Chain-melting phase transition in dipalmitoylphosphatidylcholine foam bilayers

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CHEMISTRY AND PHYSICS OF LIPIDS

Chemistry and Physics of Lipids 83 (1996) III 121

Chain-melting phase transition in dipalmitoylphosphatidylcholine foam bilayers A. Nikolova a, R. Koynova b, B. Tenchov b, D.

E x e r o w a ",*

~lnstitute of Physical Chemistry, Bulgarian Academy of Sciences, Sofia 1113, Bulgaria blnstitute of Biophysics, Bulgarian Academy of Sciences, Sofia 1113, Bulgaria Received 18 March 1996; revised 10 June 1996; accepted 22 June 1996

Abstract

Microscopic horizontal foam bilayers (Newton black films) from dipalmitoylphosphatidylcholine (DPPC) have been studied by the microinterferometric method of Scheludko and Exerowa. The foam bilayers were formed from DPPC dispersions in water and in a water/ethanol (52.5:47.5, v/v) mixture in the presence of 0.15 M NaC1. The high ethanol concentration strongly facilitates their formation. The bilayer thickness and the critical bulk concentration C~ of DPPC required for the formation of a stable microscopic foam bilayer from the water/ethanol mixture were measured in the range 35-45°C. Both parameters indicate cooperative changes in the state of the foam bilayers. These changes take place at the temperatures of the bulk chain-melting phase transitions, as determined by differential scanning calorimetry (DSC) for both aqueous and water/ethanol DPPC dispersions. The critical DPPC concentration Cc for the water/ethanol dispersions changes between 55 and 140 /~g ml ~ in the range 35-45°C. However, measurements by DSC show that decreasing the lipid concentration to 2.5/~g ml ~ in both water and water/ethanol mixtures does not affect the enthalpy, temperature and width of the bulk phase transition of DPPC. This is an indication for a mechanism of foam bilayer formation which involves adsorption of whole vesicles to the air-solution interface, followed by their subsequent spreading on the surface. A concentration-temperature phase diagram of DPPC foam bilayers that defines the regions of gaseous (ruptured), gel and liquid crystalline foam bilayers has been constructed.

Keywords: Lipid; Liposome; DSC; Microinterferometry; Bilayer thickness; DPPC

1.

* Corresponding author, Tel: + 359 2 719206; e-mail: [email protected],

Introduction

T h e f o a m bilayers ( N e w t o n b l a c k films) consist o f two m u t u a l l y a d s o r b e d , densely p a c k e d m o n o layers o f a m p h i p h i l i c molecules which are in con-

0009-3084/96/$15.00 © 1996 Elsevier Science Ireland Ltd. All rights reserved PH S0009- 3084(96)02600-X

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A. Nikolova et al./ Chemistry and Physics of Lipids 83 (1996) 111 121

tact with a gas phase. Balmbra et al. [1] first noticed the structural correspondence between foam bilayers and lamellar mesomorphic phases. Our recent studies show that foam bilayers from dimyristoylphosphatidylcholine (DMPC) undergo a chain-melting phase transition with characteristics similar to those of the main phase transition in bulk DMPC dispersions [2,3]. This was the first proof that foam bilayers are able to change their thickness due to occurrence of phase transitions in contrast to the generally accepted opinion that these bilayers do not change their thickness under variation of experimental conditions. The similarities between foam bilayers and lipid dispersions contribute to a successful application of the former system as a model for investigations of membrane fusion [4], interlamellar interactions [5,6], membrane permeability [7], alveolar surfactant stability [8], vesicle destruction at the air/solution interface [9], longitudinal electroconductivity at surfactant/water interfaces [10]. Formation of foam films from phospholipid dispersions poses certain problems emerging from the very low solubility of these amphiphiles in water and from the slow kinetics of spreading of the lipid vesicles on the air-water interface. The aim of this work is to study dipalmitoylphosphatidylcholine (DPPC) foam bilayers in the temperature range of the main phase transition of DPPC and compare their thermal behaviour with that of the corresponding lipid dispersions. The foam bilayers were formed under different conditions, in particular, from DPPC dispersions in water and water/ ethanol mixtures. Thus, it was possible to evaluate the influence of ethanol on their phase state. The foam bilayers were studied by the microinterferometric method of Scheludko and Exerowa [11,12]. Two independent experimental parameters, the critical bulk lipid concentration C~ required for formation of a stable foam bilayer and the foam bilayer thickness, were measured, similar to the experiments with DMPC foam bilayers [2,3]. A parallel study of the DPPC dispersions at low lipid concentrations was carried out by high-sensitivity differential scanning calorimetry [13].

2. Materials and methods

l, 2 - dipalmitoyl glycero - 3 - phosphocholine (DPPC) from Avanti Polar Lipids, Inc., Birmingham, AL, was used. The half-width of 0.2°C, measured by DSC, of the main phase transition in the aqueous lipid dispersions provided a guarantee that the lipid purity was comparable with the claimed value of 99%. Quartz-bidistilled deionized water (specific conductivity 10 6 f~ l cm ~, pH ~5.5) was used throughout the experiments. NaC1 (Merck) was roasted at 500°C for 2 h to remove surface active impurities. Three types of lipid dispersions were used: (i) multilamellar lipid vesicles (MLV) were prepared by dispersing the lipid in 0.15 mol dm 3 NaCI solution in water. Such electrolyte content guarantees the formation of foam bilayers in the case of dense packing of DPPC at the surface [6]. The dispersions were hydrated overnight at 20°C and for 1 h at 50°C. The samples were vortexed several times at the latter temperature for 1-2 rain; (ii) DPPC dispersions in water/ethanol mixtures containing 47.5 vol% ethanol (pa) and 0.15 mol dm 3 NaC1 were prepared by the same procedure as that for the aqueous MLV dispersions. The addition of ethanol causes an increase in the critical micelle concentration [14,15] and fusion of the small liposomes [16]. The ethanol concentration ensures maximum adsorption of DPPC at the air/solution interface [3]. The same ethanol concentration was used in previous investigations of foam bilayers from phospholipids [2,3] and natural lipid-protein mixtures [17]; (iii) dispersions of small unilamellar vesicles (SUV) were prepared from aqueous MLV dispersions by sonication in a bath-type sonicator for 90 min under nitrogen at 50°C. The foam bilayers were studied by the microinterferometric method of Scheludko and Exerowa (e.g. Refs. [11,18]), applied in its recent modification. A microscopic horizontal foam bilayer of radius 0.1 mm was formed in a glass measuring cell presented schematically in Fig. 1. A biconcave drop (b) is set up in the glass tube (a). The microscopic foam film (c) is formed in the middle of the biconcave drop by sucking out the solution from the drop through the capillary tube sn

-

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A. Nikolova et al. / ChemLs'try and Physics O! Lipi& 83 (1996) 111 121

(d). T h e o b s e r v a t i o n o f the f o a m film is carried o u t t h r o u g h the o p t i c a l l y flat glass b o t t o m (f) o f the cell c o v e r e d with the s o l u t i o n (e) used for f o r m a t i o n o f the f o a m film. T h e f o a m b i l a y e r thickness was d e t e r m i n e d by using an optical system for r e g i s t r a t i o n o f the reflected f r o m the film light [12,18]. T h e equivalent thickness o f the f o a m bilayers was c a l c u l a t e d from the d a t a for reflected light intensity with an a c c u r a c y o f _+ 3% [12]. A t least 10 thickness m e a s u r e m e n t s were m a d e at each t e m p e r a t u r e . T h e c a l o r i m e t r i c study o f the lipid d i s p e r s i o n s was carried out with high-sensitivity differential a d i a b a t i c scanning m i c r o c a l o r i m e t e r s D A S M - 1 M a n d D A S M - 4 ( B i o p r i b o r , Pushchino, Russia) with sensitivity better t h a n 4 x 10 6 cal K ~ a n d a noise level less t h a n 5 x 10 7 W [13]. H e a t i n g runs were p e r f o r m e d at 0.5°C rain ~. T h e ther-

d

.

l

.

e-

"7

80

E u

4" b" 60

2

I

40

20 0

,

,

L

,

k

100

200

300

400

500

_

TIME, rain

Fig. 2. Surface tension kinetics of fully hydrated dispersions of DPPC containing 0.15 tool dm ~ Na(l: curves I and 2 dispersion of MLV and sonicated dispersion o1' S[V, containing 70 itg cm ~ DPPC at 36°C, respectively: curves 3 and 4 dispersion of MLV containing 140 /~g cm ~ DPPC and sonicated dispersions of SUV containing 30 /tg cm ~ DPPC at 44°(7, respectively; curves 5 and 6 water-alcohol dispersions of DPPC containing 47.5 w)l% ethanol and 70/tg cm DPPC at 36°C and 140/zg cm ~ DPP(" at 44°(7. respectively. m o g r a m s were c o r r e c t e d for the i n s t r u m e n t a l baseline. T h e c a l o r i m e t r i c e n t h a l p y o f the transition was d e t e r m i n e d as the area under the excess heat c a p a c i t y curve. The lipid c o n c e n t r a t i o n varied in the range 2.5 1000/~g ml The surface tension o f the lipid d i s p e r s i o n s was m e a s u r e d with a digital t e n s i o m e t e r K 10T (Kruss) with a c c u r a c y o f 0.1 m N m ~ The refractive indices o f the a q u e o u s and w a t e r - e t h a n o l dispersions were m e a s u r e d in the t e m p e r a t u r e range 35 -45°C with an A b b e r e f r a c t o m e t e r (Carl Zeiss).

3. Results

3.1. smJace tension qf diluted D P P ( ' rfispersions

f

Fig. 1. Scheme of a measuring cell for the study of microscopic foam films: (a) glass tube, (b) biconcave drop, (c) microscopic foam film, (d) glass capillary, (e) surfactant solution, (f) optically flat glass.

The coverage o f the a i r / s o l u t i o n interface with D P P C molecules can be followed by m e a s u r i n g the kinetics o f decrease o f the surface tension a o f the lipid dispersions. Results o f such m e a s u r e m e n t s are given in Fig. 2. Curves I 4 show the

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A. Nikolova et al. / Chemistry and Physics o[' Lipids 83 (1996) I 11 121

surface tension kinetics of MLV and SUV aqueous dispersions at two temperatures below and above the temperature of the chain-melting phase transition of DPPC. It is clear that the surface tension of both SUV and MLV dispersions decreases faster at temperatures above the chain-melting transition. Sonication of the dispersions also accelerates the lipid adsorption to the interface. The surface tension of water/ethanol dispersions of DPPC was slightly lower than that of the water/ethanol mixture without DPPC ( 30 dyn cm-~) and after the first 15 min was practically time-independent (curves 5 and 6 in Fig. 2). These data were used to choose appropriate conditions for formation and characterization of the foam bilayers. 3.2. Critical concentration Jor f o r m a t i o n of f o a m bilayers

The critical bulk DPPC concentration C~ required for formation of foam bilayers was determined by measuring the probability W for observation of a foam bilayer at a given temperature [2,3,18,19]. This probability was found on the basis of the final state reached by the foam film as a result of its drainage, namely, rupture at a certain critical thickness without formation of black spots or formation of black spots which grow in size and finally convert the foam film into foam bilayer [11]. The rupture of the films takes place at bulk concentrations of DPPC below Cc and formation of black spots and foam bilayers takes place at DPPC concentrations equal to or above Cc. Two W ( C ) dependencies measured at 37°C and 42°C for foam bilayers formed from water// ethanol dispersions of DPPC are shown in Fig. 3. Sharp changes of W take place at C~= 75 tlg cm 3 (curve 1 at 37°C) and at Cc = 125/2g cm (curve 2 at 42°C). At concentrations below Cc the probability W for observation of a foam bilayer is practically zero and at concentrations equal to or higher than C~ it is practically unity. It is not possible, within the experimental limitations, to distinguish a concentration range with values of W intermediate between zero and unity. About 25 foam films were measured for each concentration

and temperature and the resulting accuracy of Cc determination was + 5%. The values of C~ for water/ethanol dispersions of DPPC obtained in this way at different temperatures are plotted in Fig. 4 in Arrhenius coordinates. A change in the slope of the Arrhenius plot takes place at about 39°C. The experiments with aqueous MLV and SUV dispersions of DPPC showed that the values of C~ for aqueous dispersions of DPPC are lower than the values of Cc for water/ethanol dispersions. For example, SUV dispersions with DPPC concentration of 70 /2g c m - 3 produced stable tbam bilayers at all temperatures in the range 35 45°C. At DPPC concentrations about 30 j2g cm 3 formation of black spots and foam bilayers was observed only at temperatures above the main phase transition. It should be noted that it was not possible to obtain reproducible results for the critical ooncentration Co, particularly at temperatures below the chain-melting transition. One possible reason for this is the slow coverage of the foam film surfaces with DPPC molecules at low amphiphile concentrations in the range of C~ (see Fig. 2, curves I and 2). k

W 1.0

0.5

2

0

~

50

'

I

t

100

150

C, ],49 cm -3 Fig. 3. Dependence of the probability W for observation of foam bilayer on the bulk concentration C of DPPC in waterethanol dispersions containing 0.15 mol dm- 3 NaCI and 47.5 voW,, ethanol: curve I 37°C, curve 2 42°C.

A. Nikolova et al. / Chemistry and Physics o/Lipids 83 (1996) 111 121

about 38°C from a constant value of 6.4 nm above this temperature to 7.0 nm below it.

t,°C

45

5.0

40

35

3.4. Microcalorimetry qf diluted DPPC dispersions

150

"•

i

100 uE c

4.5

4.0 50 3.!0

3.15

3.20

3.25

I15

3.30

103/T,K -1 Fig. 4. Arrhenius plot of the critical concentration C~ for formation of foam bilayer from water-alcohol dispersions of DPPC containing 0.15 mol dm - 3 NaCI and 47.5 vol% ethanol: circles experimental data, the straight lines are calculated from Eq. (1)under the assumption C~ = C~.

3.3. Equivalent thickness of the foam bilayers The equivalent thickness hw of the foam bilayers was calculated from the intensity of the light reflected from the film assuming that the foam bilayer is an optically homogeneous system with refractive index n2 equal to that of the bulk dispersion [12]. The values of hw of foam bilayers obtained from SUV dispersions (n2= 1.33) and from water/ethanol dispersions of DPPC (n2 = 1.35) are shown in Fig. 5. The thickness measurements for SUV dispersions were carried out after an equilibration for 2 - 3 h at 45°C ensuring formation of long-living foam bilayers. Then the temperature was lowered to the required value and after 0.5 h equilibration the thickness was determined. The values of hw for this system are significantly higher in comparison to the hw values of the foam bilayers obtained from the water/ ethanol system. A gradual change in hw takes place in a narrow range at about 40°C for SUV dispersions from a value of 8.5 nm above this temperature to 9.2 nm below it. Similarly, the equivalent thickness hw of the foam bilayers obtained from water/ethanol dispersions changes at

DSC experiments were performed at low DPPC concentrations comparable with those used for formation of foam bilayers. The calorimetric data for the temperature Tm and enthalpy A H of the chain-melting phase transition are summarized in Table 1 and a selection of thermograms is presented in Fig. 6. MLV dispersions in water and in water/ethanol mixture display highly cooperative phase transitions in contrast to the lower cooperativity of the transition in SUV. A pretransition was detected only for MLV in water. Its absence at ethanol concentration of 47.5 vol% was reported also for water/ethanol dispersions of D M P C [3]. In SUV dispersions the temperature Tm is slightly lower compared to M LV, in agreement with published data [20]. The enthalpy of the transition did not change with decrease of the

10 E Jr-

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38

0

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35

~

i

J

i

I

I

J

i

i

40

I

~

45 t, °C

Fig. 5. Temperature dependence of" the equivalent thickness h~ of foam bilayers obtained from dispersions of DPPC containing 0.15 tool dm ~-3 NaCI: C) foam bilayers obtained from sonicated aqueous dispersion of SUV containing 70 #g cm DPPC; • - - foam bilayers obtained from water-ethanol dispersion containing 160 #g cm --3 DPPC and 47.5 vol% ethanol.

116

A. Nikolova et al./ Chemistry and Physics of Lipids 83 (1996) 111-121

Table 1 Thermodynamic parameters of the chain-melting phase transition of fully hydrated dispersions of DPPC containing 0.15 mol dm 3 NaC1 Dispersion

Lipid concentration (/xg cm 3)

T,n (°C)

3 H (kcal mol ~) 3Til 2 (°C)

MLV in water

2.5 10 20 30 55 75 100 200 500 1000 55 1000 2.5 10 20 30 55 75 100 200 500 1000

41.66 41.51 41.49 41.49 41.51 41.54 41.46 41.46 41.51 41.49 40.17 39.37 39.99 40.01 40.22 40.14 40.32 40.32 40.50 40.62 40.55 40.32

9.34 7.65 6.40 6.96 7.06 6.98 6.46 7.20 6.92 7.98 4.72 6.08 11.80 9.97 10.21 9.14 9.45 9.37 10.54 8.56 9.23 8.93

SUV in water Dispersion in H20/ethanol (52.5:47.5, v/v)

0.35 0.18 0.23 0.23 0.18 0.25 0.23 0.25 0.24 0.20 2.74 2.43 0.35 0.22 0.33 0.25 0.22 0.22 0.25 0.25 0.25 0.23

Note: our experiments did not show significant influence of the addition of 0.15 M NaC1 on the temperature, enthalpy and cooperativity of the main phase transition of DPPC both for the water and water/ethanol dispersions.

lipid concentration down to the lowest concentration of 2.5 /~g cm -3 DPPC accessible with the calorimeter used. The insertion in Fig. 2 presents the effect of ethanol concentration on the temperature of the chain-melting transition. Besides the well-known biphasic effect within the concentration range up to 20 vol% ethanol [21 23], a regular decrease in the transition temperature was observed at higher ethanol contents.

4. Discussion

4. I. Conditions for formation of foam bilayers from diluted DPPC dispersions A necessary condition for the formation of a foam bilayer is the close packing of the amphiphilic molecules in its two monolayers [24,25]. For insoluble surfactants such as DPPC this requirement poses a certain problem emerging from

the very low solubility of the lipid. The calorimetric data in Table 1 show a constant enthalpy of the chain-melting phase transition within a broad concentration range from 2.5-1000 /~g cm -3 DPPC. This indicates that DPPC is present predominantly as vesicles in the dispersions from which the foam films were formed. That is why the transfer of DPPC molecules from the vesicles to the air/solution interface (i.e. the surface tension kinetics) is important for the foam bilayer formation. Fig. 2 shows rather high values of the surface tension at 36°C even 7 h after the start of the measurement for both MLV and SUV dispersions. These high values reflect incomplete coverage of the surface with lipid molecules. They are probably due to a slow disintegration of the surface vesicles while their diffusion towards the air/solution interface is of minor importance [26,27]. Fig. 2 shows that at temperatures above the chain-melting transition the surface tension decreases faster than at temperatures below this

A. Nikolova et al..i Chemistry and Physics o[Lipids 8,7 (1996) //1 /21

transition, especially in the case of SUV dispersions. These observations agree with previous findings that the spreading rate of D P P C and D M P C liposomes at the air/solution interface depends on the phase state of the lipid [27 29]. The much faster surface tension kinetics of the water/ ethanol dispersions (curve 5 and 6 in Fig. 2) is probably due to accelerated spreading of the vesicles in the presence of ethanol. The fast coverage of the air/solution interface with D P P C molecules

117

in this case ensures precise and reproducible determination of the critical concentration C~ required for formation of foam bilayers. The measured critical concentrations of the D P P C foam bilayers (Fig. 4) for the water/ethanol system are about 2 - 3 times lower than the corresponding values for D M P C foam bilayers [2,3]. This appears to reflect the higher adsorption affinity of D P P C to the air/solution interface adequately to its longer hydrocarbon chains. 4.2. Phase state o f the D P P C Jbam bilayers

a= m

2. o

o ¢ i h-

• Ii

•

Ethanol

20

•

concentration

30

n

[ v o l %]

40

Temperature [°C] Fig. 6. DSC thermograms of fully hydrated dispersions of DPPC containing 0.15 M NaCI: (a) non-sonicated aqueous dispersion of MLV containing 55 #g cm 3 DPPC; (a') nonsonicated aqueous dispersion of MLV containing 2.5/~g cm- 3 DPPC; (b) water/ethanol dispersion containing 55 /~g cm-3 DPPC and 47.5 vol% ethanol; (b') water/ethanol dispersion containing 2.5 /~g cm- ~ DPPC and 47.5 vol% ethanol; (c) sonicated aqueous dispersion of SUV containing 55/~g cm - 3 DPPC. Thermograms (a') and (b') are averages over three sequential measurements; the vertical scale is enlarged 3 times for these thermograms. Inset: the chain-melting transition temperature of DPPC dispersions in water/ethanol solutions as a function of the ethanol concentration as determined by DSC at 0.5°C rain -' scan rate; lipid concentration 0.5 mg cm-3.

The results from the microinterferometric study of the foam films and microcalorimetry of the dispersions from which they were formed present an opportunity to compare the phase states of the foam bilayers and the respective dispersions. The thermal behaviour of the foam bilayers was characterized by measuring the temperature dependence of two parameters --- the critical concentration Cc and the bilayer thickness. The determination of C~ as a function of temperature provides a possibility to apply the hole-nucleation theory of bilayer rupture for calculation of the defined binding energy Q of an amphiphile molecule in the foam bilayer [7,18,30]. This theory considers the two monolayers of a foam bilayer as two-dimensional lattices with nearest-neighbour interaction. These monolayers are in equilibrium with a bulk reservoir (the meniscus of the biconcave drop) with concentration C of amphiphile monomers. The rupture of the loam bilayer is regarded as a result of a two-dimensional phase transition of a 'gas of vacancies' into 'condensed phase of vacancies'. The gaseous and the condensed phases of vacancies are in equilibrium at bulk concentration C~. The relation between the binding energy Q and the equilibrium concentration C~ is given by: C,, = (7<, exp( - Q / 2 k T )

(1)

where T is the absolute temperature, Co is a reference concentration and k is the Boltzmann constant. The concentrations C~ and C~ are characteristic constants for each particular system. The foam bilayer is thermodynamically stable when C_> C~. It is metastable within the concen-

118

A. Nikolova et al./ Chemistry and Physics of Lipids 83 (1996) 111-121

tration range C~ < C < Ce. A sharp increase in the W(C) dependence from zero to unity as in Fig. 3 indicates that the metastable region is absent. Then the critical concentration should be regarded as equal to the equilibrium one (C~ = C¢). Such behaviour was previously detected also for foam bilayers from D M P C and egg lecithin [2,3]. The absence of a metastable region allows the calculation of Q from the Arrhenius dependence of Cc from Eq. (1) under the assumption C~ = Ce. Eq. (1) is derived under the assumption that the foam bilayer is in equilibrium with a solution of monomer amphiphiles [30]. On the other hand, the calorimetric data for the water/ethanol system indicates that DPPC is present predominantly as vesicles in the dispersions from which the foam bilayers were formed. That is why the energy Q for DPPC foam bilayers should be considered not only as the binding energy of a DPPC molecule in the foam bilayer but also as including other energetic contributions such as the adsorption energy, the energy for vesicle destruction, etc. The break in the slope of the Arrhenius plot in Fig. 4 coincides in temperature with that recorded by DSC bulk chain-melting transition for the water/ethanol dispersion (Fig. 6, Table 1). We therefore consider this effect a manifestation of a melting phase transition in the foam bilayer, similar to the transition in the bulk phase. A similar break in the Arrhenius dependence o f C~ was also found for D M P C foam bilayers [3]. The values of Q determined from the slopes of the two straight lines in Fig. 4 are (7.1 + 0.2) x 10 20 j and (4.9 _+ 0.3) X 1 0 - 1 9 j per DPPC molecule for the liquid crystalline and gel foam bilayers, respectively. The corresponding values of Q for D M P C foam bilayers are 8 . 0 x 10 2o j and 1 . 9 × 1 0 - ' 9 j [3]. The values of Q for the liquid crystalline foam bilayers from DPPC, D M P C and sodium dodecylsulphate (Q = 7.0 x 10 -2o J [19]) are rather similar. Since the sodium dodecylsulphate foam bilayers are formed from solution of monomeric amphiphiles~ on the basis of this similarity we assume that the binding energy is the major energetic contribution in Q for liquid crystalline DPPC foam bilayers. For now we cannot propose a satisfactory explanation for the considerably higher value of Q for gel DPPC foam bilayers in comparison to gel DMPC foam bilayers.

Air

h1~ nl

h2p n2 h i,n I

Air Fig. 7. Schematic representation of a foam bilayer according to the triple-layer model.

The temperature dependence of the foam bilayer equivalent thickness hw also provides an indication that a phase transition takes place in DPPC foam bilayers obtained from both water/ ethanol and SUV dispersions (Fig. 5). We consider the decrease in hw by about 0.6-0.7 nm at the temperature of the main transition as a result of the phospholipid acyl chain melting. It is noteworthy that h w changes gradually in a range of 2 3°C about the temperature of the main phase transition. Since the thickness determination is based on measurements of the light intensity reflected from a foam film area of about 2 x 10 ~' cm 2, the gradual shift of hw may be considered as due to averaging over the microheterogeneous foam bilayer structure in the transition range. It is well-known that the equivalent thickness of a foam bilayer is higher than its real thickness [31]. The difference between them can be estimated by using the triple layer model of the film structure [32]. It considers the foam bilayer as being constituted of three layers of different refractive indices (Fig. 7). We assume that the outer layers of thickness h 1 and refractive index n, include the DPPC molecules and the inner layer of thickness h2 and refractive index n 2 includes the aqueous core between them. The bilayer thickness (h = 2h, + h2) can then be calculated with the help of the following equation for h2 [32]:

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A. Nikolova et al. / Chemist O' and Physics t~/ Lipids 83 (1996) I11 121

h~ = h , - 2h,[(nl - 1)/(n2 - 1)]

(2) ~5

The right-hand side of Eq. (2) contains the parameters hw and n2 measured in the present work. The other two parameters (h~ and n~) must be estimated from published data. We accept n~ = 1.464, as measured previously for black egg lecithin membranes [33]. The values of h~ were estimated from the data about the size of the DPPC molecule in different phases. The chain length was taken as 1.315 nm and 1.875 nm in the liquid crystalline and gel states, respectively [34]. A tilt of 30 ° with respect to the normal of the bilayer surface was assumed for the gel state, thus producing a value of 1.624 nm for the hydrocarbon layer thickness. Such tilt is known to exist in the lamellar gel phase of fully hydrated DPPC dispersions [35] and in the liquid-condensed state of DPPC monolayers at air/water interfaces [36]. A thickness of 0.7 nm was accepted for the head group region [37,38]. Table 2 summarises the data for h,, and the values of h~, h2 and h calculated as explained above. Evidently the presence of ethanol results in a considerably decreased thickness h~ of the core of water molecules bound to the phospholipid polar head groups [6,39], in accord with the decrease in the hw value. The structural characteristics h 2 and h of the liquid crystalline ethanol-free foam bilayers agree well with those of fully hydrated liquid crystalline DPPC dispersions [40]. However, the calculated value of h~ for gel ethanol-free foam bilayers Table 2 Calculated values of the equivalent thickness hw, the thickness th of the phospholipid region, the thickness h 2 of the aqueous core and the thickness h of the foam bilayers obtained from fully hydrated aqueous dispersions containing 70 pg cm ~ DPPC, 0.15 tool dm ~ NaCI with and without addition of ethanol h,,. (nm) hi (nm)

h 2 (nm)

h (nm)

I. Liquid crystalline foam bilayers 0 vol% ethanol 8.50 2.02 47.5 vol% ethanol 6.40 2.02

2.54 0.84

6.57 4.87

2. Gel foam bilayers 0 vol% ethanol 9.20 47.5 vol% ethanol 7.00

2.32 0.59

6.97 5.24

2.32 2.32

ot.9 ...,"

liquid -

40

g

O

~

.~

35

i

50

~

gel

J

~

[

c r.yi?to~ b ne

~

~

biloyer

J

L

J

I00

I

,

150 C,

]vlg

cm

-3

Fig. 8. Phase diagram of DPPC foam bilayers obtained from water/ethanol dispersions of DPPC containing 0.15 mol dm 3 NaC1 and 47.5 vol% ethanol.

exceeds by about 0.6 nm the interbilayer water thickness in the gel phase of DPPC dispersions [40]. One possible reason for this difference might be a higher refractive index nt of the gel foam bilayer.

4.3. Phase diagram of DPPC Joam bilavers

The measured temperature dependence of C,. allows the construction of a temperature-concentration phase diagram of the foam bilayers obtained from the water/ethanol dispersions of DPPC (Fig. 8). The solid line shows the lowest bulk DPPC concentration at which stable liquid crystalline or gel foam bilayers can exist at the given temperature. It corresponds to a two-dimensional phase transition of condensation of DPPC molecules at the foam film surfaces. The horizontal dashed line represents the two-dimensional chain-melting phase transition between the gel and liquid crystalline foam bilayers taking place at about 39°C. This diagram strongly differs from the corresponding phase diagram for DPPC dispersions. As shown by the DSC measurements (Table 1), the horizontal transition line in a corresponding phase diagram for bulk dispersions must be extended to much lower DPPC concentration.

120

A. Nikolova et al./ Chem&try and Physics of Lipids 83 (1996) 111 121

5. Conclusions

References

F o a m bilayers o b t a i n e d f r o m w a t e r a n d wat e r / e t h a n o l d i s p e r s i o n s o f D P P C vesicles und e r g o a c h a i n - m e l t i n g p h a s e transition. T h e i r p h a s e state is c h a r a c t e r i z e d by two i n d e p e n d e n t p a r a m e t e r s - - the critical c o n c e n t r a t i o n r e q u i r e d for f o r m a t i o n o f a f o a m b i l a y e r a n d the b i l a y e r thickness. T h e t r a n s i t i o n t e m p e r a t u r e s in the f o a m bilayers a n d the c o r r e s p o n d i n g S U V a n d water/ethanol dispersions of DPPC practically coincide. This result is in a g r e e m e n t with the theoretical c o n s i d e r a t i o n s o f N a g e l [41] for the decisive role o f van der W a a l s a t t r a c t i o n between h y d r o c a r b o n chains o f p h o s p h o l i p i d molecules for the c h a i n - m e l t i n g p h a s e transition in the systems c o n t a i n i n g bilayer structures. T h e thickness m e a s u r e m e n t s show a struct u r a l similarity between the f o a m bilayers a n d the b u l k D P P C dispersions f r o m which they form. A p h a s e segregation in the f o a m bilayers was f o u n d in the region o f the c h a i n - m e l t ing p h a s e transition. T h e presence o f 47.5 v o l % o f e t h a n o l s t r o n g l y decreases the a m o u n t o f w a t e r b o u n d to the p o l a r head g r o u p s o f p h o s p h o l i p i d molecules in the f o a m bilayers. T h e A r r h e n i u s p l o t o f Cc has been used to calculate, within the f r a m e w o r k o f the hole-nuclea t i o n t h e o r y [7,18,30], the b i n d i n g energy Q o f a D P P C m o l e c u l e in the f o a m bilayer (7.1 x 10-2° j a n d 4.9 x 10-19 j for liquid crystalline a n d gel f o a m bilayers, respectively). T h e determ i n a t i o n o f the p h a s e state o f the f o a m bilayers is o f i m p o r t a n c e for the studies o f the interactions a n d stability o f bubbles, vesicles a n d d r o p l e t s in m i c r o h e t e r o g e n e o u s systems. In this respect, the e x p e r i m e n t a l l y d e t e r m i n e d p h a s e dia g r a m o f D P P C f o a m bilayers is o f p a r t i c u l a r value.

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Acknowledgements W e are grateful to Prof. D. K a s h c h i e v for v a l u a b l e discussions a n d a c k n o w l e d g e financial s u p p o r t f r o m the B u l g a r i a n N a t i o n a l Science Fund.

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