nonadsorbed state more closely, to determine if the

Figure 1. Adsorption of calcium ions charges the bilayers and increases the water spacing, dw, between them, hence the lamellar repeat distance, D. Reprinted with permission from ref. 22. Copyright 2011 American Chemical Society.

and 2) that may be either charged, polar, or dipolar (zwitterionic). In this paper, we focus on the interaction of membranes containing dipolar headgroups with salt solutions.14−22 These interactions are relevant to regulatory processes in cell membranes. Previous studies showed that in the presence of multivalent salt solutions (for example, CaCl2) multilamellar vesicles, composed of zwitterionic lipids with phosphatidylcholine (PC) headgroups, swell.23−34 The lamellar repeat distance can grow from ca. 2 to 3 nm (before salt was added) to a few dozens of nanometers (Figure 1). Lipid molecules with PC headgroups and saturated tails may adsorb calcium ions and form charged © 2016 American Chemical Society

Special Issue: Ronnie Kosloff Festschrift Received: March 15, 2016 Revised: April 28, 2016 Published: April 29, 2016 3390

DOI: 10.1021/acs.jpca.6b02708 J. Phys. Chem. A 2016, 120, 3390−3396

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The Journal of Physical Chemistry A

developed in our lab.44−46 The data were fitted to a formfactor model of a stack of infinite flat uniform slabs with varying electron density, corresponding to the electron density profile of the bilayers. Details about this model were provided elsewhere.44 The extruded samples contained unilamellar vesicles, so that form-factor scattering curves could be measured, without the contribution from the structure-factor of the lamellar phase. The form-factor curves were analyzed using a stack of infinite flat uniform slabs.45,46 The model parameters were in agreement with our earlier study.22 When lamellar phases formed, Gaussian line-shape correlation peaks were added to the form-factor model and revealed the lamellar repeat distance, D = 2π/q001, where q001 is the magnitude of the first harmonic correlation peak. D = dw + δ, where dw is the water spacing and δ is the bilayer thickness (Figure 1). δ was estimated by the head-to-head distance obtained by the form-factor model and was assumed to remain constant under the different salt and lipid concentrations.13 The peak intensity, I, in the case of infinite flat slabs, is proportional to the product of the concentration of the lamellar phase, C; the orientation average of the form-factor squared, ⟨FF2(q)⟩; and the square of the average number of repeats within the lamellar phase, N (using Warran’s approximation)47

transition is indeed sharp or gradual. We carefully searched for a critical adsorption concentration, which may provide an indirect evidence for a nucleation-based adsorption mechanism. We then characterized the behavior of the bilayers below and above the critical concentration and measured the effect of time and temperature on the adsorption process. A critical minimum calcium concentration of ca. 0.6 mM for adsorption to the membranes was established. This rather low concentration falls in a range of potential biological relevance. It is therefore important to understand the underlying physical mechanism associated with the adsorption process.

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MATERIALS AND METHODS Materials. Highly purified water (Barnstead Nanopure Diamond) with resistivity of 18.1 MΩ·cm and total organic compounds (TOC) of 5 ppb or less was used for preparing solutions. Salt solutions were prepared by diluting 1 M standard CaCl2 salt solutions, purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). The lipid (>99% pure), 1,2-dilauroyl (C12:0) phosphatidylcholine (DLPC), was purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA), in lyophilized form (Figure 2). To avoid humidity condensation, the lyophilized DLPC was equilibrated at room temperature, for 30 min after it was taken out of the freezer, before it was opened and used. Salt solutions at the required concentrations were added directly to the lyophilized lipid to reach 30 mg/mL (ca. 48 mM), and the samples were vortex mixed for 30 min. The solutions were then centrifuged at ca. 10 000g for 30 min. To obtain unilamellar vesicles, several samples were extruded through a porous membrane with 0.4 μm hole diameter and then through 0.1 μm diameter. Each sample was then transferred to quartz capillaries, flame-sealed, and centrifuged for 2 h at 8000 rpm using a SIGMA 1-15PK centrifuge at 25 °C. Methods. Solution Small-Angle X-ray Scattering. The lamellar phases formed in the lipid solutions have been characterized by small-angle X-ray scattering (SAXS). Full details of our in-house setup were provided elsewhere.12 The 2D scattering images were recorded on a MAR345 image plate detector and azimuthally integrated, using FIT2D,43 to obtain a 1D curve of the scattering intensity as a function of the magnitude of the momentum transfer vector q (Figure 3). Data analysis was done using X+ software

⎛ (q − q )2 (ND)2 ⎞ i ⎟ I(q) = C·⟨|FF(q ⃗)|2 ⟩·N 2·∑ exp⎜⎜ − ⎟ 4 π ⎝ ⎠ i

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RESULTS Figure 4 shows that 10 min after preparing the sample containing 30 mg/mL (ca. 48 mM) of DLPC and 1 mM CaCl2 a second lamellar phase appeared, which adsorbed the calcium ions and swelled to a large lamellar repeat distance, D. The fact

Figure 4. Scattering curves of 30 mg/mL of DLPC in 1 mM CaCl2 as a function of time after preparing the sample, as indicated. The low q peak (left) strengthens and sharpens with time. The second harmonic appears after 30 min (broken arrow). The curves are shifted for clarity of presentation.

Figure 3. Example of an integrated scattering intensity as a function of the q vector magnitude. 3391

DOI: 10.1021/acs.jpca.6b02708 J. Phys. Chem. A 2016, 120, 3390−3396

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The Journal of Physical Chemistry A that a new phase appeared is consistent with Gibbs’ phase rule, which allows up to three phases when there are three independent components: DLPC, CaCl2, and water. Because DLPC was at large excess, the calcium ions could form a calcium-rich phase and a calcium-poor phase. Similar lipidphase coexistences have been observed in the presence of added salts or buffers in earlier studies.5,8,10,20,22,48 Within the first 70 min, D increased from 27 to 43 nm, suggesting that more calcium cations adsorbed within this time. As more ions adsorbed, the concentration of free ions in the bulk decreased, by ca. 35% (based on the measured binding constant of ca. 11 M−1 of calcium to DLPC22). Hence, both the membrane charge density and the screening length increased. As a result the electrostatic repulsive force became stronger, and the lamellar phase, which adsorbed the calcium ions, continued to swell in the direction normal to the bilayer plane. The observed D values of the adsorbed phase were similar to earlier measurements of charged lipids.10,22 In the presence of other salts or buffers, however, the D values may alter.49 The exact balance of forces leading to these D values were discussed and explained.10 Briefly, our charged membranes are rigid enough to ideally swell when immersed in pure water or dilute salt solutions. Below a critical lipid concentration (of ca. 150 mg/mL), however, charged membranes do not swell ideally; rather, they condense into lamellar phases with repeat distances that are much shorter than the bilayers were able to assume, if swelled ideally, and filled the available space. This behavior is reversible as a function of temperature and lipid concentration, suggesting that equilibrium is achieved with respect to the flow of water in and out of the lamellar phase. This behavior is attributed to a microphase separation that was observed by cryo-TEM and optical microscopy, at which a disordered phase, composed of vesicles and nanotubes, is formed. Owing to the negative Gaussian modulus of charged membranes, the formation of the disordered phase does not cost much in elastic energy50,51 but increases the configurational entropy of the system. The disordered phase is depleted from the lamellar phase and applies an osmotic stress to it. The lamellar spacing is determined by the point at which the pressures and the water chemical potential in the two phases are balanced. Increasing the temperature increases the entropy of the disordered phase more than the repulsion between the charged membranes, and therefore the lamellar phases are further condensed at higher temperatures.52 Figure 5 shows the DLPC lamellar repeat distance, D, as a function of CaCl2 concentration, 1 h, 1 week, and 2 weeks following the addition of CaCl2. The D value of DLPC in water is 5.8 nm. This spacing corresponds to the nonadsorbing phase.21,22 The nonadsorbing phase was observed in all the concentrations measured in this study. At 0.6 mM or higher CaCl2 concentrations appeared a second lamellar phase with much larger D values. During two weeks experiments, the minimum adsorption concentration did not change. The exact D values fluctuated over time. In this concentration range there was no clear trend in the dependence of the D spacing on calcium concentration. In our earlier study, we showed that above 1 mM D decreases with CaCl2 concentrations.22 Measurements in the range between 0.5 and 0.7 mM CaCl2 with 0.025 mM intervals revealed that the critical adsorption concentration was 0.575 mM. Figure 6 shows the peak amplitude ratio, R, which is the ratio of the adsorbed (001) peak amplitude, if existed, and the nonadsorbing (001) peak amplitude. The reason for choosing

Figure 5. DLPC lamellar repeat distance, D, as a function of CaCl2 concentration, measured at ambient room temperature, over 2 weeks following mixing CaCl2 with 30 mg/mL of DLPC. At 0.4 mM and 0.5 mM a single phase was observed, which is the nonadsorbing phase. The calcium-adsorbing phase appeared at 0.6 mM CaCl2 or higher concentrations.

Figure 6. Ratio, R, between the adsorbed peak amplitude (if observed) and the nonadsorbing peak amplitude, as a function of CaCl2 concentration. Samples were equilibrated for 2 weeks, and both peaks were the first harmonic (001) of each lamellar phase.

this parameter over direct adsorbed peak intensity is to have a form of normalization, as the intensity of the scattering may vary from sample to sample. The R values increased with concentration indicating a larger cation adsorbing phase. Up to 2 weeks the R values fluctuated possibly owing to a dynamic process before reaching equilibrium. The R values estimate the relative surface fraction occupied by the calcium-rich domain surface as a function of calcium concentration. We then measured the effect of temperature on the lamellar repeat distance of the adsorbed phase. All the temperatures in the experiments were above the melting point of DLPC (−1 °C).21 The temperature was first increased and then decreased. D spacing values were measured at least twice at each temperature to verify that equilibrium was attained. Figure 7 presents a set of measurements of 30 mg/mL of DLPC in 0.7 mM CaCl2. The “u” and “d” notation on the temperature axis state whether measurements were taken during the heating (“u” - up) or the cooling (“d” - down) stages. The D spacing decreased with temperature. Similar trend, although with 3392

DOI: 10.1021/acs.jpca.6b02708 J. Phys. Chem. A 2016, 120, 3390−3396

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Figure 8. Lamellar repeat distance, D, as a function of the time elapsed following cooling 30 mg/mL of DLPC with 1.1 or 0.7 mM CaCl2 from 50 °C down to 4 °C. The incubation time at 50 °C was 3 h. Two phases coexisted, one that adsorbed calcium ions (squares) and the nonadsorbing DLPC phase (circles).

Figure 7. Lamellar repeat distance, D, as a function of temperature for 30 mg/mL of DLPC in 0.7 mM CaCl2.

significantly smaller variations in D spacing, was observed at a higher concentration of 1.3 mM CaCl2. D spacing decrease with temperature was observed with membranes that contained charged lipid molecules, with charged groups that are covalently attached to the lipid molecules.9,10 Increasing the temperature increases the entropy of the disordered phase more than the repulsion between the charged membranes, and therefore the lamellar phase is more condensed at higher temperatures by up to ca. 15%. This effect, however, is not enough to explain the data in Figure 7. In an earlier study, we found that the area per DLPC lipid increases with temperature by up to ca. 5%, and the membrane thickness decreases by up to ca. 10%.13 Both effects contribute to stronger repulsive undulation forces, hence to an increase of the D spacing. The observed decrease of D spacing with temperature by up to ca. 50% is primarily attributed to enhanced calcium desorption at higher temperature (or increased calcium adsorption at lower temperature). As more cations desorbed when the temperature was increased, the membrane charge density decreased, and the concentration of free ions increased. As a result the screening length decreased; the electrostatic repulsion decreased; and so did the D spacing. The weaker adsorption at higher temperatures is owing to the increased ion solution-entropy and enhanced lipid-fluctuations in the bilayer compared with the ion−dipole interaction between the calcium cation and the PC headgroup dipole. The peak of the adsorbed phase narrowed, and its amplitude decreased with temperature, suggesting there were fewer lipid molecules in the lamellar phase with the larger D spacing at higher temperatures. The D spacing of the nonadsorbing phase did not change with temperature. Following incubation of 30 mg/mL of DLPC with 0.7 mM CaCl2 at 50 °C for 3 h the calcium ions desorbed. We then cooled the suspension to 4 °C (within 5 min) and measured its scattering every 10 min. Figure 8 shows that D increased with the time elapsed after the suspension was cooled, suggesting the calcium ions adsorbed to the membranes. When the same experiment was repeated with 1.1 mM CaCl2, only partial calcium desorption was observed at 50 °C. Additional calcium ions, however, adsorbed following incubation at 4 °C. At 0.7 mM the lamellar repeat distance gradually grew with time, whereas at 1.1 mM stable values were obtained within 10 min. Figure 9 shows that higher calcium concentration leads to higher adsorption rate both at low and high temperatures. It

Figure 9. Ratio, R, between the adsorbed peak amplitude and the nonadsorbing peak amplitude, as a function of the time elapsed following cooling 30 mg/mL of DLPC with 1.1 or 0.7 mM CaCl2 from 50 °C down to 4 °C.

should be noted that for the 0.7 mM experiment we repeated the heating and cooling cycles two more times: the second cycle showed a strong desorbed peak and a weak and wide adsorbed peak that did not change with time; the third cycle did not show any adsorbed peak.

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DISCUSSION Upon addition of CaCl2, the lamellar repeat distance did not gradually grow with Ca2+ concentration but rather jumped from D = 5.8 nm to D = 35 ± 5 nm at 0.6 mM CaCl2 (Figure 5). Discontinuous phase transition may imply that the phase transition involves a nucleation step, as demonstrated in Figure 10. Near the minimum adsorption concentration, the lamellar repeat distance fluctuated with time and Ca2+ concentration (Figures 4−6). Above the critical adsorption concentration, the ratio, R, between membranes in the adsorbed phase and the nonadsorbed phase increased monotonically with CaCl2 concentration (Figure 6). Our earlier wide-angle X-ray scattering measurements were insensitive to the changes in the area per lipid, following calcium adsorption, even at much higher calcium concen3393

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constant (Figure 10C). At the critical point, domains containing Nc adsorbed ions are formed. The mean area of a domain is ALNc, where AL = 0.68 nm2 is the area per lipid.13 Assuming the domain has a circular shape, its radius is

AL Nc π

,

and its circumference is 2 πAL Nc . Hence, there are 2 πNc lipids at the circumference and Nc − 2 πNc lipids inside a domain. The energetic cost for calcium adsorption on a domain containing N c ions, is therefore given by (Nc − 2 πNc )εion in domain + 2 πNc εion in edge . When the total ion−dipole energy gain, Ncεion‑dipole, is large enough to overcome the above energetic cost and the solution entropy of the calcium ions, the adsorption free-energy becomes negative, and a stable nucleus is spontaneously formed, after which further adsorption continues and spreads (Figure 10D). The binding constant of calcium ions to DLPC is ca. 11 M−1, which implies a net gain of ca. 0.8kBT in standard chemical potential per adsorbed ion.20 If calcium ions were uniformly adsorbed, about 35% of the calcium ions should have been adsorbed at the concentrations used in our experiments. Under those conditions, at least ca. 0.3% of the DLPC molecules should have adsorbed calcium ions. This implies a membrane charge density that is equivalent to a membrane containing at least ca. 0.6% monovalent charged lipid molecules (in other words, 1e per 113 nm2). This membrane charge density was enough to significantly increase the D spacing, already at our lowest calcium concentration.10,62 As this increase was not observed below 0.6 mM CaCl2, our results support a phase transition and a critical adsorption CaCl2 concentration. The observed critical concentration may vary with solution conditions.49 At the concentrations used in our experiments, near and above the critical concentration the two lamellar phases coexisted. Earlier studies showed that at higher CaCl2 concentrations the only phase at equilibrium is the adsorbed phase.20,22,32,33,53 Ion−dipole interaction between the dipolar PC headgroup and the calcium cations facilitated the adsorption. Higher temperature increased the fluctuations in the DLPC bilayer, hence increased the values of εisolated ion, εion in domain, εion in edge, and the solution entropy of the ions that competed with the ion−dipole interactions (that depend weakly on temperature). Therefore, increasing the temperature led to desorption of calcium ions (Figure 7). The lamellar distance depended strongly on two main factorsthe membrane calcium cation density that created the electrostatic repulsion and the amount of free ions in the bulk solution that contributed to electrostatic screening that reduced the repulsion. Earlier studies20,22,32,33,53 showed that at high CaCl2 concentrations the lamellar distance decreases rapidly because even though the lipid layer charge density increases the concentration of free ions in the bulk keeps rising, and thus the screening effect weakens the electrostatic repulsion. A similar trend can be seen in the temperature gradient experiment at 0.7 mM. Whereas the initial concentration of Ca2+ ions in the system remained constant, the desorption of the calcium ions with temperature increased the number of free ions in the solution, contributed to the screening of the electrostatic repulsion, and decreased the lamellar repeat distance (Figure 7). The lamellar repeat distance values reflected the calcium adsorption/desorption behavior. The partial reversibility of the heating and cooling cycles (Figure 7) suggests that the

Figure 10. Suggested ion adsorption mechanism. (A) Schematic top view of a lipid layer; each blue sphere represents a lipid headgroup. (B) Few ions (red spheres) adsorbed onto the bilayer, causing a local deformation, associated with line tension (pink lines). Counterions are shown as green spheres. (C) Ion-adsorbed lipid nucleus has formed, in which the energy gain from adsorption of another ion is greater than the energy associated with the elongation of the line tension (pink line). (D) Fully ion-adsorbed layer. (E) Side view of the dipolar lipids in the nonadsorbed state. (F.) Side view of the adsorbed state.

trations than used in this paper.22 This suggests that the changes in the area per lipid were very small after adsorption of calcium ions. At low calcium concentrations (10 mM), the surface density of the calcium in the adsorbing domains was similar to the lipid density in the bilayer, which is one Ca2+ ion per 0.68 nm2.13 In the zwitterionic PC headgroup, the positively charged N(CH3)3+ group resides closer to the water phase, whereas the negatively charged PO3− group is located in the inner part of the lipid headgroup (Figure 2). The negatively charged chlorine ions are likely to be near the N(CH3)3+ groups that are quite accessible. To reach the PO3− group, the calcium cations must pass through the potential barrier of the N(CH3)3+ groups. Once a calcium cation reaches the negatively charged phosphate group, to return to the bulk solution, it must dissociate from the phosphate and pass the potential barrier of the N(CH3)3+ group again. Therefore, the polar headgroups are likely to hold the calcium cations for longer times. As calcium is a divalent cation, and it may simultaneously neutralize two phosphate groups.14,18,26,32,33,53 It is likely that adsorption causes two nearby headgroups to get slightly closer around the calcium cations (Figure 10E and F) and thus to locally deform the membrane (Figure 10B). This deformation comes with an energetic cost εisolated ion. At least for the concentration used in this study, εisolated ion and the solution entropy of the calcium ions are likely to be greater than the ion−dipole interaction energy, εion‑dipole, gained by adsorbing calcium cations on the dipolar headgroup.14,18,20,22,26,32,33,53 Therefore, a single adsorbed ion should be released readily after its entrapment. If, however, many ions are adsorbed simultaneously next to one another, the deformation energy cost in the domain, εion in domain, and the deformation energy cost required to add one more ion at the edge of the adsorbing domain, εion in edge, are both likely to be lower than εisolated ion. Hence, the total energy cost decreases as the area of the ionadsorbing domain increases,54−61 whereas the electrostatic energy gain εion‑dipole per adsorbed ion, most likely, stays roughly 3394

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(5) Dvir, T.; Fink, L.; Schilt, Y.; Raviv, U. Charging and Softening, Collapse, and Crystallization of Dipolar Phospholipid Membranes by Aqueous Ionic Liquid Solutions. Langmuir 2014, 30 (49), 14725− 14733. (6) Schilt, Y.; Berman, T.; Wei, X.; Barenholz, Y.; Raviv, U. Using Solution X-Ray Scattering to Determine the High-Resolution Structure and Morphology of PEGylated Liposomal Doxorubicin Nanodrugs. Biochim. Biophys. Acta, Gen. Subj. 2016, 1860 (1), 108−119. (7) Turjeman, K.; Bavli, Y.; Kizelsztein, P.; Schilt, Y.; Allon, N.; Katzir, T. B.; Sasson, E.; Raviv, U.; Ovadia, H.; Barenholz, Y. NanoDrugs Based on Nano Sterically Stabilized Liposomes for the Treatment of Inflammatory Neurodegenerative Diseases. PLoS One 2015, 10 (7), e0130442. (8) Dvir, T.; Fink, L.; Asor, R.; Schilt, Y.; Steinar, A.; Raviv, U. Charged Membranes under Confinement Induced by Polymer-, Salt-, or Ionic liquid Solutions. Soft Matter 2013, 9 (44), 10640−10649. (9) Moshe, L.; Saper, G.; Szekely, O.; Linde, Y.; Gilon, C.; Harries, D.; Raviv, U. Modulating the Structure and Interactions of LipidPeptide Complexes by Varying Membrane Composition and Solution Conditions. Soft Matter 2013, 9 (29), 7117−7126. (10) Steiner, A.; Szekely, P.; Szekely, O.; Dvir, T.; Asor, R.; YuvalNaeh, N.; Keren, N.; Kesselman, E.; Danino, D.; Resh, R.; Ginsburg, A.; Guralnik, V.; Feldblum, E.; Tamburu, C.; Peres, M.; Raviv, U. Entropic Attraction Condenses Like-Charged Interfaces Composed of Self-Assembled Molecules. Langmuir 2012, 28 (5), 2604−2613. (11) Zucker, D.; Andriyanov, A. V.; Steiner, A.; Raviv, U.; Barenholz, Y. Characterization of PEGylated Nanoliposomes co-Remotely Loaded with Topotecan and Vincristine: Relating Structure and Pharmacokinetics to Therapeutic Efficacy. J. Controlled Release 2012, 160 (2), 281−289. (12) Nadler, M.; Steiner, A.; Dvir, T.; Szekely, O.; Szekely, P.; Ginsburg, A.; Asor, R.; Resh, R.; Tamburu, C.; Peres, M.; Raviv, U. Following the Structural Changes during Zinc-Induced Crystallization of Charged Membranes Using Time-Resolved Solution X-ray Scattering. Soft Matter 2011, 7 (4), 1512−1523. (13) Szekely, P.; Dvir, T.; Asor, R.; Resh, R.; Steiner, A.; Szekely, O.; Ginsburg, A.; Mosenkis, J.; Guralnick, V.; Dan, Y.; Wolf, T.; Tamburu, C.; Raviv, U. Effect of Temperature on the Structure of Charged Membranes. J. Phys. Chem. B 2011, 115 (49), 14501−14506. (14) McLaughlin, A.; Grathwohl, C.; McLaughlin, S. The Adsorption of Divalent Cations to Phosphatidylcholine Bilayer Membranes. Biochim. Biophys. Acta, Biomembr. 1978, 513 (3), 338−357. (15) Lau, A. L. Y.; McLaughlin, A. C.; MacDonald, R. C.; McLaughlin, S. G. A. The Adsorption of Alkaline Earth Cations to Phosphatidyl Choline Bilayer Membranes: A Unique Effect of Calcium. In Bioelectrochemistry: Ions, Surfaces, Membranes; American Chemical Society: Washington, D. C., 1980; pp 49−56. (16) Klein, J. W.; Ware, B. R.; Barclay, G.; Petty, H. R. Phospholipid Dependence of Calcium Ion Effects on Electrophoretic Mobilities of Liposomes. Chem. Phys. Lipids 1987, 43 (1), 13−23. (17) Tatulian, S. A. Binding of Alkaline-Earth Metal Cations and some Anions to Phosphatidylcholine Liposomes. Eur. J. Biochem. 1987, 170 (1/2), 413−420. (18) Satoh, K. Determination of Binding Constants of Ca2+, Na+, and Cl− Ions to Liposomal Membranes of Dipalmitoylphosphatidylcholine at Gel Phase by Particle Electrophoresis. Biochim. Biophys. Acta, Biomembr. 1995, 1239 (2), 239−248. (19) Sabín, J.; Prieto, G.; Ruso, J.; Sarmiento, F. Fractal Aggregates Induced by Liposome-Liposome Interaction in the Presence of Ca2+. Eur. Phys. J. E: Soft Matter Biol. Phys. 2007, 24 (2), 201−210. (20) Szekely, O.; Schilt, Y.; Steiner, A.; Raviv, U. Regulating the Size and Stabilization of Lipid Raft-like Domains and Using Calcium Ions as Their Probe. Langmuir 2011, 27 (24), 14767−14775. (21) Szekely, P.; Asor, R.; Dvir, T.; Szekely, O.; Raviv, U. Effect of Temperature on the Interactions between Dipolar Membranes. J. Phys. Chem. B 2012, 116 (11), 3519−3524. (22) Szekely, O.; Steiner, A.; Szekely, P.; Amit, E.; Asor, R.; Tamburu, C.; Raviv, U. The Structure of Ions and Zwitterionic Lipids

equilibrium was not fully attained in those experiments. Figures 8 and 9, however, indicate that following a rapid decrease in temperature from 50 to 4 °C most of the change was observed within ca. 30 min, suggesting that changes after several tens of minutes occur at a much slower rate. The slower rate may be attributed to the rate of ion diffusion across bilayers in the multilamellar phase, which are likely to go through defects in the bilayer. High temperature enhances thermal fluctuations that enable faster diffusion across bilayers. In the bulk, ions gain solution entropy, hence high temperatures favor diffusion of ions to bulk solution.

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CONCLUSIONS The lamellar repeat distance values characterized the behavior of calcium adsorption/desorption onto DLPC. Our observations support a critical calcium adsorption concentration of ca. 0.6 mM. Near the critical concentration there was a coexistence between membranes that adsorbed calcium and membranes that did not adsorb calcium ions. The adsorption was dynamic, and the amount of adsorbed ions on the membranes fluctuated with time. Higher temperature facilitated calcium desorption to the bulk solution, and lower temperature favored adsorption of calcium ions to the bilayers. These observations are consistent with an adsorption nucleation mechanism, in which there is a critical domain size that is required to enable calcium adsorption. Had uniform adsorption occurred, a large separation between bilayers should have been observed already at the lowest calcium concentration used in our experiments.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +972-2-6586030. Present Address †

Applied Physics Department, Hebrew University of Jerusalem, Jerusalem, Israel. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project was supported by Israel Science Foundation (1372/13) and by the Focal Technology Area - Hybrid Nanomaterials program of the Planning and Budgeting Committee of the Israel Council of Higher Education.

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REFERENCES

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DOI: 10.1021/acs.jpca.6b02708 J. Phys. Chem. A 2016, 120, 3390−3396

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DOI: 10.1021/acs.jpca.6b02708 J. Phys. Chem. A 2016, 120, 3390−3396