Monolayers and bilayers of lipids

Colloids and Surfaces, 53 (1991) 135-145 Elsevier Science Publishers B.V., Amsterdam

Monolayers E. Margheri,

135

and bilayers of lipids

A. Niccolai, G. Gabrielli’

and E. Ferroni

Dipartimento di Chimica, Universitci de&i Studi di Firenze, Via Gino Capponi, g-50121 Firenze (Italy) (Received 2 1 November

1989; accepted 25 May 1990)

Abstract The spreading monolayers of ceramides alone and in mixtures with monooleoyl glycerol were studied at the air/water interface in the 15-30°C temperature range. The bidimensional phases were determined from the spreading isotherms of the pure components, whereas the interfacial miscibility of the compounds being studied was deduced from the spreading isotherms of the mixtures. The specific resistance and capacitance of black lipid membranes containing monooleoyl glycerol and monooleoyl glycerol/ceramides in squalene were studied. Langmuir-Blodgett monolayers and bilayers obtained by the transfer of spreading monolayers at the air/water interface onto chromated glass slides, were prepared. Vesicles of dioleoylphosphatidylcholine and of mixtures of dioleoylphosphatidylcholine/ceramides were also prepared. The results obtained for the different types of aggregates were compared in order to find the relations that connect the characteristics of the various systems. Results showed that the possibility of obtaining flat and curved bilayers of pure components and of mixtures was closely connected to the phase of the corresponding monolayers at the air/water interface.

INTRODUCTION

In this paper four possible biological membrane models were studied, remembering that these systems approximate some aspects of corresponding biological systems [ 11. The study of monolayers is very useful because it provides information about the molecular orientation of the amphiphilic molecules at the air/water interface and about the compatibility of different amphiphilic molecules at the interface [2]. The study of black lipid membranes (BLMs) offers useful indications about the ion transport and electrical properties of the membrane [3]. Langmuir-Blodgett (LB) films [4] and curved bilayers, such as vesicles, are other important membrane models. These latter aggregates are useful because they model the real cellular membrane structure (an inner solution and an outer solution separated by a hydrophobic bilayer ). Furthermore, their study offers indications concerning membrane permeability to ‘To whom correspondence

0166-6622/91/$03.50

should be addressed.

0 1991-

Elsevier Science Publishers

B.V.

136

solutes and solvents and concerning the processes of membrane-membrane fusion [ 11. Lastly, the parallel study of various types of aggregates of the same amphiphilic substances is extremely interesting, since it can lead to the determination of relationships that connect the properties of the different systems. The substances examined in this study were ceramide (CER), alone or mixed with either monooleoyl glycerol (MON) or dioleoylphosphatidylcholine (DOPC ) . CER is biologically interesting since it is a component of natural membranes, as is DOPC, whereas MON has a simple chemical formula but the same glyceric skeleton as the phospholipids. Given the importance of the Na+ ion in the biological environment, the CER monolayers were studied on 0.1 M NaCl. Miscibility between CER and MON was verified, and it was found that these mixtures are able to give flat bilayers (BLMs) . Then DOPC, which forms individual vesicles, was studied, and the possibility of obtaining mixed CER/DOPC vesicles was verified. EXPERIMENTAL

The following substances were used: 1-monooleoyl glycerol (MON) , with a purity of 99%, and ceramides (CER) from bovine brain with the following composition in carboxylic fatty acids, as determined by gas chromatography C16:0=1.9%; C18:0=32.3%; C20:4=3.7%; by the supplying firm: C24 : 0 = 10.9%; C24 : 1 = 34.4%; others = 16.8%. Dioleoylphosphatidylcholine (DOPC), with a purity of 99%, and squalene, with a purity between 98 and lOO%, were also used. CER, MON, DOPC and squalene were supplied by Sigma Chemie GmbH, Darmstadt, F.R.G. Monolayer-s

The spreading solvent was pure chloroform supplied by Farmitalia Carlo Erba S.p.A., Milan, Italy. All solutions were stored in a freezer. NaCl (0.1 M) was used as the substrate, and was supplied by C. Erba (purity 99%). The solutions were prepared using doubly distilled water freed from colloidal impurities by a Milli-Q (Millipore) ultrafiltration apparatus; water thus obtained had a resistivity of 18 MS2 cm. Measurements of the surface pressure z and the molecular area A were carried out using a Lauda balance, interfaced with a Digital PDP 11/23 computer and an apparatus described elsewhere [ 51. The determination of n was carried out according to the Langmuir method [ 61. Once the surface cleanliness had been verified, the spreading isotherms were recorded 10 min after spreading of solutions, and the surface pressure versus area curves were obtained with discontinuous compressions of the monolayer. Measurements were taken at 15,25 and 30°C. The accuracy was determined to be: for n 2 0.07 mN m-‘, for A 2 0.01 m2 mgg’ and for temperature ? 0.05’ C.

137

Langmuir-Blodgett films The LB films of CER were obtained at room temperature by transferring spread monolayers on NaCl (0.1 M) onto a solid support made of a glass slide covered with vacuum-evaporated chrome. Transfer was carried out using a Langmuir trough 4 balance (Joyce Loebl), which uses the Wilhelmy method to determine surface pressure. Surface pressure was established at 36.5 mN m -l, with a dipping speed equal to 4.76*10m3 cm s-l. The thickness of the transferred films was measured ellipsometrically [ 7 ] using a thin-layer ellipsometer 43702-2003 (Rudolph Research, U.S.A. ), and the data obtained were processed on a program set up by the Department of Chemistry, University of Florence, and based on the program of McCrackin and Colson [8]. Measurements of the film thickness were carried out with an a priori error of 5%. BLMs BLMs were obtained from gently heated dispersions of 8 mg total lipids in 1 ml squalene. The BLMs were prepared using the technique of Mueller et al. [ 91, i.e. by placing a drop of dispersion on a circular hole (1.2 mm in diameter) in a Teflon partition separating two saline solutions contained in two semicells of about 16 ml volume each. Two Ag/AgCl electrodes were introduced. Solutions of NaCl and of CaCl, (0.1 M) were used. The two salts (purity 99% ) were supplied by Farmitalia. BLM formation was facilitated by blowing air bubbles through a Pasteur pipette into the system in the hole immediately after deposition. The BLMs were studied at room temperature. Measurements of their electrical properties were performed using a voltage clamp circuit. The applied potential was varied in the 20-280 mV range. The area of the BLM was determined by means of an ocular micrometer with an error of 5 10e4 cm’. The membrane capacitance (C) was measured at 30 Hz with a 0.2-2% accuracy a.c. impedance bridge. The resistance (R) measurements were performed using a square wave of variable potential at a frequency of 300 mHz. The signal was visualized on a Tectronic digitizing oscilloscope and plotted by a HewlettPackard recorder. The specific capacitance values (e) were obtained from the relation C= C/A, and the specific resistance values Rfrom the relation I?= RA, where A was the BLM area. Vesicles DOPC and DOPC/CER vesicles were obtained by sonication [lo] of aqueous dispersions, using a Sonifier B 12 Cell Disruptor sonicator (Branson Sonic Power Company) at 70 W for 1 h at 60°C. Aqueous dispersions of the amphiphile were prepared in buffer solutions (E. Merck, Darmstadt, G.D.R.) at pH 7.

138

The dispersions contained 20 mg of total lipids and had a volume of 5 ml. The vesicles thus prepared were observed by means of scanning electron microscopy (SEM) using a Philips 30 kV electronic microscope. ESR measurements were carried out with a Bruker spectrometer model 200D, operating in the X band ( = 9.5 GHz). RESULTS AND DISCUSSION

Monolayer-s Pure components Figure 1 shows the spreading isotherms of MON and CER as a function of temperature. Table 1 reports the values of the compressibility module C,-‘. The surface phases were characterized by these values [ 61, and the eventual presence in the isotherms of discontinuities associated with phase transitions further helped to characterize the phases. At 15°C MON isotherms show the presence of a plateau, which could be attributed to a transition from a liquid expanded (LE) phase to a liquid inter-

40.00

0.00 0.00

0.70 A (m’/v)

1.40

0.00

0.50 A (m’/ms)

1

) 30°C. TABLE

1

Parameters of MON monolayers at the air/water interface T(K)

C,-’ (mN m-‘)

A (pmol-‘)

a, (A’ mol-‘)

288 298 303

18.1~80.6 52.8G78.9 50.0 f 66.8

53.3 f 35.5 43.8G33.7 44.4 + 36.7

-8.2 - 22.8 - 17.3

139 TABLE 2 Parameters

of CER monolayers

at the air/water

interface

T(K)

C,-’ max (mN m-l)

AIi, (A* mol-‘)

n=a+bA

288 298 303

164.7 187.0 270.0

50.5 55.6 50.5

71= 133.9-377.54 7c= 124.1-187.5A n= 133.9-233.64

mediate (LI) one, even if we did not try a further study of this transition. In any case, a large range of the areas per molecule correspond to an LI phase, at the three temperatures, in agreement with the literature [ 11,121. This phase can he described by the following state equation [ 111: 71A=kT(1+al/A+az/A2+...)

(1)

Table 1 reports the first virial coefficient values a,. Since these were negative and close to zero, they indicated the presence of weak attractive chain-chain interactions, in agreement with that reported elsewhere [ 111. The values of C,-’ reported in Table 2 indicated that CER was in the liquid condensed phase (LC) at the three temperatures, as reported previously [ 131. This phase can be described by the following state equation: n=a+bA

(2)

Table 2 also reports the state equations of the LC phase for CER at the three temperatures. A mean molecular weight of 608.78 was used to calculate the molecular areas. In summary, MON showed principally an LI phase which was characterized by slightly interacting hydrophobic chains of the amphiphile molecules. These were therefore probably arranged with wide angles randomly distributed with respect to the perpendicular to the interface. Instead, CER showed principally an LC phase, where the aliphatic chains of the molecules interacted strongly and were considered to have an almost vertical orientation to the surface. MON/CER mixtures The spreading isotherms of MON/CER mixtures in the ratios l/l, 2/l, 3/l and l/3 at 1525 and 30” C are reported in Fig. 2. Figure 3 shows the molecular areas as a function of the weight fraction of CER in the mixtures. The dimension of the symbols gives the experimental error and the trend is representative of a positive deviation from additivity, especially at 25 and 30’ C, as a function of the composition of the mixtures. The eventual presence of repulsive forces among the hydrophobic chains of the two components in the bidimensional phase was deduced from this. The miscibility of the two components at all weight ratios was confirmed by applying the bidimensional phase rule [ 141, since the collapse pressure llcoll varied with the composition of the mixtures,

0.00

0.70

0.00

0.00

1.40

1.40

0.70

0.00

0.70

1.40

A (m’/ms)

Fig. 2. Spreading isotherms of MON (0 ), CER (* ), MON/CER= 1 (o), MON/CER=2 MON/CER=3 (A) andCER/MON=3 (A),at (a) 15, (b) 25and (c) 30°C.

(*),

C

0.90

* ---.

--_.--A

i

0.40

3---_

‘-_*

*

---

--.. - -

_\

I

b

GO.90

5 L 4:

i-

= _ _$ -- _ --*> -_-< * _ - =-_>

o,40 0.00 Weight

0.50 Fraction

1.

I

(CER)

Fig. 3. Molecular area versus CER weight fraction calculated for x=25 mN m-’ at (a) 15, (b) 25 and (c) 30°C.

as reported in Fig. 4. Once the miscibility of MON and CER was ascertained, we considered a thermodynamic analysis to be possible and useful; hence we determined dG,i,, the excess free energy of mixing, dH,i,, the excess of enthalpy of mixing, and A!&,, the excess of entropy of mixing, according to Bacon and Barnes [ 151; the values are shown in Fig. 5. The values of dG,i, were always positive, although very near zero; this indicated that the mixtures

141

-

20.00

b

< ~40.00

-

2

Y

20.00

--

40.00

-

a

20.00

Fig.

4.

0.00

v I I I I I 7 I 1 , I I I I I I 1 f I 1 .OQ 0.50 Weight Fraction (CER)

Collapse pressure versus CER weight fraction at (a)

15,

(b)

25

and (c ) 30 ’ C.

C

cl+=

0.0

10.0 -30.0

-i

b

? 2 k 5 s

0.0

10.0 -30.0

P

:

a

0.0

-30.0 10.0

-i

,,(,

Weight Fig.

,,,,

5

0.00

0.50 Fro&ion

1 .oo (CER)

5. ( * ) dG,i,, (0 ) AH,,,, and (A ) TAS,,, versus CER weight fraction at (a) 15, (b) 25 and (c) 30°C. The a priori errorsare: k 4.1. 10e4 J g-’ for AC,,,, and? 0.06 J g-’ for AH,,, and TAS,,,.

142

were more unstable than the pure components. In particular, ASmix for the MON : CER= 1:1 and the MON : CER= 3 :1 mixtures was positive, indicating that large amounts of CER introduced disorder in the MON monolayer. Langmuir-Blodgett films It was not possible to transfer monolayers of MON with reproducible results, whereas the CER monolayer was easily transferred from the air/water interface to the solid/air interface. This was probably because only CER formed a monolayer in the condensed phase; this behaviour was in agreement with our data for other substances [ 161. Furthermore, since it easily formed a bilayer, in spite of the presence of different aliphatic chain lengths for each CER molecule, it is possible that interdigitation occurred between the molecules of the two layers. The data obtained for the thickness D and the refractive index n of the LB monolayers and bilayers of CER are reported in Table 3. BLMs Figure 6 reports the specific The BLMs of MON and of the had a specific capacitance that the applied potential. We could inside the membrane [ 171. The

capacitance Gversus mixtures, both with did not vary, within therefore deduce that specific capacitance

the applied potential Vi. NaCl and CaCl, (0.1 M) , experimental error, with squalene was not present of the membranes studied

TABLE 3 Thickness Monolayer Bilayer

D and refractive index n for LB films of CER D=25 ii D=52A

nz1.5 n=1.6

o~500.-

vi W) Fig. 6. Specific capacitance CaCl, (0).

eversus

applied potential

Vi for MON BLMs in 0.1 IV NaCl ( * ) and

143

is reported in Table 4. The area occupied by a single aliphatic chain in the presence of NaCl (0.1 M) was obtained from the following equation, valid for BLMs without solvents: A = 2~v,,,/e,

en

(3)

where e0 = 8.85. lOpa FF cm- ‘, en is the dielectric constant of the hydrocarbon core of the MON chain and Vmol is the volume occupied by a single aliphatic chain. Using the values calculated by other authors [ 181 for en and Vmol, we obtained A = 36.11 t 0.01 A”; this corresponded to the area occupied by one molecule of MON in the LI phase in the monolayer at the air/water interface (Fig. 1). Owing to the use of NaCl (0.1 M) as substrate in the spreading isotherms, we could calculate the value of rc corresponding in the monolayer to the area per molecule obtained in the bilayer. This was 28.3 mN m-l at 15°C 25 mN m-l at 25°C and 27.7 mN m-’ at 30°C. This result is similar to the one obtained by Marcelja [ 191, who compared the calculated and measured values of the order parameters for lecithins [ 201 and deduced that at surface pressures equal to 25-15 mN m-’ are comparable for monolayers and bilayers. The same calculations [ 3] were performed using CaCl, solutions. Here the value of A was much lower, i.e. 31.72 2 0.01 A”. We deduce that the BLM was more condensed in the presence of CaCl, than in the presence of NaCl. Presumably, the aliphatic chains form a smaller angle with respect to the normal line to the interface in the bilayer in CaCl, than that in NaCl. Hence the Ca2+ ions have a condensing effect. BLMs containing CER alone were not prepared because the lipid is slightly soluble in squalene; hence if the solvent varies it is not possible to compare the results obtained for each system. For the same reason only MON/CER mixtures with weight ratios greater than 1 were studied. In NaCl (0.1 M) the mixed BLMs had lower evalues than the MON BLMs. TABLE

4

Specific capacitance

C and resistance I? for MON and MON-CER

Mix MON (NaCl) CER:MON=l:l CER:MON=l:2 CER:MON=1:3 CER:MON=1:5 MON (CaCl,) CER:MON=1:2 CER:MON=1:5

(NaCl) (NaCl) (NaCl) (NaCI) (CaCl,) (CaCl,)

BLMs

c (.uF cm-‘)

R (@cm”)

0.74 k 0.010 0.61+ 0.006

107 106

0.64 0.63 0.64 0.65 0.66 0.65

107 106 106 106 106 106

k k k 5 k +

0.004 0.005 0.003 0.005 0.002 0.003

144

-50.00

0.00

2.00 Weight Ratio

4.00 (MON/CER)

6.00

Fig. 7. Natural logarithm of the specific conductibility g, (Q-l cm-‘) ratios in 0.1 M NaCI.

versus MON/CER weight

This suggests that the addition of CER produced by itself a condensing effect which then masked the effect of the Ca2+ ions. The specific resistance values R are reported in Table 4 for each system. Furthermore, a plot of In g, (see Fig. 7), where g, is the specific conductance at zero current, versus the composition of the BLM in the presence of NaCl, revealed the absence of CER microdomains inside the bilayer. In fact, the specific conductance at zero current did not change with the composition of the BLM, and therefore CER and MON did not have differential permeabilities to the Na+ ions [22]. We also prepared closed bilayers such as vesicles, observing that, as for BLMs, CER did not form vesicles in its pure state, in agreement with the mean value of the critical packing parameter [23], which was calculated to be 1.17. Vice versa, we could prepare vesicles using CER mixed with DOPC, which is able to form such aggregates alone. DOPC and DOPC/CER vesicles were stable for about 20 days. The presence of CER within the bilayer was deduced using the ESR technique, and the results obtained agreed with the interpretation discussed above; these results will be discussed in a future paper. CONCLUSIONS

Our paper confirms that a correspondence between monolayers and bilayers of a substance or of a mixture of substances can be found. In fact we found that: (1) The possibility of obtaining bilayers is closely connected to the phase of the single or mixed monolayer at the air/water interface. Therefore, to obtain fluid bilayers, such as BLMs and vesicles, it is necessary to use substances that form liquid monolayers or, when this condition is not possible, to add quantities of substances that spontaneously form such aggregates. (2) Since fluid bilayers were obtained under our experimental conditions [ 24,251, there was a net correspondence between fluid bilayers and fluid monolayers at the same temperatures. (3 ) As a direct consequence of points (1) and (2 ) , we could hypothesize that

145

the hydrophobic chains of the substances studied here have the same orientation both in the monolayer and in the bilayer; the effect of different interfaces is then negligible with respect to the chain-chain interaction. Thus the monolayer at the air/water interface has properties similar to those of the monolayers composing the bilayers. The latter, at least for our study, can be considered as overlapping monolayers. ACKNOWLEDGEMENTS

The authors wish to express their gratitude to Dott. G. Pratesi for writing the program for the ellipsometer. Financial support of this work by the C.N.R. (PROGETTO 10, Sottoprogetto 2.) and by the M.P.I. is gratefully acknowledged.

REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

18 19 20 21 22 23 24 25

J.H. Fendler, Membrane Mimetic Chemistry, J. Wiley and Sons, London, 1982. D.A. Cadenhead, Structures and Properties of Cell Membranes, CRC Press, Boca Raton, FL, 1985, Chap. 2. A. Gliozziin R. Rolandi, in G. Colombetti and F. Lenci (Eds), Membranes and Sensory Transduction, Plenum Press, New York, 1984, Chap. 1. G.G. Roberts, Adv. Phys., 34 (1975) 475. F. Bonosi, G. Gabrielli, G. Martini and M.F. Ottaviani, Langmuir, 5 (1989) 1037. J.T. Davies and E.K. Rideal, Interfacial Phenomena, Academic Press, New York, 1963. F.L. McCrackin, E. Passaglia, R.R. Stromberg and H.L. Steimberg, J. Res., 67A(4) (1963) 363. F.L. McCrackin and J.P. Colson, Natl. Bur. Stand. Misc. Publ., 256 (1964) 61. P. Mueller, D.D. Rudin, H.T. Tien and W.C. Wescott, Circulation, 26 (1962). W.D. Harkins, The Physical Chemistry of Surface Films, Reinhold, New York, 1952. A. Niccolai, P. Baglioni, L. Dei and G. Gabrielli, Colloid Polym. Sci., 267 (1989) 262. G. Gabrielli, P. Lo Nostro and A. Niccolai, Colloids Surfaces, 39 (1989) 335. G.D. Fidelio, B. Maggio and F.A. Cumar, Biochim. Biophys. Acta, 854 (1986) 231. D.J. Crisp, Surface Chemistry Suppl. Research London, (1949) 17. K.J. Bacon and G.T. Barnes, J. Colloid Interface Sci., 67 (1) (1978) 70. G. Gabrielli, A. Niccolai and L. Dei, Colloid Polym. Sci., 264 (1986) 972. 0. Alvarez and R. Latorre, Biophys. J., 21 (1978) 1. J. Requena and D.A. Haydon, Proc. R. Sot. London, Ser. A, 47 (1975) 161. S. Marcelja, BBA, 367 (1974) 165. M.C. Phillips and D. Chapman, Biochem. Bbiophys. Acta, 163 (1968) 301. J.F. Nagle, Faraday Discuss. Chem. Sot., 81 (1986) 000. F. Gambale, M. Robello, C. Usai and C. Marchetti, Biochem. Biopphys. Acta, 693 (1982) 165. B. Maggio, BBA, 815 (1985) 245. R.E. Pagano, R.J. Cherry and D. Chapman, Science, 81 (1973) 557. R. Lawaczeck, M. Kainosho and S.I. Chan, Biochem. Biophys. Acta, 443 (1976) 313.