Interactions Between Liposomes and Cations in Aqueous Solution

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Interactions Between Liposomes and Cations inAqueous SolutionJuan M. Ruso a; Lina Besada a; Pablo Martínez-Landeira a; Laura Seoane a;Gerardo Prieto a; Félix Sarmiento aa Biophysics and Interfaces Group, Department of Applied Physics, Faculty ofPhysics, University of Santiago de Compostela, santiago de composa, Spain

Online Publication Date: 01 May 2003

To cite this Article: Ruso, Juan M., Besada, Lina, Martínez-Landeira, Pablo,Seoane, Laura, Prieto, Gerardo and Sarmiento, Félix (2003) 'Interactions BetweenLiposomes and Cations in Aqueous Solution', Journal of Liposome Research, 13:2,131 — 145

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08 ©2003 Marcel Dekker, Inc. All rights reserved. This material may not be used or reproduced in any form without the express written permission of Marcel Dekker, Inc.

MARCEL DEKKER, INC. • 270 MADISON AVENUE • NEW YORK, NY 10016

JOURNAL OF LIPOSOME RESEARCH

Vol. 13, No. 2, pp. 131–145, 2003

Interactions Between Liposomes and Cations in

Aqueous Solution

Juan M. Ruso, Lina Besada, Pablo Martınez-Landeira,

Laura Seoane, Gerardo Prieto, and Felix Sarmiento*

Biophysics and Interfaces Group, Department of

Applied Physics, Faculty of Physics, University of

Santiago de Compostela, Santiago de Compostela, Spain

ABSTRACT

An investigation on the dependence of electrophoretic mobilities of unilamellar

vesicles of phosphatidylcholine–cholesterol–phosphatidylinositol (PC–Chol–PI)

on the concentration of several cations with variations in the relation charge/

radius in the range Naþ, Kþ, Csþ, Mg2þ, Ca2þ, Ba2þ, Al3þ, and La3þ has been

realized. Plots of zeta potential against ion concentration exhibit a maximum for

all the cations under study, the position of the maximum is greatly affected by the

charge of the ion. From the feature of these plots two phenomenon were

observed: an initial binding of cations into the slipping plane for ion concentra-

tion below the maximum and a phenomenon of vesicle association for concentra-

tion above the maximum. To confirm these observations measurements on

dynamic light scattering were performed to obtain the corresponding size

distribution of the liposomes at different ion concentrations. Finally the ability

of the Stern isotherm to describe the adsorption of the cations to vesicles was

tested by two methods. The two main parameters of the theory: the total

number of adsorption sites per unit area, N1, and the equilibrium constant, K;

*Correspondence: Felix Sarmiento, Biophysics and Interfaces Group, Department of

Applied Physics, Faculty of Physics, University of Santiago de Compostela, E 15782

Santiago de Compostela, Spain; Fax: þ34 981 520 676; E-mail: [email protected].

131

DOI: 10.1081/LPR-120020316 0898-2104 (Print); 1532-2394 (Online)

Copyright & 2003 by Marcel Dekker, Inc. www.dekker.com

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(and consequently the free energy of adsorption, �G0ads) were calculated for the

different ions, showing good agreement. The equilibrium constants of adsorption

have been found to obey a linear relationship with ion radius the slope of which

decreases with the ion charge.

Key Words: Zeta potential; Cation-liposome interactions; Free energy of

adsorption.

INTRODUCTION

Liposomes have been considered to be excellent models of cell membranes andhave been used as targetable drug carriers in which various materials such as drug,proteins, enzymes are encapsulated. In fact phospholipids are the main structuralcomponents of biological membranes and determine their specific bilayer structure.Knowledge of interactions with the surrounding medium is paramount in under-standing the functionality of biological membranes (1), so in this sense there aremany works devoted to studying the interactions between liposomes and a widevariety of substances from polymers (2), proteins (3), dendrimers (4), surfactants(5), and ions (6).

The most remarkable effect of adsorption of ions is to screen the charge ofphospholipid head groups with an effect which is qualitatively similar to protona-tion. It was observed how the main transition temperature Tc (temperature abovewhich the lipid bilayer exhibits a liquid-like phase and below which several gel phasesare observed), increase with the valence of the ions. Some ions can cause precipita-tion of vesicles at different concentrations and some of them seems to have a specificbinding site (7).

Many liposome characteristics depend on and some are even governed by theelectrostatic and hydration membrane potential. The electrostatic potential is ofparamount importance for the colloidal stability of the liposomal suspensions.Measuring and controlling the electrostatic properties of lipid vesicles therefore iscrucial for the basic understanding and practical applications of liposomes (8).Stability of vesicles has become of great interest, as an example, large vesicles arecleared from the bloodstream more rapidly than small one. In this sense cations wereused to induce vesicle aggregation (9) also grafted polymers were used successfully tostabilizing them (10).

The use of the Stern adsorption isotherm (a combination of the Langmuiradsorption isotherm, the Boltzmann equation and the Gouy Chapman theory ofthe diffuse double layer) has been widely applied in different ways to study theadsorption of ions onto liposomes. These include zeta potential measurements,nuclear magnetic resonance (11,12), and absorbance measurements (13) orequilibrium dialysis (14).

Alargova et al. tried many models to interpret successfully the dependence of thezeta potential of latex particles covered by a monolayer of amphoteric surfactant ondifferent ionic strength. Only one works properly, this model was based on theassumption that Hþ and Naþ satisfy the Stern adsorption isotherm (15). Tatulian(16) used the framework of Gouy–Stern theory to obtain the intrinsic binding con-stant of alkaline-earth metal cations to multilamellar liposomes. Apparent binding

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constants and number of binding sites per unit area are calculated from maximum inabsolute values of zeta potential against salt concentration.

Now we present a wide study of the adsorption of different cations onto vesiclesformed by phosphatidylcholine (PC), cholesterol (Chol) and phosphatidylinositol(PI), in an attempt to observe and analyzed the behavior and energies of adsorptionas a function of electric charge and ion radius. For this purpose we used zetapotential measurements and dynamic light scattering. Zeta potential measurementis a useful technique to study the electrokinetic behavior of liposomes (17,18).Knowledge of the zeta potential of liposome can help to predict the fate of theliposomes in vivo.

MATERIALS AND METHODS

Materials

L-a-Phosphatidylcholine (product number P-3556), L-a-phosphatidylinositolsodium salt (product number P-0639) and cholesterol (product number C-8667)were purchased from Sigma Chemical Co. All the other reagents used were ofanalytical grade and the water was doubly distilled.

Preparation of the Liposomes

Small Unilamellar Vesicles (SUV) were prepared by the sonicationtechnique (19). The desired amounts of stock solutions of lipids (PC–Chol–PI65:25:10 wt%, 10mg) in chloroform methanol 4:1 (5mL) were evaporated by theuse of a rotary evaporator to form a thin dry film on the round bottom flask.The resulting lipid film was flushed with nitrogen to eliminate trace solvent andthe required quantity of nitrogen-saturated aqueous medium added. Deionizedwater (25mL) was added to disperse the film by gentle shaking. The mixture washeated above the lipid phase transition temperature for 30min (heating temperature:41�C for PC). The cooled suspension was subjected to vortex mixing for 1min. It wasdisrupted by sonication for 30min with a Branson 5200 bath sonicator at full power.The clear suspension was allowed to stand for 2 h above the phospholipid transitiontemperature. After cooling to room temperature, the solution was filtered through a0.22 mm filter. To have the security of liposome stability, only samples that retained aconstant size for a period of at least 6 h prior to the spectroscopic measurements wereused (20).

Zeta Potential Measurements

The zeta potential measurements of the liposomes were made using a MalvernInstruments Ltd. Zetamaster 5002 by taking the average of five measurements at thestationary level. The cell used was a 5mm� 2mm rectangular quartz capillary.

Liposomes and Cations in Aqueous Solution 133

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The temperature of the experiments was 25.0�C controlled by a Heto proportionaltemperature controller.

The zeta potential were calculated from the electrophoretic mobilities, �E, usingthe Henry equation (21)

� ¼3

2

�E�

"0"r

1

f ð�aÞð1Þ

where the permittivity of vacuum ("0), relative permittivity ("r), and viscosity ofwater (�) were taken as 8.854� 10�12 J�1 C2m�1, 75.8, and 8.904� 10�4Nm�2 s,respectively. The function f (�a) depends on the product of particle radius, a, (deter-mined by dynamic light scattering, see Table 1) and Debye–Huckel parameter, �,calculated from the equation:

� ¼2e2NAcz

2

"0"rkBT

!1=2

ð2Þ

where kB is the Boltzmann’s constant, T the absolute temperature, c the electrolyteconcentration, e the elementary charge, z the valence of the ion and NA theAvogadro’s number. Values of �a for the whole range of particle radius and electro-lyte concentrations were calculated, giving values in the range 90–1100, for thesevalues of �a� 1 the equation of Henry is a consistent approach to study thesesystem. Therefore, the Henry factor for our system was determined by

f ð�aÞ ¼3

2�

9

2�aþ

75

2�2a2�

330

�3a3ð3Þ

valid for �a>1. Trying to obtain the best accuracy of the measurements we usedthe recommendations offered by Cevc for measuring the electrostatic potential ofliposomes under optimal measuring conditions (8).

Table 1. Liposome sizes obtained from light scattering in different electrolytes at

different concentrations.

Liposome diameters (nm)

Electrolyte a b c d

NaCl 130±11 430±50 370±54 505±80

KCl 125±8 175±50 248±41 453±50

CsCl 120±12 136±15 144±22 271±56

MgCl2 145±12 440±50 280±22 450±50

CaCl2 130±10 176±19 170±18 440±68

BaCl2 122±10 227±35 458±38 549±47

AlCl3 145±16 400±50 385±52 656±92

LaCl3 137±22 375±65 546±70 575±86

(a) low electrolyte concentrations; b) concentration corresponding to maximum

zeta potential; c) when zeta potential is decreasing; d) at electrolyte concentrations

corresponding to the plateau.

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Dynamic Light Scattering

Dynamic light scattering measurements were made at 298 0.1K and at a scat-tering angle of 90� using a Coherent DPSS 532 laser light-scattering instrumentequipped with a 0.5W solid state laser, operating at 532 nm with vertically polarizedlight. Time correlation was analyzed by an ALV-5000 multiple-tau correlator (ALV,Langen, Germany). Diffusion coefficients were determined from a single exponentialfit to the correlation curve. Hydrodynamic radii were calculated from measureddiffusion coefficients by means of the Stokes–Einstein equation.

RESULTS

Figures 1, 2, and 3 show zeta potential measurements of sonicated PC–Chol–PI(65:25:10 wt%) vesicles as a function of concentration of ions of different charge/radius ratio (results were plotted in three different graphs to improve clarity). Similarbehavior could be observed for all the systems: the initial negative value ofPC–Chol–PI vesicles (�58 5mV) decrease with increase in ion concentration, sug-gesting the binding of cations to the phospholipid headgroups, until a maximum isreached. After the maximum a decrease and a final plateau at high ion concentra-tions is observed. We must recall that the errors in measurements increase withelectrolyte concentration from 8% at low concentrations to 20% at high. Reverse

Figure 1. Zeta potentials of phosphatidylcholine (PC)–cholesterol (Chol)–phosphatidylino-

sitol (PI) (65:25:10 wt%) liposomes as a function of the ion concentration in water at 25�C:

(g) Naþ; (f) Kþ; (m) Csþ.


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(g) Mg2þ; (f) Ca2þ; (m) Ba2þ.



(g) Al3þ; (f) La3þ.

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of negative to positive values of zeta potentials is observed for all electrolytes exceptfor Naþ and Kþ. Plots obtained for Al3þ and La3þ, show a maximum at very lowelectrolyte concentrations which could be due to their high charge.

The decrease in the zeta potentials as the concentration is increased can beexplained in one of two ways: adsorption of anions or fusion and aggregation ofthe vesicles. To solve this problem dynamic light scattering measurements werecarried out to determine the size distribution, showing that vesicle size remainsconstant and quite equal to the naked vesicles until the maximum is reached. Sizemeasurements above the maximum show a great increase in the vesicle size of two orthree times the initial size. This increment is different for the different electrolytesused and there is no clear a relationship with radii or electrical charge (Table 1). Themost remarkable fact is the increment in the dispersion obtained for the samples onincreasing the ion concentration, this increase of polydispersity could be attributedto an inhomogeneous vesicle aggregation resulting in clusters of different size. Theseresults indicate to us that two different events are present in these systems: first, anadsorption of cations onto the liposomes at concentrations below the respectivemaximum and second, an aggregation of vesicles at concentrations above it.

We assume that the charge distribution is uniform in the vesicle surface. Sinceliposomes are large compared to the thickness of the electric double layer, theinfluence of the liposome curvature on the structure of the electric double layercan be considered negligible.

A detailed study of the structure of the electric double layer only requires aknowledge of the surface potential and zeta potential with indifferent electrolyte(22). Adsorption of cations onto the liposomes could be classified in terms ofinner-sphere complexes, where the ligand is inserted into the first coordinationsphere of the cation and outer-sphere complexes where the charged ligand and thefully-hydrated cation form an ion-pair (23). These two phenomena are related to thesurface potential and zeta potential. Normally nuclear magnetic resonance is used todetermine the number of cations bound to the liposome surface including the ions upto the slipping plane (i.e., the zeta potential plane).

In the present study we considered the electrical double layer like an adsorptionsurface, we do not distinguish between the cations adsorbed onto the liposomesurface and those adsorbed up to the slipping plane. With these simplifications wetry to monitor subsequent modification of the cation adsorption by measurement ofthe zeta potential.

The net charge per unit area over the slipping plane is calculated from zetapotential measurements by using the equation (21):

� ¼ 2"0"rkBTXi

ni0 exp �zie�

kBT

� �� 1

� �( )1=2

ð4Þ

for unsymmetrical electrolytes and equation

� ¼4n0ze

�sinh

ze�

2kBT

� �ð5Þ

for symmetrical electrolytes. In these equations, n0 is the number of ions per unitvolume.


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Zeta potential measurements will give information about the overall surfacecharge of the particles and how this is affected by changes in the environment,i.e., when the electrolyte concentration is increased, therefore from the change inzeta potential, �� ¼ � � �0, where � is the value corresponding to each electrolyteconcentration and �0 the value of the liposomes in the absence of ions, the netvariation of charge, �� could be obtained. By using the Stern adsorption isotherm:

�� ¼ zex ¼zexN1K

1þ cKð6Þ

where x is the molar fraction of electrolyte, N1 is the total number of adsorption sitesper unit area, K, the equilibrium constant, c, the electrolyte concentration, and z, thevalence of the ion. The equilibrium constant is related to the free energy of adsorp-tion by the equation

�G0ads ¼ �kBT lnð55:56K Þ ð7Þ

where 55.56 converts concentration in mol L�1.To obtain N1 and K the experimental data were fitted by using the Levenberg–

Marquardt algorithm which has proved to be an effective way to solve nonlinearleast squares problems (24,25). A nonlinear regression method based on thisalgorithm, starting from some initial parameter values, minimizes chi-square byperforming a series of iterations on the parameter values and evaluating chi-square at each iteration. Results are listed in Table 2. In Figs. 4–6 we show thecorresponding fittings.

Adsorption constants could be calculated another way as proposed by Tatulian(16). The theoretical background begins again with an expression for the surfacecharge density adsorbed, �ads:

�ads ¼N1Kc01þ Kc0

ð8Þ

where the symbols are known and c0 is the ion concentration at themembrane solution interface. Assuming that these ions are distributed according

Table 2. Total number of adsorption sites per unit area, N1, equilibrium constant, K,

and free energy of adsorption, �G0ads, obtained from fitting of the Stern isotherm.

Cation N1 (m�2) K (mol�1) �G0

ads (kJmol�1)

Naþ 2.41� 1016 4.59 �13.72

Kþ 3.47� 1013 7.16 �14.82

Csþ 10.52� 1013 7.98 �15.09

Mg2þ 3.70� 1017 9.02 �15.39

Ca2þ 4.89� 1016 9.16 �15.43

Ba2þ 6.83� 1013 10.17 �15.69

Al3þ 5.75� 1017 15.43 �16.72

La3þ 8.62� 1017 15.58 �16.72

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Figure 4. Net variation of charge, ��, calculated by the Stern adsorption isotherm (Eq. 5) on

the interaction between phosphatidylcholine (PC)–cholesterol (Chol)–phosphatidylinositol

(PI) (65:25:10 wt%) liposomes and different ions in aqueous solution at 25�C: (g) Naþ;

(f) Kþ; (m) Csþ.



(PI) (65:25:10 wt%) liposomes and different ions in aqueous solution at 25�C: (g) Mg2þ;

(f) Ca2þ; (m) Ba2þ.


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to the Boltzmann’s law

c0 ¼ c exp�ze 0

kBT

� �ð9Þ

where c is the concentration of the ion in the bulk solution.Then the total surface charge density, �, will amount to:

� ¼ �0 þ �ads ð10Þ

From the theory of the electrical double layer and combination of Eqs. (4) and(8)–(10) yields

�0 þNAezc0 expð�ze�=kBTÞ

1þ Kc0 expð�ze�=kBTÞ� ½2"0"rNAkBTc0fexpð�ze�=kBT Þ � 1g 1=2 ¼ 0

ð11Þ

from curves of zeta potential which exhibit a maximum which provide the possibilityof determining the parameter N1 using experimental values of �max and cmax by usingthe equation:

N1 ¼2

ze2"0"rNAkBTcmax exp

�ze�maxkBT

� �� 1

� � �1=2� �0 ð12Þ



(PI) (65:25:10 wt%) liposomes and different ions in aqueous solution at 25�C: (g) Al3þ;

(f) La3þ.

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and

K ¼expð�ze�max=kBT Þ

cmaxð13Þ

Theoretical curves of Figs. 1 and 2 were obtained using Eq. (11). The results arelisted in Table 3.

DISCUSSION AND CONCLUSIONS

Two methods have been here evaluated to obtain the main parameters ofadsorption. While the results show differences, they show a similar variation withradius and charge, which give us some confidence in the results. Both methods haveadvantages and disadvantages. The first method is related more directly with thebasic theory of adsorption, it is more intuitive but with the problem of an excessivedependence of the initial conditions. In the second method values of equilibriumconstant and number of adsorption sites are calculated directly from the maximumin zeta potential plots, making the characterization of the system easier but movingaway from the basic theory of the adsorption isotherm.

Both methods exhibit common simplifications and do not distinguish betweencations adsorbed onto the liposome surface and cations adsorbed in the electricaldouble layer, this fact is due to the use of zeta potential instead of surface potential,however it leads to a simplification of the experimental resources needed (i.e., nuclearmagnetic resonance to can distinguish between the two classes of cations beforementioned).

The differences obtained for the equilibrium constants and the number ofadsorption sites by the two methods, not only in this work and previously citedones but in all the literature relating to this subject in general, seem to indicatethat we are a long way from obtaining a complete characterization and understand-ing of the adsorption of ions onto liposomes. In this way Ermakov (26) indicatedthat there is no attempt to assume that the surface density of binding sites has thesame fixed value under all experimental conditions. The value is determinedmostly by the surface area per lipid molecule which is usually obtained from the

Table 3. Total number of adsorption sites per unit area, N1, equilibrium constant, K,

and free energy of adsorption, �G0ads, obtained from the method of Tatulian (16).

Cation N1 (m�2) K (mol�1) �G0

ads (kJmol�1)

Naþ 2.18� 1016 2.18 �11.88

Kþ 2.16� 1016 2.22 �11.93

Csþ 1.85� 1016 3.03 �12.69

Mg2þ 1.65� 1016 3.82 �13.27

Ca2þ 1.62� 1016 3.95 �13.35

Ba2þ 1.93� 1016 2.81 �12.51

Al3þ 9.28� 1015 12.10 �16.12

La3þ 9.17� 1015 12.39 �16.18


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measurements using crystalline samples, monolayers or multilayers of lipids andthere is no guarantee that the state of ionization and packing of lipid molecules inthese systems are identical to those in bilayer lipid membranes. On the other handTatulian’s (16) results indicate that identical lipid molecules are able to exhibitdistinct functional properties.

From our results alkaline-earth metal cations are in the series Ca2þ>Mg2þ>Ba2þ for their affinity for PC–Chol–PI liposomes. This sequence agrees with thoseobtained for the adsorption of the same ions onto different liposomes (16,27–29).For the alkaline metal cations the order is Csþ>Kþ>Naþ, and La3þ>Al3þ forthe final series. Curiously no bibliography was found to corroborate this sequencealthough some works show results of adsorption for La3þ (30) and for Al3þ (31),obtaining result in the same order.

The overall free energy change for complex formation between ions andliposomes, is conveniently written as the sum (31,32)

�G0ads ¼ �G0

coul þ�G0solv þ�G0

chem ð14Þ

where �G0coul is the change in coulombic energy due to adsorption, �G0

solv is thechange due to displacement of the hydration shell associated with the ion and thesurface and �G0

chem is any interaction term not included in �G0coul such as hydrogen

bonding or covalent bonding. We are going to assume that the attraction betweeneach metal and the liposome is mainly coulombic, so according to Coulomb’s law

�G0coul ¼

zþz�e2

4"0"rrð15Þ

where zþ and z� are the charges on cation and anion respectively, r is the distancebetween charges and the other constants are known. We are going to consider r asthe crystal ion radius to obtain an estimation of the coulombic energy. Values ofionic radii taken from (33) and the results obtained are shown in Table 4. Thesevalues can be compared with those listed in Tables 2 and 3 showing how the differ-ences between them increase with the radii and this seems to indicate that coulombicinteraction are predominant for small ions. However, it should be noted that thecalculations are based on crystal (unhydrated) radii. For hydrated radii the Gibbsenergies would be numerically smaller.

Table 4. Ion radii and change in coulombic energy, �G0coul,

for different cations.

Cation Radius (m) �G0coul (kJmol

�1)

Naþ 9.70� 10�11 9.33

Kþ 1.33� 10�10 6.85

Csþ 1.67� 10�10 5.45

Mg2þ 8.2� 10�11 22.21

Ca2þ 1.18� 10�10 15.44

Ba2þ 1.53� 10�10 11.91

Al3þ 5.1� 10�11 53.58

La3þ 1.39� 10�10 19.66

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In Fig. 7, we show a plot of the equilibrium constants (data from Table 2) vs.cation radii. These were grouped as a function of their charge, so each linear fittingcorresponds to monovalent, divalent, and trivalent cations respectively. It is clearthat the slopes decrease with charge. Trying to obtain a correlation as a function ofthe radius we have calculated the ratio between size of ions for each ion chargefinding the values 1.72, 1.86, and 2.73 for Csþ/Naþ, Ba2þ/Mg2þ, and La3þ/Al3þ

respectively. These values are inversely related to the slopes in Fig. 7. This resultindicates that the binding constants of ions of a fixed charge with liposomes aredetermined principly by their charge and to a lesser degree by their size.

ACKNOWLEDGMENT

We thank Dr. Malcolm N. Jones (School of Biological Sciences, The Universityof Manchester, U.K.) for many helpful discussions of the manuscript.

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Interactions Between Liposomes and Cations in Aqueous Solution

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