Interfacial orientation of DOPC liposomes spread at the air-water interface

147

Advances in ColZoid and Interface Science, 40 (1992) 147-156 Elsevier Science Publishers B.V., Amsterdam 00102

A

Interfacial

orientation

I.PANAIOTOV

of DOPC liposomes water interface. *,Tz.IVANOVA**

spread

at the

air-

and A.SANFELD***.

*Visiting Professor at University Aix-Marseille 3,France. Biophysical Chemistry Laboratory, University of Sofia, Anton Ivanov str. 1, 1126 Sofia **Pulma Laboratory SoBa,Bulgaria. ***Universities

of Brussels, Belgium and Aix-Marseille 3, France.

Abstract. The kinetics

of surface

spread at constant surface pressure describing electrical

film formation

from DOPC small unilamellar

surface is studied, by measuring

and of the surface potential.

the interfacial

orientation

affinity of orientation

The thermodynamical

process

shows

and of the electrical

vesicles

the time evolution

of the

approach

the importance

of the

surface pressure.

Introductioa The mechanism

of formation

of a surface

film from liposomes

spread

at the

air-water interface was recently studied by several authors [l-43. The behavior following

of the liposomes

processes

i) diffusion

at an air-water

single

this phenomenon,

the diffusion

transformation

constant

This rough

theoretical

vesicles

scheme

allows

A better

process is however required.

In this

field

Sanfeld

[5,6]

0 1992 -

into

mesophases. consists

summarized

open

to obtain

understanding

Especially,

I41 in a

surface-active a global

kinetic

of the interfacial

it is interesting

to evaluate

of the complex process of orientation.

has developed

in order to study the interfacial

0001~6666/92/$15.00

in a slow

a first approximation

closed

the role of the pure electrical contribution

approach

resulting

into superficial

process from the other processes

of perfectly

of the transformation.

orientation

interaction

of closed bilayer structures

quantitatively

structures.

by the

from the interface to the bulk;

transformation

to distinguish

is governed

:

ii) a more complex liposome-surface

To approach

interface

an irreversible dipole orientation.

thermodynamics He has shown

Elsevier Science Publishers B.V. All rights reserved.

the relationship between the affinity of orientation of dipoles and the rate of orientation.

L.Lavielle

et al. [7-91 applied this last theory to interfacial

properties of grafted polyethylene in contact with water. The aim of the present work is i) to follow the kinetics of the surface film formation from DOPC small unilamellar vesicles spread at constant surface area, by recording the time evolution of surface parameters : surface pressure x and surface potential AV ii) to give the theoretical frame to describe this inter-facial orientation process.

Materials Preparation

Dioleoylphosphatidylcholine Company.

and Methods.

of the DOPC liposomes.

(DOPC)

is purchased

from Sigma

Analytical grade chloroform is purchased from Merck.

NaCl solution is made from triple-distilled

Chemical The 0.15m

water and Merck purest quality

NaCl roasted at 700°C. Liposomes are prepared by the Bangham method [lo] with sonication at 20°C (above the phospholipid phase transition temperature) using a probe with a Branson Sonifer cell disruptor B30 (350 W, 20 kHz) for several 5 min periods (resting time 5 min) until the solution becomes almost clear. The liposomal suspensions are filterd through a 0.22 pm Millex GV single-use filter unit (Millipore).

The phospholipid

concentration

is equal to 10 mg/ml.

The

suspensions are stored at +4”C during two weeks.

so 60-

PARTICLE UNIRDDAL

DIAMETER (NM) DISTRIBUTION BROAD

Fig.1 Distribution of mean particle diameter for DOPC liposomal suspensions.

149

The liposomes size measurements analyzer

Coulter

are performed with a submicron particle

model N4 MD (Coulter

Electronics)-temperature

20’33,

detection angle 90°, measurement time 5 min. The mean particle diameter d is about 50 nm with monopeak dispersion (Fig.1). The liposomal concentration

Co of the suspension can be calculated assuming

that all the phospholipids are contained liposomes : Co = 2.4 1014liposomes/cms. Measurements

in unilamellar

50 nm diameter

of surface parameters.

The different volumes of the liposomal suspension are spread at constant surface area of 186 cm2 . Spreading is performed by using an Agla syringe, during about l-2 min. The subphase is 0.15 m NaCl at T= 295°K. The kinetics of surface film formation after spreading is studied by recording the evolution of the surface pressure 7~ and of the surface potential AV with time t. The time t = 0 corresponds to the end of the spreading procedure. The surface pressure x is measured by the Wilhelmy plate method by using a platinum plate and the electronic computered balance KSV with a precision 0.02dyn cm-r. The surface potential

AV is measured by using a gold coated Am241 ionizing

electrode, a reference calomel electrode and an electrometer VA-J-51 connected to a Sefram chart recorder with an accuracy f 15mV on the initial surface potential Ve. Model and theoreticalapproach

After spreading of the liposomal suspension at the air-water interface, the kinetics can be described by two simultaneous processes (Fig.%) : i) an irreversible diffusion process of intact liposomes to the liquid bulk phase, ii) a progressive transformation

of perfectly closed structures (non surface active

liposomes without any effect on the surface potential) into open structures (surface active mesophases increasing the surface potential). It was shown [4] that for large amounts of liposomal suspensions spread at the air-water interface, the diffusion of liposomes towards the liquid bulk phase did not affect the liposomal concentration Co at the subsurface. The number of

150 Surface potential = 0 Surface pressure = 0

I Fig.2

diffusion

Scheme

surface

Surface potential ( t ) Surface pressure ( t )

film

describing after

the two processes

the spreading

involved

of liposomal

in the formation

suspension

of a

at the air-water

interface.

liposomes

in the first subsurface

thus constant.

layer able to be continuously

In this case, the

x(t)

and the

continuous

transformation

phospholipid

molecules

AV(t) of

the

into destroyed

kinetic

curves

closed

spherical

In addition to a mechanical

an electrical process.

dipole contribution

As we will see furtheron

at constant we will describing

phospholipids

contribution

a pure

structures

to the of

the

have a dipolar character

(short range forces) there is t.hus

the reorientation

phenomenological

the orientation

are only related

which plays a role in the complex

and uniform surface concentration.

use

is

surface structures.

We notice that the surface reorganizing [ll].

transformed

One may then write f = Cod.

orientation

kinetics will be considered

In order to estimate this effect,

theory

developed

previously

[5,6]

for thin surface layers.

Neglecting

the dipolar quadratic

effects due to the second-order

(dispersion

terms),

of the interfacial

the variation

and p is given, for one single orientating do=-rdp-xA,d{m$

component

tension

:

contributions

(T , at constant

T

151

where

(T is the macroscopic

liposomes

in the first subsurface

the open structure, surface

surface tension,

v

orientation

moment per mole

M

acting

potential

transformed

of the liposomes,

on the mean projection

i

into

Ai is the

of the dipole

< mi > .

From a microscopic mechanical

layer able to be continuously

is the chemical

affinity

I- = Cc d is the number of spread

background,

we are allowed to split all the forces into both

and electrical

E contributions.

Consequently,

Eq. (1) can be

read do=da,-do,=-rd~,FAiMd(mi)+

Tdp,+

FAiEd(m$ (3)

where

(3)

d~M=-rd~M-CAiMd(mi) 1 As previously

and

do,=-

rdpa-

T AiEd(mi)

(4)

shown [5 Eq.81 , Eq.(4) leads to the relation

l-1 30,

AiE=d(llli)

_-

30,

eq 9(mi)

(5)

where the first derivative in the r-h-s of (4) is taken at orientation By using the microscopic each containing

equilibrium.

model of a compact layer of discrete dipoles in 1 cells

k neighbours,

it was shown [5 Eq.191 that

(6) where

mk] is the distance between two dipoles in a lattice characterized

parameter dipoles

a

; v

with respect

moment and

is a coefficient to the surface,

depending m

on the relative

is the arithmetic

position

by the of the

value of the dipole

E is a mean static dielectric constant of the medium.

By taking into account Eq.(6) the expression (5) reads

(7)

152 In the case

of spread

liposomes

at air-water

rotating dipoles towards final equilibrium

interface

with

state, it is reasonable

progressively to assume that

(8) Indeed,

out of equilibrium

distance

the orientation

parameter

mkl are much more sensitive to the variations

than at equilibrium.

and the dipolar

Eq.(5) reduces to

3%

*i~=qq

At constant

As a first approximation,

v

of the dipolar moment

(9)

T, p , pE ( according to Eq.9)

the integration

of Eq.(9) from (TE= 0 (t

= 0) to o,(t) leads to

(10) Let us now express called Marcelin-De

where

the electrical

orientation

affinity

Aj~ by means of the so-

Donder relation [5 Eq.111 :

d(mi)

and

vi =dt

0

are the direct rates of dipolar orientation at time t and t = 0 respectively. During

the

mesophases,

continuous AV(t)

concentrations. example

However,

constant

both

electrical

dipolar

electrical

effects.

volumes

into

orientation

spread

Co at the subsurface

able to be continuously

Consequently,

contribution,

liposomes

dipolar

in the next paragraph)

concentration

in the first sublayer (r = Co d ).

of the

electric

for large

> 1600 ~1 as considered

affect the liposomial liposomes

transformation

measures

on the

interfacial and surface surface

the diffusion

doesn’t

and the number transformed

II(t) measures

of

is thus

for this case, AV(t) only measures

whereas

(for

both mechanical

the and

153

In order to calculate the pure electrical orientation affinity AiE(Eq.(ll)) corresponding

electrical part of the surface tension

surface potential measurements

and the

(Ts (Eq.(lO)) we use the

AV(t) . Indeed, the surface potential [12] of

an uncharged monolayer is related to the vertical component i = z of the dipole moment < mi > , the dielectric constant E and the surface density F ( r = C, d). A”=O.&%E

(12)

The AV versus t curve allows to obtain and d /dt if E and I are known. From and d /dt , Ais and es are derived. The knowledge of E is however not trivial aswell as the quantitative interpretation

of the

surface potential data.

Experimental rarmlts and discussion. Figures

3 and 4 show the kinetics of the surface pressure

surface potential

n(t) and of the

AV(t) at constant surface area (186 cm*) after spreading of

different volumes of DOPC liposomal suspensions (80,180,400

and 1600 ~1).

For 80, 180 and 400 ml, the kinetics is governed by both diffusion transformation

and

processes l.41. As previously shown 141 for an upper critical

TimeIsocl Fig 3 Time variation of the surface pressure x after spreading of various volumes of DOPC liposomal suspension ( 80 ,180 , 400 and 1600 ul ; spreading area = 186 cm2 )

154

Fig.4 Time variation of the surface potential AV after spreading of various volumes of DOPC liposomal suspension ( 80 , 180 ,400 and 1600 pl ; spreading area = 186 cm2 1.

volume of spread liposomal suspension, the kinetic process is nomore controlled by diffusion. The curves obtained above this critical value (1600 pl for DOPC liposomes) are practically merged. By using Eq.(12) and with an approximate value for E (E = 2) ,we calculate the values of (t) from the AV(t) data obtained for a spread volume equal to 1600 ~1. After a graphical derivation, one gets the values of Ai, by Eq.(ll). integrating graphically, Eq.( 10) gives the corresponding values of bs.

By

Due to the unaccuracy of the initial time of the process (O-60 s), it is difficult to obtain the exact result corresponding to the beginning of the kinetics. Table 1 gives the values of the mean vertical projection of the dipole moment per molecule < mi> (d), the electrical surface orientation affinity Ai, (e ), the electrical and mechanical parts of the surface pressure xs (0 and xM (g), obtained from the experimental data x(t) (b) and AV(t) (cl.

155

Table 1. Table 1. Time evolution of the vertical electrical

component

affinity

pressure

of the surface pressure

after

A,

x , of the surface potential

of the mean dipole moment

, of the electrical

spreading

- xE

, of the orientation

and mechanical

of 1600 ~1 of DOPC

AV ,

liposomal

- ‘CM surface

suspension

with a

constant e = 2 .

a

b

c

d

e

t

n

AV

-=P

AiE

M

[dynhml

[mVl

ID/molec I

0

0 60

0

3000

0 0.80 2.50 3.00 4.90 6.45 7.80 10.75 14.00 IS.00

3600

15.85

440

4600

17.00

440

60 120 150 300 450 600 1200 2400

200 325 360 373 395 422 433

g =M

Idynkml [dyn/cm]

0.92 2.08 3.08 5.00 5.54 5.74 6.08 6.49 6.66 6.77 6.77

135

I-=E

0.280

0.33 0.58 0.71 0.84 0.85 0.85 0.85 0.85 0.85 0.85 0.85

0.150 0.100

0.030 0.010 0.004 0.002 0.001 0.001 0 0

0.47 1.92

2.29 4.06 5.60 6.95 9.90 13.15 14.15 15.00 16.15

eq The

electrical

surface

transformation process.

Indeed,

at

60s).

t = 60s ,

Ai,= 2 kT. After

of the process

It decreases

equilibrium.

affinity

the

mentioned,

the of the

t = 15Os, Ai, < kT .Similarly,

at the

orientation

present results (Table 1) is conditioned

during

acts at the beginning

xE is of the order of magnitude

during

As already

AiE decreases

It essentially

of the liposomes.

rotation

beginning

orientation

process

of xM (41% of x for t= to reach

the quantitative

5% of x at

interpretation

of the

to the value of E . However, instead of E

equal to 2 we assumed a rough time evolution of E (=l at t=O and =3 at t= t), the contribution important.

of

Ai,

at the beginning

of the process

For example, at t= 12Os, Ais --0.575

equilibrium

xE is 15% of I[. That goes without

quantitative

analysis

required.

of the results,

then becomes

more

(= 4kT) and xE is 78% of K. At saying that to give a better

the relationship

between

E and is

156

Conclusion.

After spreading a sufficiently large amount of liposomal suspension at the airwater interface, a progressive transformation of perfectly closed vesicles into open surface-active

structures

The time evolution

occurs.

of the surface

pressure and of the surface potential allows to show the importance of the electrical orientation affinity by means of the thermodynamics dipole layers.

of interface

The opening process is mainly governed by the mechanical

contributions due to short range forces. However, at least at the beginning of the dynamical transformation,

the electrical part of the surface pressure is

larger than 40% of the total surface pressure. Although a precise quantitative analysis of the results is conditioned

to the knowledge of the relationship

between the dielectric constant and the dipole moment, the present study permits one to compare the driving forces due to short and middle range interactions. References

1. F.Pattus, P.Desnuelle, R.Verger, BBA, 507 (1978) 62. 2. H.Schindler, BBA, 555 (1979) 316. 3. M.Obladen, D.Popp, C.Scholl, H.Schwartz, F.Jahnig, BBA, 735 (1983) 215. 4. Tz.Ivanova,

G.Georgiev,

I.Panaiotov,

M.Ivanova,

M.A.Surpas,

J.Proust,

F.Puisieux,Prog. Colloid Polym. Sci., 79 (1989) 23 5. A.Sanfeld, Nuovo Cimento, 12D,N”7 (1990) 901. 6. A.Sanfeld, “Introduction

to the thermodynamics

of charged

and polarized

layers”, Wiley Interscience, London (1968).

7. L.Lavielle, G.Lischetti, A.Sanfeld, J.Schu1tz,J.Co11.1nt.Sci.,138,N01(1990) 134 8. L.Lavielle, JSchultz, J.Coll.Int.Sci.,106,N02 (1985) 438. 9. L.Lavielle, J.Schultz,A.Sanfeld,

J.Coll.Int.Sci.,106,N”2 (1985) 446.

10. A.D.Bangham, M.M.Standish, J.C.Watkins, J.Moll.Biol.,lS (1965) 238. 11. H.Mohwald, Ann.Rev.Phys.Chem., 41 (1990) 441. 12. G.L.Gaines,“Insoluble

monolayers

Interscience, New York (1966).

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interfaces”,

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