Aggregation of Dipalmitoylphosphatidylcholine Vesicles Induced by

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Aggregation of Dipalmitoylphosphatidylcholine Vesicles Induced by Some Metal Ions with High Activity for Hydrolysis Hideyuki Minami† and Tohru Inoue* Department of Chemistry, Faculty of Science, Fukuoka University, Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan Received December 7, 1998. In Final Form: May 17, 1999 AlCl3, YCl3, and Pb(NO3)2 induced a reversible aggregation of vesicles prepared from dipalmitoylphosphatidylcholine (DPPC). The dependence of the aggregation behavior on both the salt concentration and temperature was similar to that previously reported for BeSO4. A nature common to these four salt species is that the metal ions have a strong tendency to be hydrolyzed in aqueous solutions. The mechanism of the DPPC vesicle aggregation induced by these metal ions is considered as follows. When these metal ions are bound to the vesicular surface, they could cause a partial destruction of the hydration shell on the surface of DPPC vesicles because of their high activity for hydrolysis. This would result in the reduction of a repulsive hydration force which is responsible for the stability of phosphatidylcholine vesicles, and accordingly, would lead to an aggregation of vesicles. The dehydration of hydrated water around DPPC headgroups is suggested by the phase-transition behavior of hydrated DPPC bilayer in the presence of these metal ions.

Introduction Phospholipids form molecular assemblies called liposomes or vesicles when dispersed in aqueous media. The vesicles prepared from acidic phospholipids readily undergo an irreversible aggregation in the presence of multivalent metal ions, because these vesicles have negative charges and are stabilized by electrostatic repulsion.1-9 On the other hand, phospholipid vesicles composed of phosphatidylcholines (PC) are usually stable against aggregation in the presence of multivalent metal ions because of their electroneutrality. PC vesicles exist stably despite the lack of electrostatic repulsion. The stability of PC vesicles has been attributed to a so-called hydration force instead of an electrostatic repulsive interaction.10,11 The molecular mechanism of the repulsive hydration force acting between PC vesicles has not yet been fully elucidated; it has been ascribed to a steric origin such as bilayer undulation, bilayer thickness fluctuations, and the motion of hydrated lipid headgroups,12,13 or to the † Present address: Department of Chemistry, Faculty of Science, Kitasato University, Sagamihara 228-0829, Japan. * To whom all correspondence should be addressed. E-mail: [email protected].

(1) Ohki, S.; Du¨zgu¨nes, N.; Leonards, K. Biochemistry 1982, 21, 2127. (2) Rosenberg, J.; Du¨zgu¨nes, N.; Kayalar, C¸ . Biochim. Biophys. Acta 1983, 735, 173. (3) Furusawa, K.; Kakoki, H.; Matsumura, H. Bull. Chem. Soc. Jpn. 1984, 57, 3413. (4) Ohki, S.; Zscho¨rnig, O. Chem. Phys. Lipids 1993, 65, 193. (5) Walter, A.; Siegel, D. P. Biochemistry 1993, 32, 3271. (6) Carrio´n, F. J.; Maza, A.; Parra, J. L. J. Colloid Interface Sci. 1994, 164, 78. (7) Nakashima, T.; Shigematsu, M.; Ishibashi, Y.; Sugihara, G.; Inoue, T. J. Colloid Interface Sci. 1990, 136, 447. (8) Minami, H.; Inoue, T.; Shimozawa, R. J. Colloid Interface Sci. 1993, 158, 460. (9) Minami, H.; Inoue, T.; Shimozawa, R. J. Colloid Interface Sci. 1994, 164, 9. (10) Gamon, B. L.; Virden, J. W.; Berg, J. C. J. Colloid Interface Sci. 1989, 132, 125. (11) Inoue, T.; Minami, H.; Shimozawa, R.; Sugihara, G. J. Colloid Interface Sci. 1992, 152, 493. (12) Marra, J.; Israelachvili, J. Biochemistry 1985, 24, 4608. (13) Israelachvili, J. N.; McGuiggan, P. M. Science 1988, 241, 795.

electrostatic origin resulting from polarization charges induced by zwitterionic headgroups of PC.14 In any case, it may be sure that the water molecules hydrated around PC headgroups participate in this repulsive interaction. Contrary to a general aspect of the insensitivity of PC vesicles to metal ions, it was recently found that Be2+ ion induces the aggregation of PC vesicles.15 The Be2+-induced aggregation of PC vesicles has the following characteristic features: (i) there exists an optimum concentration range of Be2+ for the aggregation to occur, (ii) the effect of Be2+ ion becomes most pronounced at the temperature corresponding to the bilayer phase-transition temperature of the vesicle membranes, and (iii) the aggregation is reversible with respect to the Be2+ concentration. The reversibility of the aggregation implies that PC vesicles within the aggregates are trapped in a secondary minimum of intervesicular potential. This in turn lead to the postulation that the repulsive hydration force characteristic of PC vesicles is weakened by the action of Be2+. It is likely that the reduction of hydration force results from a partial dehydration of PC bilayers caused by the addition of Be2+. The dehydration of PC headgroup induced by Be2+ was also supported by observing the effect of Be2+ on the phase-transition behavior of hydrated dipalmitoylphosphatidylcholine (DPPC) bilayer.16 The remarkable feature of Be2+ is its strong tendency to undergo a hydrolysis in aqueous solution. The unique effect of Be2+ to induce the aggregation of PC vesicles may be attributed to this high activity of Be2+ for hydrolysis. It is likely that the hydrated water molecules around the PC headgroups are consumed to hydrolyze the Be2+ ion bound near the bilayer surface, which in turn results in the partial destruction of the hydration shell formed on the bilayer surface. If this is the case, it may be expected that the metal ions with high activity for hydrolysis also induce the aggregation of PC vesicles similarly to Be2+. (14) Jo¨nsson, B.; Wennerstro¨m, H. J. Chem. Soc., Faraday Trans. 2 1983, 79, 19. (15) Minami, H.; Inoue, T.; Shimozawa, R. Langmuir 1996, 12, 3574. (16) Minami, H.; Inoue, T. J. Colloid Interface Sci. 1998, 206, 338.

10.1021/la981687s CCC: $15.00 © 1999 American Chemical Society Published on Web 07/23/1999

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In the present work, we investigated the effect of Al3+, Y3+, and Pb2+ on the colloidal stability of DPPC vesicles. These ions are known to undergo hydrolysis in an aqueous solutions.17 Experimental Section Materials. Synthetic DPPC (>99%) was obtained from Nippon Oil and Fats Co. and was used without further purification. MgCl2, LaCl3, AlCl3, YCl3, and Pb(NO3)2 were of analytical grade (Wako Pure Chemicals Co.), and used as received. Water was purified by deionization followed by distillation twice. Unilamellar vesicles with controlled size distribution, about 90 nm in diameter, were obtained by applying the extrusion method18,19 as described previously.11 Vesicle Preparation. The vesicles were prepared in pure water without any buffer solution. Dust-free water for the preparation of solution sample for the light scattering experiments was obtained by filtration through a 0.1-µm membrane filter. The concentration of DPPC was kept at 0.2 mM for the turbidity measurements. For the dynamic light-scattering measurements, the DPPC concentration varied from 0.2 to 0.02 mM; this was due to obtaining the appropriate scattered-light intensity for particle size determination, and also, to the experimental requirement for the case of dilution experiments. Turbidity Measurements. Turbidity is a simple and convenient tool to detect the aggregation of vesicles, because the change in size of particles dispersed in solution is reflected sensitively in the turbidity. Time course of the vesicle aggregation at constant temperature was followed by monitoring the change in optical density (turbidity) at 400 nm after mixing of a vesicle preparation with a salt solution of various concentration, by 1:1 in volume, using a spectrophotometer equipped with a temperature-controlling accessory. The temperature was varied from 10 °C to 60 °C with an accuracy of (0.2 °C. Dynamic Light-scattering (DLS) Measurements. The vesicle aggregation was also followed by the increase in particle size after the addition of salts to vesicle preparations. In addition, the reversibility of the aggregation was examined by monitoring the change in the size of the aggregates associated with the change in salt concentrations or with the change in temperature. The size of the aggregates was evaluated in terms of a mean hydrodynamic diameter obtained by DLS measurements using a NICOMP Submicron Particle Sizer Model 370 with an argonion laser (λ ) 488.0 nm) with a maximum power of 75 mW. For some selected samples, the measurements were repeated two or three times under the same conditions in both the turbidity experiments and the size measurements. A reproducibility within approximately (10% was obtained in the repeated experimental runs. Differential Scanning Calorimetry (DSC). DSC measurements were performed using a Seiko Denshi Model SSC5200. DPPC of about 5 mg was weighed in a sealable sample pan made from aluminum, to which 20 µL of water or salt solution of various concentrations was added, and then the pan was sealed. As a reference material, alumina was used, the weight of which was about 20 mg. The sample was held at 90 °C for about 1 h in the oven of the DSC apparatus to ensure homogeneous mixing of the lipid and water. Then, a cooling/heating cycle was repeated several times at the rate of 2 °C/min at temperatures ranging from 10 to 90 °C. Good reproducibility was obtained for the DSC thermograms recorded by the repeated scan.

Results Turbidity Measurements. Figure 1 shows the plot of optical density (OD) of DPPC vesicle preparations as a function of time after the addition of AlCl3 at various concentrations at 25 °C. OD increases with the time, and reaches a constant value, which depends on the salt (17) Baes, C. F.; Mesmer, R. E. The Hydrolysis of Cations; John Wiley & Sons: New York, 1976. (18) Hope, M. J.; Bally, M. B.; Webb, G.; Cullis, P. R. Biochim. Biophys. Acta 1970, 812, 55. (19) Mayer, L. D.; Hope, M. J.; Cullis, P. R. Biochim. Biophys. Acta 1986, 858, 161.

Figure 1. Time course of the optical density change at 400 nm after mixing the DPPC vesicle preparations with AlCl3 solutions of various concentrations at 25 °C. AlCl3 concentrations (mM) are 100 (n), 200 (0), 300 (B), 600 (x), 800 (2), and 1000 (!). The salt concentrations are those after mixing.

Figure 2. Variation of OD80 with AlCl3 concentration at different temperatures. OD80 is the optical density at 80 min after the mixing of the DPPC vesicle preparation with AlCl3 solution. Temperatures are 10 °C (b), 25 °C (O), 40 °C (9), and 60 °C (0).

concentration, within about 30 min. This increase in OD is ascribed to the aggregation of DPPC vesicles as described previously15 and as demonstrated by the growth of the particle size in the vesicle preparations (see below). The AlCl3-induced aggregation of DPPC vesicles requires rather high salt concentrations on the order of a few hundred millimolar and a long time scale of a few tens of minutes. This characteristic is common to that observed for the aggregation of PC vesicles caused by BeSO415 and contrast remarkably with the aggregation of acidic phospholipid vesicles induced by multivalent cations; the acidic phospholipid vesicles usually aggregates within a few seconds after the addition of divalent cations at a concentration of a few millimolar.7-9, 11 The dependence of the increase in OD on AlCl3 concentration is not monotonic but biphasic. This is shown more clearly in Figure 2, where OD80, the optical density at 80 min after the addition of AlCl3, is plotted against the salt concentration at different temperatures. At each temperature, the OD80 exhibits a maximum at the AlCl3 concentration of about 300 mM. Figure 3 depicts the temperature dependence of OD80 obtained at 300 mM

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Figure 3. Temperature dependence of OD80 for DPPC/AlCl3 system. The AlCl3 concentration is 300 mM.

AlCl3. This figure shows that the OD80 increases with temperature up to about 45 °C, and then it decreases steeply; the aggregation is suppressed completely above 60 °C. This demonstrates that DPPC vesicles become most unstable against Al3+ at the temperature corresponding to the bilayer phase-transition temperature of DPPC vesicle membrane. The aggregation behavior of DPPC vesicles caused by the addition of AlCl3 with respect to the concentration dependence and temperature dependence is similar to that observed for Be2+-induced aggregation of PC vesicles.15 The values of OD80 obtained for DPPC/YCl3 system are shown in Figure 4. The concentration dependence of OD80 exhibits a maximum at about 300 mM YCl3 (Figure 4a). In the variation of OD80 with temperature, a maximum appears at about 45 °C (Figure 4b). The behaviors of DPPC vesicle aggregation induced by YCl3 are almost the same as those for the DPPC/AlCl3 system. Figure 5 shows the effect of Pb(NO3)2 on OD80 for DPPC vesicle preparations. Pb(NO3)2, AlCl3, and YCl3 increase OD80. However, some differences are seen among these salt species. The increment of OD80 caused by Pb(NO3)2 is relatively small compared with the cases of AlCl3 and YCl3, and the appearance of a maximum in the concentration dependence of OD80 is not so clear for Pb(NO3)2 as for AlCl3 and YCl3 (Figure 5a). The most pronounced difference is the temperature dependence of OD80. In DPPC/Pb(NO3)2 system, OD80 decreases with temperature rather monotonically (Figure 5b) instead of appearance of a maximum around 45 °C, which is the case for AlCl3 and YCl3 (Figures 3 and 4b). These differences in the salt species for the aggregation behavior of DPPC vesicles might be attributed to the difference in anionic species of the salts. However, the effect of anionic species on the vesicle aggregation is rather weak.20 In particular, the difference among monovalent anions such as chloride, bromide, nitrate, and perchlorate ions is negligible in its effect on the vesicle aggregation of acidic phospholipids.21 For phosphatidylcholines as well as for acidic phospholipids, no difference is also appreciable between Cl- and SO42-, as will be shown later regarding the pH effect on the DPPC vesicle aggregation. In addition, the effect of inorganic salts on the phase-transition behavior of hydrated DPPC bilayer is quite insensitive to anionic species (20) Minami, H.; Inoue, T.; Shimozawa, R. J. Colloid Interface Sci.. 1996, 178, 581. (21) Minami, H.; Inoue, T. unpublished results.

Figure 4. (a) Variation of OD80 with YCl3 concentration at different temperatures. Temperatures are 10 °C (b), 25 °C (O), 40 °C (9), and 60 °C (0). (b) Temperature dependence of OD80 obtained at 300 mM YCl3.

involved in the salts.20,21 Considering these facts, it may be concluded that somewhat different behavior of DPPC vesicle aggregation observed for AlCl3, YCl3, and Pb(NO3)2 is attributed to the intrinsic property of individual metal species. DLS Measurements. A DLS technique was applied to prove the vesicle aggregation in terms of the increase in particle size. In the DLS measurements, the salt concentration was kept at rather low levels to obtain an appropriate scattered-light intensity for size determination. Figure 6 shows the effect of several inorganic salts on the mean hydrodynamic diameter, 〈Dh〉, and the polydispersity of the size distribution for vesicle particles measured after the incubation for 48 h at 25 °C. The polydispersity was evaluated in terms of the standard deviation derived by assuming the Gaussian distribution of the particle size. Figure 6a shows that MgCl2 and LaCl3 exhibit no essential effect on 〈Dh〉 within the measured concentration range being in accordance with the turbidity results reported in previous paper.15 On the other hand, the 〈Dh〉 values obtained with AlCl3, YCl3, and Pb(NO3)2 increase with the increase in the salt concentration (Figure 6b) concomitantly with the widening of the size distribution (Figure 6c). This demonstrates that the aggregation of DPPC vesicles is induced by these salt species and is consistent with the prediction from turbidity experiments. In Figure 6b, the increase in 〈Dh〉 is more appreciable at lower salt concentrations than the increase in turbidity (Figures 2, 4a, and 5a). This may be attributed to the higher sensitivity of DLS for the detection of the change in particle size, as well as the prolonged incubation time for DLS measurements.

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Figure 5. (a) Variation of OD80 with Pb(NO3)2 concentration at different temperatures. Temperatures are 10 °C (b), 25 °C (O), 40 °C (9), and 60 °C (0). (b) Temperature dependence of OD80 obtained at 300 mM Pb(NO3)2.

The DLS also was used to examine the reversibility of the DPPC vesicle aggregation induced by AlCl3, YCl3, and Pb(NO3)2. If the aggregation process is reversible, the size of the aggregates formed at high salt concentration, say 150 mM, would be reduced by diluting the salt concentration, say down to 30 mM (see Figures 2, 4a, 5a, and 6b). Similar reduction of the particle size is also expected by changing the temperature of the sample with the fixed salt concentration, especially for AlCl3 and YCl3 (see Figures 3 and 4b). Figure 7a shows the dilution effect on 〈Dh〉 of the aggregates for the three salt species. The addition of the salts to DPPC vesicle preparations increases 〈Dh〉 of the particles, indicating that the vesicle aggregation occurs. At 20 min after the addition of the salts, the samples were diluted by 5-fold to reduce the salt concentration. The reduction of salt concentration decreases 〈Dh〉 down to the size of the original vesicles. This demonstrates that the aggregation of DPPC vesicles induced by Al3+, Y3+, and Pb2+ is completely reversible with respect to the concentration of these metal ions. The time course of the polydispersity of particle size distribution is shown in Figure 7b for the DPPC/YCl3 system as an example. Although the data points are somewhat scattered because of the poor reproducibility of the standard deviation of Dh derived from DLS measurements, it can be recognized that the polydispersity increases with the progress of the aggregation, and is returned to the original level by dilution. Similar tendency was also seen for other two salt species. The change in 〈Dh〉 associated with a stepwise change of temperature is shown in Figure 8 for DPPC/AlCl3 and

Figure 6. Effect of salt concentration on the particle size (a and b) and the polydispersity of the size distribution (c) in the DPPC vesicle suspension. The mean hydrodynamic diameter, 〈Dh〉, and the standard deviation of Dh were measured by a dynamic light-scattering method after the 48-h incubation at 25 °C. The salt species are MgCl2 (O) and LaCl3 (0) for panel a, and AlCl3 (O), YCl3 (0), and Pb(NO3)2 (4) for panels b and c. The DPPC concentration is 0.2 mM.

DPPC/YCl3 systems. In the experiments presented in Figures 8a and 8b, the vesicle preparation containing 120 mM AlCl3 was kept at 25 °C for 20 min, and then the temperature was raised to 40 °C; after an incubation at 40 °C for 20 min, the temperature was increased again to 60 °C. The temperature rise from 25 °C to 40 °C leads to the increase in 〈Dh〉 due to the promotion of the vesicle aggregation as predicted from turbidity results (see Figure 3). When temperature is changed from 40 °C to 60 °C, the value of 〈Dh〉 decreases, but the size expected for vesicles incubated at 60 °C, i.e., nonaggregated vesicle size, is not recovered. The variation of the polydispersity of particle size distribution, which is shown in Figure 8b, is parallel with the variation of 〈Dh〉. These results demonstrate that the aggregation of DPPC vesicles promoted by the temperature change under the presence of Al3+ is not fully but partially reversible with respect to the temperature reversal. The behavior of 〈Dh〉 associated with temperature change observed for DPPC/YCl3 system (Figure 8c) is almost the same as that for DPPC/AlCl3 system. The reversibility of DPPC vesicle aggregation described here is similar to the case previously reported for the PC/ Be2+ system with respect to both the salt concentration (perfectly reversible) and temperature (partially reversible).15

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Figure 7. Effect of the dilution of salt concentrations on the particle size (a) and polydispersity of the size distribution (b) in DPPC vesicle preparations at 25 °C. The salt species are AlCl3 (O), YCl3 (0), and Pb(NO3)2 (4) for (a), and YCl3 for (b). The salt concentrations before and after the dilution are indicated in the figure. The initial DPPC concentration is 0.15 mM.

DSC Measurements. The extent of hydration of PC headgroups in the bilayer is reflected in the behavior of gel-to-liquid-crystalline phase transition of the bilayer, as demonstrated by Kodama et al.22 They have revealed that the thermograms obtained by DSC experiments vary stepwise depending on the water content added to carefully prepared unhydrated DPPC sample. In our previous study,16 it was shown that the addition of Be2+ to hydrated DPPC brings about a quite similar effect on the bilayer phase transition to that observed when the water content is decreased. This suggested that Be2+ ion causes partial dehydration of DPPC headgroup, which is probably relevant to the aggregation inducing potency of the ion. In the present work, effects of AlCl3, YCl3, and Pb(NO3)2 on the bilayer phase transition behavior of hydrated DPPC were studied by DSC measurements. The sample conditions for DSC experiments are different from those for experiments of vesicle aggregation. In DSC experiments, DPPC was swollen by aqueous salt solutions rather than dispersed in aqueous media as a form of vesicle (20 µL of salt solution was added to 5 mg of DPPC), because of facilitating the calorimetric detection of the bilayer phase transition by using a high lipid concentration. The hydrated phospholipid bilayers formed in water-swollen samples mimic well the vesicle membranes. Thus the observation for the hydrated lipid bilayers would reproduce, at least qualitatively, the event occurring in the bilayer membranes of vesicles dispersed in aqueous media. Figure 9 depicts the DSC thermograms obtained for hydrated DPPC containing the three salt species at various (22) Kodama, M.; Kuwabara, M.; Seki, S. Biochim. Biophys. Acta 1982, 389, 567.

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Figure 8. Variation of the particle size (a and c) and polydispersity of the size distribution (b) associated with a stepwise change in temperature for DPPC vesicle preparations containing 120 mM AlCl3 (a and b) and 150 mM YCl3 (c). The DPPC concentration is 0.1 mM.

concentrations. The salt concentration is expressed in terms of the salt-to-DPPC molar ratio. In the thermogram obtained for salt-free DPPC, two endothermic peaks appear, i.e., the smaller peak at about 35 °C and the larger one at 41 °C. They are ascribed to the bilayer phase transition from Lβ′ to Pβ′ (pretransition) and that from Pβ′ to LR (main phase transition), respectively.23,24 Figure 9 shows that the addition of the salts leads to the appearance of another endotherm at higher temperatures in addition to the original endotherm of the main phase transition. For AlCl3 and YCl3, with the increase in the salt concentration, this peak moves toward the higher temperature side up to about 60 °C, growing more and more at the expense of the lower temperature peak. For Pb(NO3)2, the effect is small, but a similar tendency is appreciable. The variation of the main phase transition of the hydrated DPPC bilayer caused by the addition of the salts, particularly AlCl3 and YCl3, is similar to that observed by decreasing the water content in the DPPC/water mixture. According to Kodama et al.,22 when the water content is decreased from 80 wt % to 47 wt %, another endotherm appears as a shoulder at the higher temperature side in addition to the endothermic peak of the main phase transition of the fully hydrated DPPC bilayer. With a further decrease in the water content in the mixture, the relative intensity of the higher temperature endotherm (23) Small, D. M. The Physical Chemistry of Lipids; Plenum Press: New York, 1986. (24) Tenchov, B. Chem. Phys. Lipids 1991, 57, 165.

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Figure 9. DSC thermograms obtained for hydrated DPPC bilayer in the presence of (a) AlCl3, (b) YCl3, and (c) Pb(NO3)2 at various concentrations. The salt concentrations expressed in terms of the salt-to-DPPC molar ratio are indicated in the figure.

becomes progressively larger, and a single endothermic peak appears at about 60 °C when the water content is decreased to 7.6 wt %. The behavior of the main phase transition of the DPPC bilayer caused by the addition of the salt species into the hydrated bilayer and that caused by the removal of water from the hydrated bilayer closely resemble each other. This correspondence implies that the partial dehydration of the hydrated DPPC bilayer is induced by the action of AlCl3, YCl3, and Pb(NO3)2. Discussion This study revealed that the aggregation of DPPC vesicles is induced by Al3+, Y3+, and Pb2+. The characteristics of this aggregation are summarized as follows. (i) The aggregation requires high concentrations of the metal ions on the order of a few hundred millimolar and a long time scale on the order of a few tens of minutes. (ii) There is a maximum concentration for the efficiency of the metal ions to induce the aggregation. (iii) The aggregation-inducing effect of the metal ions becomes maximum at the temperature corresponding to the bilayer phase transition temperature except for Pb2+, in which it decreases monotonically with temperature. (iv) The aggregation is fully reversible with respect to the salt concentration and is partially reversible with respect to temperature. These features are common to those observed for the aggregation of PC vesicles induced by Be2+. This suggests that the aggregation of PC vesicles induced by these metal ions proceeds through the same mechanism, at least for Al3+, Y3+, and Be2+. The common feature of these four metal ions, Al3+, Y3+, Pb2+, and Be2+, is their strong tendency to be hydrolyzed.17 This can be seen by referring to the pH of the solution containing these metal ions (Figure 10a). When the metal ions are hydrolyzed, the pH of the aqueous salt solution is expected to decrease according to the following general reaction scheme:

xMz+ + yH2O h Mx(OH)y(xz-y)+ + yH+ This is the case for AlCl3, YCl3, and Pb(NO3)2 as is shown in Figure 10a. On the other hand, no essential change

Figure 10. (a) Variation of pH with salt concentration. The salt species are MgCl2 (x), LaCl3 (]), AlCl3 (O), YCl3 (0), and Pb(NO3)2 (4). (b) Effect of pH on 〈Dh〉 of DPPC vesicles. The pH was adjusted by HCl (0) or H2SO4 (O), and 〈Dh〉 was measured after the 48-h incubation at 25 °C at the respective pH values. The DPPC concentration is 0.2 mM.

occurs in pH for solutions of Mg2+ and La3+ which exhibit weak activity for hydrolysis; for example, the equilibrium constants for the formation of M(OH)(z-1)+ for Mg2+ and La3+ are smaller than those for Al3+, Y3+, and Pb2+ by four or more orders of magnitude (Mg2+) and one or more orders of magnitude (La3+).17 The major hydrolysis products of these three metal ions are as follows: Al(OH)2+, Al2(OH)24+, and Al3(OH)45+ for Al3+; Y(OH)2+, Y2(OH)24+, and Y3(OH)54+ for Y3+; Pb(OH)+, Pb2(OH)3+, and Pb4(OH)44+ for Pb2+.17

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In Al3+, the polymeric species expressed by Al13O4(OH)247+ is known to be stable. However, the formation of this hydrolysis product requires a prolonged time of several weeks.17 Thus, the possibility that this polymeric product interfered the present DLS experiments is eliminated. The distribution of each species including nonhydrolyzed free metal ions depends on various conditions such as the salt concentration, the ionic strength of the medium, the pH of the solution, and so on. In the present experimental conditions of rather high salt concentration and acidic pH, the polynuclear species such as Al2(OH)24+ and Al3(OH)45+ are dominant as hydrolysis products.17 As mentioned above, the pH of the solutions of AlCl3, YCl3, and Pb(NO3)2 is decreased because of the hydrolysis of the metal ions. Then, a possibility exists that the origin of DPPC vesicle aggregation in the presence of these metal ions may be attributed to the decrease in the solution pH, or in other words, the aggregation is caused by H+. Therefore, the effect of pH on the vesicle size was examined adjusting the pH by HCl and H2SO4. The results are given in Figure 10b. As shown in this figure, the vesicle size is essentially unaltered by the reduction in pH to 1.5, which indicates that the aggregation of DPPC vesicles occurring in the presence of the metal ions under consideration is not due to the decrease in pH. Furthermore, the metal ions with weak activity for hydrolysis, e.g., Mg2+, Ca2+, La3+, and so on, do not induce aggregation of DPPC vesicles, although they are highly effective for the aggregation of acidic phospholipid vesicles. Thus, the aggregation-inducing ability of these metal ions for DPPC vesicles may be attributed to an intrinsic nature of the metal ions themselves, i.e., their high activity for hydrolysis. Phosphatidylcholines are zwitterionic molecules, and hence, the PC vesicles have no net charge. Despite the lack of electrostatic repulsion, the PC vesicles exist stably in an aqueous medium for a long time without causing to aggregation. The origin of the stability of PC vesicles has been attributed to the hydration force acting between two approaching vesicles,10,11 which is a short-range repulsive force originating from water molecules strongly hydrated around PC headgroups.12,13,25-29 It is plausible that when the metal ions with high activity for hydrolysis are bound to the surface of PC vesicles, the partial dehydration of PC headgroups occurs, because the hydrated water would be consumed to hydrolyze the metal ions. This may result in the reduction of the repulsive hydration force between PC vesicles, and hence induce the aggregation. The postulation that the partial dehydration of PC headgroups is caused by these metal ions is supported by their effect on the phase-transition behavior of hydrated DPPC (Figure 9). It should be considered that the metal ions bound on the vesicular surface bring about positive charges on the vesicular surface concomitantly with the partial destruction of the hydration repulsion. Then, the intervesicular interaction potential between PC vesicles is composed mainly of the following components: (1) the van der Waals attraction, (2) the hydration repulsion reduced by the metal ions adsorbed on the vesicular surface, and (3) the electrostatic repulsion, which is also caused by the binding (25) Leneveu, D. M.; Rand, R. P.; Parsegian, V. A. Nature 1976, 259, 601. (26) Leneveu, D. M.; Rand, R. P.; Parsegian, V. A.; Gingell, D. Biophys. J. 1977, 18, 209. (27) Lis, L. J.; McAlister, M.; Fuller, N.; Rand, R. P. Biophys. J. 1982, 37, 657. (28) Afshar-Rad, T.; Bailey, A.; Luckham, P.; MacNaughtan, W.; Chapman, D. Faraday Discuss. Chem. Soc. 1986, 81, 239. (29) Marsh, D. Biophys. J. 1989, 55, 1093.

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Figure 11. Schematic illustration of intervesicular potential representing the creation of shallow potential well (secondary minimum) with a potential barrier between primary and secondary minima. The total potential (Vtotal) is the sum of the van der Waals (Vvdw), hydration (Vh), and electrostatic (Ve) interaction potential. These potential curves were calculated according to the models described in the literature10,11 by the use of appropriate parameter values.

of the metal ions. Of these components, the first two are short-range, whereas the third is a long-range interaction. Thus, combination of these three components creates the so-called secondary minimum in the intervesicular interaction potential with a high potential barrier separating the primary and secondary minima.10,11 This situation is illustrated schematically in Figure 11. If the intervesicular interaction is ascribed to the van der Waals interaction and the strong hydration force, no potential minimum appears, and hence, the vesicles exist stably without undergoing aggregation. When the hydration force is reduced and the electrostatic potential is added, the combination of the three components produces a shallow potential minimum and a high potential maximum, as shown in Figure 11. This potential maximum becomes a barrier between primary and secondary minima, which locate at the left and right sides of the potential maximum, respectively. The primary minimum is a deep potential well created at a short-separation distance between the two approaching colloidal particles. On the other hand, the secondary minimum is a shallow potential minimum occurring at a long-separation distance between the two particles. This potential minimum appears as a result of the balance among the attractive and repulsive interactions at that distance, and hence, is sensitive to the external conditions surrounding the particles. When the primary and secondary minima are separated by a sufficiently high potential barrier, the aggregation would occur at the secondary minimum, because the barrier prevents the particles from approaching each other to settle down in the primary minimum. Therefore, in this case, the individual vesicles in the aggregated entities can be considered trapped in the secondary minimum of the intervesicular potential curve. The aggregation occurring at the deep primary minimum essentially is irreversible, whereas the aggregation associated with the secondary minimum is usually reversible. The aggregates formed at the secondary minimum can dissociate readily into original particles when the external condition is returned to the initial condition, because the secondary minimum is shallow and sensitive to the external conditions. Thus, if the aggregation of the PC vesicles induced by metal ions with high activity for hydrolysis is attributed to the partial destruc-

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tion of the hydration repulsion, then it is predicted that the aggregation process is reversible. This prediction agrees with the experimental observations shown in Figures 7 and 8. The aggregates of DPPC vesicles formed at high salt concentrations are dissociated by diluting the salt concentration (Figure 7) and those formed at low temperatures are partly dissociated by increasing the temperature (Figure 8). The occurence of PC vesicle aggregation induced by these metal ions at the secondary minimum of the intervesicular potential is also consistent with a slow aggregation rate. Because the secondary minimum is shallow, the dissociation of dimeric aggregates would take place rapidly. Thus, the net rate for the aggregation would become much slower than that for irreversible, diffusion-controlled aggregation as in the cation-induced aggregation of acidic phospholipid vesicles. A slow rate for the aggregation occurring at a shallow potential minimum has been reported for other vesicle systems.10 However, the possibility that the aggregation takes place at the primary minimum might not be excluded completely. If the potential barrier lying between primary and secondary minima is not high enough and the primary minimum and the potential barrier disappear when the external condition is returned to the initial condition, the aggregation at the primary minimum would also become reversible. If this is the case, the rather slow aggregation rate observed in the present system would be attributed to the potential barrier existing between the primary and secondary minima. The appearance of a maximum in the profile of DPPC vesicle aggregation against metal ion concentration may be explained qualitatively in terms of the balance of the two counteracting effects of the metal ions: one is the destruction of hydration force, which destabilizes the vesicles, and the other is the creation of an electrical charge on the vesicular surface, which stabilizes the vesicles. When the concentration of the metal ion becomes higher and higher, the electrical charge on the vesicular surface is increased more and more, because the metal ions adsorbed on the vesicular surface increase with the increase in the bulk concentration. Then, above a certain salt concentration, the electrostatic repulsive term would overcome the attractive effect of the destruction of the hydration force. This makes the secondary minimum more shallow, and hence, the vesicles become relatively stabilized again. Accordingly it may be reasonable to assume that the action of these metal ions appears as a result of their binding on the vesicular surface rather than a saltingout effect occurring in the bulk aqueous phase, although it is difficult to know the extent of the binding of these metal ions to the vesicular surface. The temperature dependence of PC vesicle aggregation caused by metal ions with high activity for hydrolysis shows that the aggregation-inducing effect of the metal ions becomes most pronounced at the temperature corresponding to the bilayer phase-transition temperature of the vesicle membrane; an exceptional case is Pb2+, for which the efficiency to induce the aggregation decreases monotonically with the increase in temperature. In any case, including Be2+, much less aggregation is induced above the bilayer phase-transition temperature; for DPPC vesicles, the aggregation is suppressed completely above 60 °C. The insensitivity of the PC vesicles with fluid-phase membrane to the aggregation compared with those of gelphase membrane is also explained by the postulation that the aggregation results from the reduced hydration force because of the action of the metal ions. Several molecular models have been proposed for the origin of the hydration force.12-14 According to any model, the hydration force is

Minami and Inoue

expected to be stronger for the fluid-phase bilayer than for the gel-phase bilayer. Some experimental evidence is consistent with the prediction for this relation between the strength of the hydration force and the physical state of lipid bilayers. Thus, it is likely that the hydration force for PC vesicles with a fluid-phase bilayer is so strong that the effect of the metal ions to destroy the repulsive interaction is not big enough to create the secondary minimum in the intervesicular interaction potential. The difference in the aggregation-inducing effect of the metal ions between gel- and fluid-state bilayers of the vesicle membrane may be attributed to the difference in the intrinsic strength of the hydration force between these two state bilayers. In the presence of the metal ions except for Pb2+, PC vesicles become most unstable near the bilayer phasetransition temperature of vesicle membranes, where both gel and fluid states coexist in the vesicle membranes. According to the interpretation above, this implies that the repulsive hydration force becomes weaker when the gel and fluid phases coexist in the hydrated PC bilayer, compared with the pure gel- or fluid-phase bilayer. However, no experimental evidence, consistent or conflicting with this implication, is available, as far as we know. Thus, at the present stage, we cannot give a reasonable explanation for the low stability of PC vesicles appearing at the bilayer phase-transition region in the presence of metal ions with high activity for hydrolysis. The aggregation of DPPC vesicles induced by Pb2+ exhibits somewhat different behavior than those observed for Al3+, Y3+, and Be2+. In Pb2+, no minimum appears in the bilayer phase-transition region for the stability of DPPC vesicles. In addition, the aggregation-inducing effect of Pb2+ on DPPC vesicles is considerably smaller than that of other metal ions, as seen by comparing Figures 2 through 7. This might mean that the perturbing effect of Pb2+ on the hydration shell of DPPC vesicular surface is weaker than that of other metal ions. The weak perturbing effect of Pb2+ is also reflected in the DSC results (Figure 9), which demonstrates that the extent of the dehydration of DPPC headgroups is much less for Pb(NO3)2 than for AlCl3 and YCl3. Then, the activity for hydrolysis of Pb2+ might be expected to be lower than that of other metal ions, because the extent of dehydration of the DPPC headgroups caused by metal ions would be correlated to the activity for hydrolysis of the metal species. However, considering the variation of solution pH with the salt concentration, the activity of Pb2+ for hydrolysis is not necessarily lower than other metal ions; it is even higher than Y3+, although it is lower than Al3+. The difference in the aggregation behavior of DPPC vesicles between Pb2+ and other metal ions would suggest that the aggregation induced by Pb2+ proceeds through a mechanism different from that applicable to other metal ions with high activity for hydrolysis, although the details presently are not clear. In conclusion, the present study revealed that the metal ions with high activity for hydrolysis usually can induce the reversible aggregation of PC vesicles, which are generally stable against the addition of common metal ions. The mechanism of this aggregation is considered as follows. The aggregation occurs as a result of a reduction of the hydration force acting as a repulsive force between PC vesicles. The reduction of the hydration force is caused by a partial dehydration of PC headgroups brought about by the action of the metal ions. The activity of the metal ions for hydrolysis is responsible for this dehydration of vesicular surface, i.e., the hydrated water around PC

Inducement of Aggregation of DPPC Vesicles

headgroups is consumed to hydrolyze the metal ions adsorbed on the vesicular surface. The reduced hydration force, combined with the electrostatic interaction produced by the adsorption of the metal ions on the vesicular surface and the van der Waals interaction, creates the secondary minimum in an intervesicular interaction potential. Individual vesicles in the aggregates are settled in this

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potential minimum. The secondary minimum is a shallow potential well, and hence, the aggregation is reversible. Acknowledgment. This work was supported in part by funds from the Central Research Institute of Fukuoka University. LA981687S