Influence of lysolecithin on surface properties of dipalmitoyl lecithin monolayer

Influence of Lysolecithin on Surface Properties of Dipalmitoyl Lecithin Monolayer P. GUTKOWSKI, J. HABER, AND J. P A W E L E K Institute o f Catalysis and Surface Chemistry, Polish Academy o f Sciences, Krakow, Poland, and National Institute o f Mother and Child, Rabka, Poland Received February 14, 1979; accepted February 27, 1980 Surface tension and surface potential vs surface concentration have been determined in static and dynamic conditions for dipalmitoyl lecithin (DPL) monolayers on water, albumin, human plasma, and lysolecithin solutions. In the dynamic system DPL always determines the properties of the film, but once the lysolecithin monolayer has been formed DPL modifies only its properties through the DPL/lysolecithin interface. INTRODUCTION

The behavior of dipalmitoyl lecithin (DPL) monolayers at the air-water interface is of particular interest because of its significance for understanding the role of pulmonary surfactant. Many studies have been devoted in recent years to the properties of these monolayers, the orientation of molecules, and interaction of their polar groups in the surface layer of the liquid phase (1-7). Of particular influence on the properties of the monolayer are the degree of hydration of the polar groups of DPL molecules and their interaction with ions (Na +, K +, Ca 2+) as well as with molecules of other substances present in the solution (8). The pulmonary surfactant is composed of several components, one of them being lysolecithin. Although its content is relatively small, lysolecithin plays a very important role in maintaining the low surface tension of the monolayer of the alveolar lining because of its similarity to DPL and solubility in water. Lysolecithin contains the same 1phosphatidylcholine structural group as DPL and differs from the latter only in that it has one palmitoyl acid chain instead of two, showing therefore higher polarity and considerable solubility in water. It thus

seemed of particular interest to study a model system composed of the monolayer of DPL spread on the surface of the water solution of lysolecithin at the subphase. It could have been expected that interactions between molecules of lysolecithin and DPL would strongly modify the properties of DPL films. EXPERIMENTAL

Materials

Synthetic L-oMecithin fl,T-dipalmitoyl (grade purity) was supplied by Calbiochem, San Diego, California. The monolayers were obtained by spreading the appropriate amount of the DPL solution in a mixture of methanol and chloroform (85:15) or chloroform and carbon tetrachloride (1:5) onto the surface of the investigated subphase. The following solutions were used: 0.9 wt% NaCl-water, solution of 10-4 M lysolecithin obtained using the 1-palmitoyl of lysolecithin produced by Koch-Light Labs Ltd., and solution of plasma obtained from human blood by centrifugation of erythrocytes. The concentration of 10-4 M lysolecithin corresponds approximately to its concentration in pulmonary surfactant.

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Journalof Colloidand Interface Science, Vol.78, No. 2, December1980

0021-9797/80/120289-06502.00/0 Copyright© 1980by AcademicPress, Inc. All rightsof reproductionin any formreserved.

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Methods Surface tension was m e a s u r e d by the Wilhelmy plate method. A platinum plate 3 × 0.5 × 0.01 cm, blasted by sanding, was used. The appropriate amount of the D P L solution was spread onto the given subphase and the surface tension was m e a s u r e d at r o o m t e m p e r a t u r e (296°K) after 20 min. In separate e x p e r i m e n t s it was found that 20 min after spreading of each portion of the D P L solution the surface tension attains the equilibrium value, which r e m a i n s constant even after 2 hr. All values given in the figures are the average of ten measurements. A c c u r a c y of the m e a s u r e m e n t of the surface tension was 0.1 dyn cm -1. The surface potential was m e a s u r e d with a radioactive plutonium 239pu air electrode against the 0.1 calomel electrode i m m e r s e d in the solution (9). The potential difference was determined by a L i n d e m a n n electrometer with an a c c u r a c y of 5 mV. All measurements were carried out after 20 min. E x p e r i m e n t s in the dynamic systems were carried out with the help of a Wilhelmy balance equipped with a sweep device, which enabled the m o n o l a y e r to be subJournal of Colloid and Interface Science, Vol. 78, N o . 2, D e c e m b e r 1980

jected to consecutive cycles of compression and expansion. The duration of one cycle was 25 sec. The area range for all m e a s u r e m e n t s was 100-20 cm 2. RESULTS Figure 1 shows the surface tension (curve 1) and surface potential (curve 2) of the solutions of lysolecithin in 0.9% NaC1 as a function of its concentration. On increasing the concentration the surface tension decreases and the surface potential rises, both attaining a constant level at the concentration of about 2 × 10 -5 M, which m a y thus be taken as the critical micellization concentration. When the surface tension of the solution of lysolecithin was m e a s u r e d as a function of time, no changes were found for concentrations higher than the CMC. Figure 2 illustrates the surface tension as a function of the concentration of D P L molecules in the m o n o l a y e r (expressed in square angstroms per molecule) spread o v e r the surface of 0.9% NaC1, albumin, h u m a n plasma, and 10-4 M lysolecithin aqueous solutions. Figure 3 shows the corresponding changes of the surface potential.

SURFACE PROPERTIES OF DPL MONOLAYER

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FIG. 2. Surface tension as a function of the surface concentration of DPL molecules in the monolayer on aqueous solutions of: (1) 0.9% NaC1; (2) 10-4 M lysolecithin; (3) albumin; (4) human plasma.

R e s u l t s o f the m e a s u r e m e n t s o f the c h a n g e s o f surface t e n s i o n in the c o u r s e o f c o m p r e s s i o n and s u b s e q u e n t e x p a n s i o n are s h o w n in Fig. 4 for the film o f D P L spread on distilled w a t e r and 0.9% NaC1 s o l u t i o n , and in Fig. 5 for 10 -5 and 10 -4 M s o l u t i o n s

o f l y s o l e c i t h i n as w e l l as f o r the films o f D P L w i t h t h e s e s o l u t i o n s as the s u b p h a s e . DISCUSSION R e s u l t s o f the e x p e r i m e n t s s h o w n in Figs. 4 and 5 m a y be interpreted b y a s s u m i n g that

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GUTKOWSKI, HABER, AND PAWELEK

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100

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FIG. 4. Surfacetension as a function of the area of DPL film on: (1) distilled water; (2) 0.9% NaCI aqueous solution. the DPL molecules in the monolayer interact with each other forming surface micelles. The interactions operate between the long hydrocarbon chains of the palmitoyl groups and apparently are hindered by the hydration of the polar groups of phosphatidylcholine, resulting in the increase of the distance between the interacting chains. Thus the aggregation of surface micelles on compression depends on the degree of hydration of polar groups. The presence of an electrolyte in the subphase decreases the degree of hydration of these polar groups and increases the interaction between DPL molecules. On the surface of distilled water the interactions between compressed micelles are only weak and they disintegrate easily on expansion, the compression and expansion branches of the surface tension vs concentration curve lying therefore not far apart and the hysteresis loop being small. When, however, Na ÷ and CI- ions are present in the solution, surface micelles become stabilized and their aggregates formed on compression are not so easily dispersed Journal of Colloid and Interface Science, Vol. 78, No. 2, December 1980

on expansion. A larger hysteresis loop is observed. Comparison of the changes of surface tension with those of surface potential, observed on increasing the surface concentration of DPL on 0.9% NaC1 solution in static conditions (curves 1 in Figs. 2 and 3), indicates that in the expression for surface potential, AV = 47rN/x, both the number of dipoles N and the dipole moment /x must change. This may be easily understood if one remembers the micellar noncontinuous structure of the DPL film. The change of surface potential observed on addition of the first portions of DPL is apparently due to the formation of the first small micelles at the surface. After attaining the surface concentration of about 60/~2 per DPL molecule, which is not much more than the area taken up by one molecule, a sudden large increase of the surface potential is observed, indicating that rearrangement of the orientation of molecules has taken place, probably related to the formation of a continuous film. Thus,

293

S U R F A C E P R O P E R T I E S OF DPL M O N O L A Y E R

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further increase of the surface concentration of DPL does not result in any further change of the surface potential. Only at very high concentrations of DPL can the reorientation of the multilayer structure, separation from the monolayer, or collapse take place, and the increase of the surface potential is again observed. Contrasting behavior is observed when DPL molecules are spread over a surface of the lysolecithin solution (cf; curves 2 in Figs. 2 and 3). After a rapid drop of the surface tension on addition of the first por~ tions of DPL (from the value of 42 N/m x 10-.3 characteristic for the CMC of lysolecithin to the value of 32.5 N/m × 10-3) it remains constant independently of the concentration of DPL, being at its high concentration much greater than that observed in the case of DPL monolayer on water (cf. curve 1). These observations may be explained in two ways: (a) the formation of mixed DPL-lysolecithin films takes place, due to penetration of DPL molecules into empty surface sites

between the lysolecithin molecules, DPL influencing the surface properties only to the extent permitted by these sites; (b) no penetration of DPL molecules into the Iysolecithin monolayers takes place, DPL forming a second film on top of the lysolecithin monolayer and influencing the surface properties through the D P L lysolecithin interface. Results of the measurements of surface potential permit discrimination between these two poss~ilities. The surface potential (Fig, 3, curve 2) remains at first constant, but after attaining the concentration of about 55 •2/molecule jumps rapidly to a new higher value, which then shows only a small steady increase. The fact that on increasing the amount of DPL a sudden increase of surface potential is observed at the same concentration, whereat a jump of potential occurs in the case of spreading DPL over the surface of water (cf. curve 1), seems to indicate that DPL forms a second film on top of lysolecithin. Further support of this conclusion is lent by the reJournal of Colloid and Interface Science, Vol. 78, No. 2, December 1980

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GUTKOWSKI, HABER, AND PAWELEK

sults of dynamic experiments shown in Fig. 5. As seen, the final surface tension attained after compression of DPL on the 10-4 M solution of lysolecithin, and the surface concentration, whereat it is observed are identical to those registered in the case of DPL on the surface of 0.9% NaC1 solution. This indicates that in both cases the surface properties are totally determined by the monolayer of DPL, which apparently has suppressed the more soluble lysolecithin from the surface into the bulk of the solution. The main difference between the dynamic and the static experiments with the DPL on lysolecithin solution consists in the fact that in the dynamic experiment both DPL and lysolecithin are present at the free surface of the solution from the beginning of the experiment, the increase of the concentration being attained by decrease of the surface, whereas in the static experiment DPL is introduced on the surface of the solution covered by the saturated monolayer of lysolecithin. By virtue of the same argument it may be concluded that in the static experiment case DPL molecules do not penetrate into the monolayer of lysolecithin but spread over it in the form of a second layer. The sudden increase of the measured surface potential may be due to the reorientation of the DPL molecules when they approach the monolayer coverage on top of the lysolecithin layer and its further slow rise to the contribution from the potential at the DPL/ lysolecithin interface, whereas the decrease of the surface tension may result from the

Journal of Colloid and Interface Science, Vol. 78, No. 2, December 1980

contraction of the lysolecithin monolayer under the influence of DPL palmitoyl chains inserted in between the less densely packed single palmitoyl chains of lysolecithin. At variance with the behavior of the D P L lysolecithin system, DPL spread over the surface of solutions of human plasma or albumin suppresses them from the surface and at higher concentrations completely determines the surface properties. It may thus be concluded that the presence of lysolecithin may strongly influence the surface properties of the DPL monolayer, but this influence depends on the ways in which both types of molecules are introduced on the surface. REFERENCES 1. Abramson, M. B., "Lipids in Water, The Chemistry of Biosurfaces," Vol. 1, Dekker, New York, 1971. 2. Clements, I. A., in "Respiratory Distress Syndrome" (C. A. Villee, D. B. Villee, and J. Zuckerman, Eds.). Academic Press, New York/ London, 1973. 3. Colacicco, G., Buckeler, A. R., and Scarpelli, E. M., J. Colloid Interface Sci. 46, 147 (1974). 4. Pawe~ek, J., Electrochim. Acta 9, 531 (1964). 5. Chatelain, P., Berliner, C., Ruysschaert, J. M., and Jaffe, J., J. Colloid Interface Sci. 51, 239 (1975). 6. Phillips, H. C., Progr. Surface Membr. Sci. 5, 139 (1972). 7. Pethica, B. A., Mingius, J., and Taylor, J. A. G. J. Colloid Interface Sci. 55, 2 (1976). 8. Hauser, H.,J. Colloidlnterface Sci. 55, 85 (1976). 9. Pawetek, J., Gutkowski, P., Glanowska, H., and Hanicka, M., "Lung Lipid Metabolism and Alveolar Surfactant, Proceedings of International Symposium, Varna, Bulgaria," 196 (1976).