the interactions between pc vesicles and the air/water and oil/water interfaces

Colloids and Surfaces

A: Physicochemical and Engineering Aspects 148 (1999) 191–198

The interactions between PC vesicles and the air/water andoil/water interfaces

Bo Yang a, Hideo Matsumura b, Kunio Furusawa a,*a Department of Chemistry of Tsukuba Uni6ersity, Tsukuba, Ibaraki 305, Japan

b Electrotechnical Laboratory, AIST, MITI, Tsukuba, Ibaraki 305, Japan

Received 27 June 1997; accepted 9 September 1998

Abstract

The interactions between egg phosphatidylcholine (EPC) vesicles (diameter 200 nm) and flat surfaces (air/water andtypes of oil/water) were investigated by measuring the change in interfacial tension after adding PC vesicles to bulkNaCl or MgCl2 aqueous solutions. Electrostatic potential and Van der Waals potential curves were applied to analyzethe effects of salt concentration and the influence of different oil interfaces on changes in interfacial tension. Theexperimental results show that the electrostatic interaction between vesicle and the interface agrees with the constantsurface charge model. The largest decrease in interfacial tension was observed with a 10−3 M MgCl2 concentration.The larger the Hamaker constant (A132), the greater the change in interfacial tension change observed. © 1999Elsevier Science B.V. All rights reserved.

Keywords: Electrostatic potential; Electrophoresis; EPC vesicle; Hamaker constant; Interfacial tension; Membrane leakage; Oil

1. Introduction

Phospholipid vesicles are extensively used invarious fields of cell biology as a model for cellmembranes. For example, the investigation of in-teraction between vesicles [1–3] is very importantto understanding the interaction between cells inbiological systems. Specifically, the study of fu-sion of phospholipid vesicles [4–7] may add toour understanding of the fusion of cells. On theother hand, vesicles are also studied for potential,use for delivery of drugs, protein and nucleic acid

[8–10]. From a theoretical point of view, cells andvesicles are regarded as colloidal particles. Theapplication of the DLVO theory to study interac-tions between cells has been reported before [11–13]. A more detailed study and a review of thesubject was presented by Nir [14]. In these studies,variation in composition, separation distances be-tween layers and layer thickness were consideredin the calculation of the interaction between cellu-lar surfaces and phospholipid vesicles for bothplanar and spherical systems.

An understanding of interactions betweenmembrane and oil is very important to help un-derstand the various physiological roles of mem-branes. Therefore, we have studied the

* Corresponding author. Tel./Fax: +81-298-53-4426; e-mail: [email protected].

0927-7757/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved.

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B. Yang et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 148 (1999) 191–198192

interactions between vesicles and various kinds ofsurfaces, such as air/water and oil/water by utiliz-ing colloid chemical techniques, such as surfacetension, electrophoresis and fluorescence measure-ment. The experimental results are interpretedusing DLVO theory.

2. Experimental

2.1. Materials

The egg yolk phosphatidylcholine (EPC) whichwas purchased from QP (Japan) was used toprepare vesicles. As described in the previouspaper [15], the egg PC sample may include a smallamount of acidic lipids, which is expected fromthe negative electrophoretic mobilities of thevesicles.

For leakage tests of fluorophore/quencher fromthe inner compartment of vesicles, 8-aminonaph-thalene-1,3,6,-trisulfonic disodium salt (ANTS)and p-xylene-bis-pyridinium bromide (DPX) wereused as the fluorophore and the quencher, respec-tively. These chemicals were obtained fromMolecular Probes (USA).

The three kinds of oil (octane, dodecane andhexadecane) and inorganic chemicals of analyticalreagent grade were supplied by Wako Pure Chem-ical (Japan).

The water phase used in the experiments waspurified by the Nanopure system and redistilled(PYREX model still-1, Iwaki Glass).

2.2. Measurements of electrophoretic mobility of6esicles

EPC vesicles were prepared by extrusionthrough two stacked Polycarbonate filters of 200nm pore size (Nuclepore®, Costar, Cambridge,MA) at 25°C. The vesicles were mixed with elec-trolyte solutions of different concentrations, andthe mixtures were incubated at room temperaturefor at least 1 h. The measurements of elec-trophoretic mobility were carried out on the elec-trophoretic light scattering apparatus (OtsukaElect. ELS-800) at 25°C and z potentials werecalculated by using the Smoluchowski equation.

2.3. Leakage test of probe molecules from 6esicles

Possible destabilization of vesicle membranes atthe oil–water interface was determined by theleakage of fluorescence dye molecules from theinside of the vesicles to the bulk dispersionmedium.

EPC was dispersed in a mixture containingboth fluorophore ANTS (12.5×10−3 mol l−1)and quencher DPX (45×10−3 mol l−1) simulta-neously. The vesicle dispersion was dialyzed toremove unentrapped fluorescence dye from thebathing solution. We used 0.1 mm pore size nucle-apore membrane for extrusion and dialysis tubing(cut off MW=10 000 Da). Dialysis was repeatedseveral times in a refrigerator until the solutiondid not show fluorescence. The emission of lightfrom the flurophore is hindered by quencher con-tained in the vesicles. When dye molecules arereleased from the interior of the vesicles to bulksolutions by destruction of membrane structure,the quenching of light is released and an increasein the intensity of fluorescence can be observed bya conventional fluorimeter with emission at 510nm and excitation at 386.4 nm. Details of thismethod are described in [16,17].

The electrophoretic mobility of the vesicles en-capsulated fluorophore/quencher was shown to befairly close to that of bare vesicles withoutfluorophore/quencher. This suggests there was nofluorescence dye on the outer surface of thevesicles.

2.4. Measurement of interfacial tension

The interfacial tension was measured using Wil-helmy plate method [18]. A scheme of the appara-tus is shown in Fig. 1.

A total of 1 ml of vesicle dispersion containing3.5 mM EPC lipid was injected into 50 ml bulksolutions consisting of various salt concentrations.Using a magnetic stirrer, the final concentrationof EPC vesicles in bulk phase was 0.07 mM atequilibrium. The concentration of lipid was ana-lyzed by the method of Bartlett [19]. Interfacialtension was measured three times in each saltconcentration at room temperature after EPCvesicles were added to the bulk solution. The

B. Yang et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 148 (1999) 191–198 193

value of interfacial tension were not exactly thesame at the same salt concentration because theinjection amounts of EPC vesicle dispersion had alittle error. Twice distilled water was used toprepare solutions and wash all glassware.

3. Results and discussion

3.1. Effect of electrolyte on the electrophoreticmobility of EPC 6esicles

The electrophoretic mobilities, UE, of EPC vesi-cles were measured under different concentrationsof various electrolytes. The magnitude of the elec-trokinetic potential was found to be influencedstrongly by the composition of the aqueous phase(Fig. 2). The z potential becomes smaller withincreasing electrolyte concentrations. This is usu-ally explained as the result of shrinkage of theelectrical double layers at high ionic strength.When the concentration of MgCl2 increasedabove a certain value, the negative z potentialbecame a positive. The reversal of the z potentialis probably due to specific adsorption of Mg2+ atthe outer surface of EPC vesicles.

3.2. Leakage of fluorescent probe molecules fromEPC 6esicles

EPC vesicles containing both fluorophoreANTS and quencher DPX were added to hexade-cane emulsion in the presence of the 10−3 M

Fig. 2. z Potential of EPC vesicles as a function of NaCl (�)and MgCl2 (�) concentrations.

MgCl2 aqueous solution. The hexadecane emul-sion was prepared using an ultrasonicator (Ultra-sonics, Japan) at 150 W for 3 min with 1/30 oil towater volume ratio. The emulsion was stored at4�10°C overnight. Because the hexadacane canbe easily formed as a stable emulsion, we usedonly the hexadecane emulsion, another octaneand dodecane emulsion were not used in thisexperiment. The intensity of fluorescence wasrecorded for the elapsed time as shown in Fig. 3.The value of relative fluorescence intensity is 21.3

Fig. 3. Time elapse of fluorescent intensity from fluorophore/quencher encapsulated in EPC vesicles after adding with thedispersion of emulsion at 10−3 M MgCl2 in the aqueousphase. �, in emulsion; , without emulsion.

Fig. 1. An apparatus for measuring interfacial tension. Theplate is glass. EPC vesicles are added from the sample en-trance.


after adding the detergent to destroy all offluorophore/quencher encapsulated vesicles. Theintensity of fluorescence increased very slowlywith the elapsed time. No difference in intensitywas observed under the same salt conditions inthe absence of hexadecane emulsion.

The fluorescence intensity in the bulk is at-tributed to the mixture of fluorophore/quencherentrapped in the vesicles. The slow increase offluorescence intensity in the solution implies thatthe fluorescent dye molecules leak from the insideof vesicles to the bulk solution. This is due todestruction of the vesicle membrane through theinteraction between EPC vesicles and the oil/wa-ter interface of emulsion droplets. The slow in-crease of fluorescence intensity suggests that EPCvesicle membrane collapsed at a very slow rate.

3.3. Changes in interfacial tension of air/waterand oil/water interface under the different saltconcentration

The interfacial tensions at the air/water or oil/water interface (oil: octane, dodecane and hexade-cane) were recorded with the elapsed time (Fig.4(a)–(f)), upon addition of EPC vesicles (diameter200 nm) to the aqueous solution containing differ-ent MgCl2 or NaCl concentrations.

For the air/water interface, as shown in Fig.4(a) and (b), the value of Dg increased slowly withthe time after injecting the EPC vesicle dispersioninto the bulk solution. In the case of oil/water,especially at a high concentration of MgCl2, Dg

decreased rapidly and irregularly on the Dg versustime curves (Fig. 4(d), (e) and (f)). This behaviormay be due to the adsorption of EPC vesicles atthe oil/water interface and their subsequent in-stantaneous collapse. Then, the concentration oflipid molecules in the monolayer increased sud-denly at the oil/water interface, which causes asharp decrease of interfacial tension.

As shown in Fig. 4(a) and (c), for both inter-faces of air/water and oil/water with NaCl in thebulk, Dg decreased gradually with increasing saltconcentration. On the other hand, for interfacesincluding MgCl2, the Dg decrease firstly with in-creasing MgCl2 concentration until 10−3 M, andthen, gradually increase again with increasing

MgCl2 concentration. This can be explained bythe behavior of z potential. As seen in Fig. 2, thenegative z potential of EPC vesicles in NaClsolution decreases monotonously with increasingNaCl concentration. The electrostatic repulsionbetween EPC vesicles and the oil/water interfacewill also decrease monotonically and EPC vesiclescan adsorb more easily as NaCl concentrationincreases. On the other hand, in systems ofMgCl2, the negative z potential of EPC vesiclesdecreased with MgCl2 concentration up to 10−3

M, approached to zero at 10−3 M, and thenincreased to a small positive value. The datasuggest the electrostatic repulsion is minimized at10−3 M, and Dg reached its minimum value. Apositive z potential at higher concentration ofMgCl2 decreases vesicle adhesion.

3.4. Analysis of interaction between EPC 6esiclesand interface by using DLVO theory

3.4.1. Analysis using electrostatic potentialThe electrostatic potential (Vr) is long range

and will contribute mainly to the interaction be-tween EPC vesicles and the interface at the initialencounter. The electrostatic interaction energiesbetween spherical particles and flat plates at con-stant charge (V r

s) and constant potential (V r8) are

given, respectively by the following equations[20,21]

V r8=

ao

4�

2f1f2 lnexp (kH0)+1exp (kH0)−1

+ (f12+f2

2) lnexp (2kH0)−1

exp (2kH0)n

(1)

V rs=

ao

4�

2f1f2 lnexp (kH0)+1exp (kH0)−1

− (f12+f2

2) lnexp (2kH0)−1

exp (2kH0)n

(2)

where o is the dielectric constant of suspendingmedium (78.5 for water at 25°C), f1, f2 are thepotentials of the particle and the plate, and f1 canbe approximated by the z potentials of the vesiclesand f2 is assumed to be zero, k is the Debye–Huckel reciprocal length parameter and given byEq. (3)


Fig. 4. Time elapse of change of interfacial tension at various concentrations of salt for air/water or oil/water interface after addedEPC vesicles in bulk. (a) air/water, NaCl (b) air/water, MgCl2 (c) octane/water, NaCl (d) octane/water, MgCl2 (e) dodecane/water,MgCl2 (f) hexadecane/water, MgCl2. �, 10−6 M; ", 10−5 M; �, 10−4 M; �, 10−3 M; �, 10−2 M.

k='4pe2(n+z+

2 +n−z−2 )

okT(3)

in which z9 is the valence of the ionic species insolution, e is the electronic charge, k is Boltz-

mann’s constant and T is the absolute tempera-ture (298 K).

Fig. 5(a) and (b) show the electrostatic poten-tial curves in the different MgCl2 concentrationsunder constant charge and constant potential con-


ditions. As shown in the figures, the electrostaticinteraction for the two conditions indicate com-pletely different dependances on the electrolyteconcentration. In the constant charge system, therepulsive potential decreases with increasing elec-trolyte concentration, becomes zero at MgCl210−3 M and finally increases again above 10−3

M. This accounts for the Dg versus MgCl2 con-centration curves, and shows that the maximumrate of change of Dg at 10−3 M MgCl2 will bebased on the zero electrostatic potential betweenEPC vesicles and octance/10−3 M MgCl2 solu-tion. On the contrary, in the system of constantsurface potential, Fig. 5(b) indicates that the elec-trostatic interaction between EPC vesicles and theinterface becomes attractive, and the attractiveenergies decrease with salt concentration. At 10−3

M MgCl2 solution, the attractive energy showsthe lowest value, which corresponds to Dg havingthe slowest rate. This is contradictory to the real

Fig. 6. Model used to calculate the Van der Waals interactionbetween planar slab and vesicles.

experimental results. Therefore, from analysis us-ing electrostatic interaction, we concluded that theinteraction of EPC vesicles with the octane/waterinterface occurs with the constant surface chargecondition.

3.4.2. Analysis using Van der Waals attractionpotential

The Van der Waals attraction potential is usedto analyze the interaction between EPC vesicles atthe zero surface potential point (10−3 M MgCl2solution) and oil (or air/water) interface. Thepotential energy of Van der Waals interactionbetween vesicle and planar interface (Fig. 6) isgiven by the methods of Vincent [22] and Vold[23] as follows:

VA= −A132

12� 1

X1

+1

X1+1+2 log

1X1+1

�+

A132

12� 1

X2

+1

X2+1+2 log

1X2+1

�(4)

A132= (A11−A33)(A22−A33) (5)

here, A11, A22 and A33 is the Hamaker constant ofoil or air, phospholipid and water, respectively.

X1=H2a

(6)

and

Fig. 5. Electrical repulsive energy between EPC vesicles andoctane/water interface at various MgCl2 concentrations. (a)constant surface charge (b) constant surface potential 1, 10−6

M; 2, 10−5 M; 3, 10−4 M; 4, 10−3 M; 5, 10−2 M.


X2=H+d

2(a−d)(7)

where H is the distance between the sheathedsphere and planar slab, a the radius of the spheri-cal particle and d is thickness of lipid bilayer.

Fig. 7 shows the VA potential curves betweenEPC vesicles and various interfaces in 10−3 MMgCl2 aqueous solutions where EPC vesicles havezero surface potential. In the calculations, we usedthe Hamaker constants as follows [24]: Aair=0,Aoctane=4.5, Adodecane=5.0, Ahexadecane=5.2,Awater=3.7, Alipid=8.0 (×10−20J). As shown inFig. 7, the interaction between EPC vesicles andthe air/water interface is quite different fromother systems. The positive Van der Waals inter-action potential means that the Van der Waalsforce is repulsive for the air/water interface. Onthe other hand, in the case of oil/water systems,VA potential energy gives negative values indicat-ing the existence of an attractive force. Interfacialtension at the oil/water interface actually de-creased more quickly than at the air/water inter-face (Fig. 8). The initial slope of the curves, (thatis, the rate vesicles are adsorbed at the interface),is nearly zero in the air/water system, but isalmost the same in three oil/water systems. Theinitial rate (Dg/Dt) can be explained by the theo-retical calculations (Fig. 7). In the case of theair/water system, Dg shows no change when the

Fig. 8. Dg Versus time curves for various interfaces in 10−3 MMgCl2 after added EPC vesicle in bulk solution. �, air/waterinterface; �, octane/water interface; ", dodecane/water inter-face; , hexadecane/water interface.

vesicles first encounter the interface. This corre-sponds to the repulsive interaction. However, Dg

changes after a long period, which may be at-tributable to the adsorption of mono-molecularlydispersed lipid molecules. This behavior cannot beexplained by the theoretical treatment we haveused. The oil/water interface has no potentialbarrier, indicating a rapid aggregation by Brown-ian motion, which gives the same rate for thethree oil/water interfaces.

Fig. 9 shows the correlation between the appar-ent Hamaker constant and the equilibrium valuesof Dg obtained from Fig. 8. It is interesting to

Fig. 9. The relationship between interfacial tension Dg ap-proaching equilibrium and Hamaker constant A132 at 10−3 Mof MgCl2 solution.

Fig. 7. Van der Waals interaction energy between EPC vesiclesand various interfaces at 10−3 M MgCl2. (1) air/water inter-face; (2) octane/water interface; (3) dodecane/water interface;and (4) hexadecane/water interface.


note that where A132=0, Dg is still 40 mJ m−2.The interfacial free energy change (Dg) is assumedto consist of three contributions: electrostatic re-pulsive energy VR, Van der Waals energy VA andshort range interaction energy VSR (e.g. hydrogenbonds) [25].

Dg=VR+VA+VSR (8)

As can be seen from Fig. 2, the z potential at10−3M MgCl2 solution is almost zero, suggestingVR is zero. This suggests that 40 mJ m−2 of Dg

can result from the sum of short range interac-tions other than Van der Waals force and theelectrostatic force. That is, the adsorption behav-ior of the vesicles to the oil/water interface, isdetermined by the DLVO force at the initial stage,but is mainly determined by the short range inter-action at the final stage.

References

[1] H. Hauser, M.C. Philips, R.M. Marchbanks, Biochem. J.120 (1970) 329.

[2] L. Weiss, S. Nir, J.P. Harlos, J.R. Subjeck, J. Theor. Biol.51 (1975) 439.

[3] A.M. Carmona-Ribeiro, J. Phys.Chem. 97 (1993) 11843.[4] D. Papahadjopoulos, G. Poste, B.E. Schaeffer, W.J. Vail,

Biochim. Biophys. Acta 352 (1974) 10.[5] J. Kim, H. Kim, Biochim. Biophys. Acta 983 (1989) 1.[6] M.G. Salgo, F.P. Corongiu, A. Sevanian, Arch. Biochim.

Bioophys. 304 (1993) 123.[7] T.D. Bradrick, A. Philippetis, S. Georghiou, Biophys. J.

69 (1995) 1999.[8] S.C. Kinsky, R.A. Nicolotti, Ann. Rev. Biochem. 46

(1977) 49.[9] D. Papahadjopoulos, Ann. New York Acad. Sci. 308

(1978) 1.[10] M.J. Ostro, Sci. Am. 91 (1986).[11] D. Gingell, J. Theor. Biol. 19 (1967) 340.[12] B.W. Ninham, V.A. Parsegian, J. Theor. Biol. 31 (1971)

405.[13] V.A. Parsegian, D. Gingell, Biophys. J. 12 (1972) 1192.[14] Shlomo Nir, Prog. Surf. Sci. 8 (1977) 1.[15] H. Matsumura, F. Mori, K. Kawahara, C. Obata, K.

Furusawa, Colloids Surf. A 92 (1994) 87.[16] M.N. Dimitrova, H. Matsumura, Colloids Surf. B:

Biointerfaces 8 (1997) 287.[17] H. Elens, T. Bentz, F.C. Szoka, Biochemistry 24 (1985)

3099.[18] R. Matuura, Mem. Fac. Sei, Kyushu Univ. ser. c. 47

(1949) 1.[19] G.R. Bartlett, J. Biol. Chem. 234 (1959) 466.[20] R. Hogg, T.W. Healy, D.W. Fuerstenau, Trans. Faraday

Soc. 62 (1966) 1638.[21] S. Usui, J. Colloid Interface Sci. 44 (1973) 101.[22] B. Vincent, J. Colloid Interface Sci. 42 (1973) 270.[23] M.J. Vold, J. Colloid Sci. 16 (1961) 1.[24] J.N. Israelachvili, Intermolecular and Surface Forces (ch.

11), Academic Press, London, 1992.[25] C.J. Vanoss, J. Chromatogr. 391 (1987) 53.

.

the interactions between pc vesicles and the air/water and oil/water interfaces

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