Langmuir 1991, 7, 318-322
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Interaction of Cyclosporin A with Dipalmitoylphosphatidylcholine at the Air/Water Interface Timothy S. Wiedmann' and Kimberly R. Jordan Department of Pharmaceutics, College of Pharmacy, University of Minnesota, Minneapolis, Minnesota 55455 Received February 9, 1990. I n Final Form: July 12, 1990 The surface pressure-surface area ( P A ) isotherms of cyclosporin A (CyA) and the whole range of mole fractions of CyA with dipalmitoylphosphatidylcholine (DPPC) have been determined. The isotherm of pure CyA rises gradually and undergoes a transition at about 15 dyn/cm. CyA forms nonideal films at low surface pressures based on analysis of the compressibility (sA/kT)as a function of surface pressure. The limiting area is calculated to be about 130 A2/molecule, which corresponds to a smaller area/amino acid than has been observed with larger, linear polypeptides. The attractive interactions were comparable to medium chain fatty acids spread at the air/water interface. The extent of hysteresis was measured by repeated compression and expansion of a monolayer of CyA. The equilibrium spreading pressure of CyA was found to be about 30 dyn/cm, which together with the observed hysteresis indicates that a combination of instability as well as molecular rearrangement occurs with monolayers of CyA above 15 dyn/cm. Comparison of the weighted average molecular areas of mixtures of CyA with DPPC reveals that deviations from simple additivity occur at both low and high mole fractions of CyA at a surface pressure below the liquid expanded to liquid condensed phase transition. The results suggest that the introduction of the second component causes a disruption in the association leading to the nonideal behavior. The additivity of the area/molecules at equal mole fractions of CyA may indicate the presence of a compensatory mechanism in the packing of the lipid and protein.
Introduction Cyclosporin A (CyA) is a hydrophobic polypeptide that has been shown to be very effective in the suppression of the immune response in patients who have undergone organ transplantation.' The circular, undecapeptide has also been shown to associate with plasma lipoproteins but the distribution in the lipoproteins and the energetics of the association have not been determined.2 To examine the nature of this interaction, the combination of CyA with model lipid bilayers has been studied in our laboratory3 as well as in others. Techniques that have been used include 31PNMR," 2H NMR,3 19FNMR,5 13C NMR,6 FTIR,7ESR* X-ray diffra~tion,~ and DSC.3p7 For phospholipids in the gel state, CyA causes a slight disordering in the average conformation of the carboncarbon bonds of the acyl chains, whereas for phospholipids in the liquid crystalline, a slight ordering effect is observed. However, few investigators have examined the effect of the lipid to protein ratio which is important for a better understanding of the interaction of small peptides with phospholipid^.^*^ One approach, which historically has proven successful for investigating lipid-protein interactions and may be easily extended for studying the whole range of lipid/
protein ratios, is analysis of the surface pressure-surface area ( s - A ) isotherms at the air/water interface.1°-19 With a known amount of lipid or protein carefully spread at the surface of an aqueous subphase, the resulting change in the surface pressure may be measured. Furthermore, the use of a Langmuir trough with a movable barrier conveniently allows quantification of the surface pressure as a function of surface concentration. Thus, the n-A isotherms of not only pure proteins but also proteins combined with lipids may be determined. The study of the surface properties of CyA has the added advantage of providing insight into the conformation of proteins at the air/water interface. Proteins possess a unique conformation in solution for purposes of physiological activity. However a t the surface, proteins are believed to unfold as a function of time to a variable extent depending inter alia on disulfide bonds.13-17 Furthermore, differences in the surface behavior may be observed depending on whether the protein is adsorbed to the surface from the bulk solution or spread at the surface.17 Ultimately, there is ambiguity as to the structure of the protein at the surface. The use of CyA, which is locked into a relatively rigid, cyclic structure, can provide quantitative information as to the effect of proteins on the surface properties. Thus the surface properties of cyclosporin A have been
* To whom corresoondence should be addressed. (1) De Bakey, M. E, Compr. Ther. 1984, 10,7-15. (2) Mraz, W.; Zink, R. A.; Graf, A.; Preis, D.; Illner, W. D.; Land, W.; Siebert, W.; Zottlein, H. Transplant. Proc. 1983, 15, 2426-2429.
(3) Wiedmann, T. S.; Trouard, T.; Shekar, S. C.; Polinkandritou, M.; Rahman, Y.-E. Biochim. Biophys. Acta 1990, 1023, 12-18. (4) Stuhne-Sekalec, L.; Stanacev, N. Z. Chem. Phys. Lipids 1988,48,
1-6. (5) Rossaro, L.; Dowd, S.; Ho, C.; Van Thiel, D. In The 13th International Conference on the Biological Applications of NMR, Madison, WI, 1988. (6) Green, P. M.; Mason, J. T.; O'Leary, T. J.; Levin, I. W. J . Phys. Chem. 1987, 91, 5099-5103.
(7) O'Leary, T. J.; Ross, P. D.; Lieber, M. R.; Levin, I. W. Biophys. J .
1986,49, 795-801. (8) Epand, R. M.; Epand, R. F.; McKenzie, R. C. J. Biol. Chem. 1987, 262, 1526-1529. (9) Polindandritou, M.; Rahman, Y.-E. Personal communication.
0743-7463/91/2407-0318$02.50/0
(10) Malcolm, B. R. In Progress in Surface and Membrane Science; Cadenhead D. A., Danielli, J. F., Rosenberg, M. D., Eds.; Academic Press: New York, 1973; Vol7, p 183-229. (11) Pearson, J. T. J. Colloid Interface Sci. 1968,27, 64-74. (12) Pearson, J. T.; Alexander, A. E. J . Colloid Interface Sci. 1968,27, 53-63. (13) Graham, D. E.; Phillips, M. C. J . Colloid Interface Sci. 1979, 70, 403-414. (14) Graham, D. E.; Phillips, M. C. J. Colloid Interface Sci. 1979, 70, 427-439. (15) Graham, D. E.; Phillips, M. C. J. Collid Interface Sci. 1979, 70, 415-126. (16) Adamson, A. W. In Physical Chemistry ofsurfaces, 4th ed.; John Wiley & Sons: New York, 1982. (17) MacRitchie, F. J . Colloid Interface Sci. 1981, 79, 461-464. (18) MacRitchie, F. ACS Symp. Ser. 1987, No. 343, 165-179. (19) MacRitchie, F. Adu. Protein Chem. 1978, 32, 283-323.
0 1991 American Chemical Society
Langmuir, Vol. 7, No. 2, 1991 319
Interaction of C y A with DPPC
assessed through measurement of the P A isotherm and equilibrium spreading pressure. Furthermore, since the interaction of CyA with lipids may be an integral factor in its action on the immune system and probably greatly influences the distribution in the body after intravenous administration,20the interaction of CyA with dipalmitoylphosphatidylcholine(DPPC) a t the air/water interface has been determined. Finally, the analysis of the phase behavior of CyA-DPPC mixture monolayers has been performed, which may lead to a better understanding of the effect of polypeptides on phospholipid monolayers.
50-
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4030-
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20-
d
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I
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Experimental Section The water used in the surface pressure measurements was passed through a carbon filter, followed by a reverse osmosis filter, and finally distilled from a basic permanganate solution in an all-glass apparatus. The dipalmitoylphosphatidylcholine was obtained from Avanti Polar Lipids. Since TLC failed to reveal any impurities, the lipid was used as received. Cyclosporin A (Sandoz) was also used as received because it was obtained in the highest purity available and eluted as a single peak with HPLC.*O The surface pressure-surface area isotherms were obtained with a KSV 5000 Langmuir-Blodgett instrument housed within a laminar flow hood. The rectangular trough (15 X 46 cm) was equipped with a circulating water bath, and the temperature was maintained a t 21.5 f 0.5 "C for all isotherms. Each isotherm was determined on a freshly poured subphase, which was checked for impurities by compression of the barrier while monitoring the surface pressure. Compression of the surface layer generally resulted in a rise of the surface pressure of about 0.1 dyn/cm, and a new subphase was poured if the level exceeded 0.4 dyn/cm. The lipid and lipid-peptide mixtures were prepared by dissolving DPPC and CyA in HPLC grade hexane/2-propanol (9:l) from which the mixtures were made by combining the appropriate volumes of each. Solutions were then spread at the surface, and 5-10 min was allowed to elapse for solvent evaporation before the start of the compression. A constant compression speed of 30 mm2/min was used. The equilibrium spreading pressure was determined in a double-walled, acid-washed glass beaker with a glass cover that was thermally controlled to the same precision stated as above. The equilibrium spreading surface pressure was measured by injecting a suspension of cyclosporine into the subphase in excess of the saturated solubility and monitoring the surface tension as a function of time until no further decrease was found (overnight). Essentially the same result was obtained by placing a few crystals a t the surface and waiting until equilibrium was achieved ( 1 4 h).
Results and Discussion Figure 1 shows the resulting surface pressure as a function of surface area per molecule for different mole fractions of CyA. The area per molecule refers to the total number of molecules, DPPC and CyA, spread at the surface. The isotherm obtained with pure DPPC (Figure 1g) is in excellent agreement with previously published data.2Q2 The "kink" in the x-A isotherm, which has been identified as the liquid expanded to liquid condensed (LELC) phase transition, occurs at a pressure of 5 dyn/cm, which is in good agreement with the work of Pallas and Pethica.22 The transition region is not flat, but rather the pressure rises gradually with a reduction in area/molecule due to the use of continuous compression rather than the spreading of discrete aliquots of DPPC a t the surface.22 (20) Gruber, S. A.; Venkataram, S.;Rahman, Y.-E. Pharm. Res. 1989, 6, 601-607.
(21) Albrecht, 0.;Gruler, H.; Sackmann, E. J.Phys. (Paris) 1978,39, 301-313. (22) Pallas, N. R.; Pethica, B. A. Langmuir 1985, 1, 509-513.
Figure 1. Surface pressure-surface area isotherms a t 21.5 "C on distilled water, pH 4.5,of mixtures of cyclosporin A with DPPC a t mole fractions of DPPC of (a) 0, (b) 0.13, (c) 0.2, (d) 0.4, (e) 0.6, (f) 0.8, and (9) 1.0.
In the liquid condensed region, the pressure rises sharply with a limiting area of 41 A2/molecule. In Figure la, the x-A isotherm of pure CyA is given. CyA has several desirable characteristics for a study of the interaction with lipids at the air/water interface including the fact that it is readily soluble in hexane/2propanol solutions and may be mixed with lipids prior to spreading. A detectable pressure is found to occur at areas of about 200 A2/molecule. The surface pressure rises more gradually than is seen with DPPC with a reduction in area. At a surface pressure of 15 dyn/cm corresponding to a surface area of 125 A2/molecule, the CyA monolayer undergoes a transition. Thereafter, the pressure changes much more slowly with changes in area/molecule yielding a plateau. Furthermore, the monolayer becomes relatively more sensitive to the rate of compression. That is, the surface pressure decreases if the compression is stopped. This does not occur with pressures below 15dyn/cm. Thus, Cya forms a kinetically, if not thermodynamically, stable monolayer a t surface pressures below 15 dyn/cm. The nature of the monolayer of CyA may be examined more carefully by a plot of the product of the surface pressure and surface area as a function of the surface pressure as shown in Figure 2.23 A linear plot was obtained at pressures below 3 dyn/cm. From least-squares linear regression, the line was xA = 113 + 135(7r). This analysis consists of a two-dimensional analogue of the threedimensional compressibility factor. In the limit of zero pressure, the intercept should be equal to kT after the appropriate corrections for the different units of length used in the surface pressure and surface area. This value is 4.07 X 10-14erg/molecule, which is considerably higher than that found for the extrapolated data of 1.13 X erg/molecule. This indicates that there are attractive forces present among the CyA molecules that lead to the observed negative deviations from ideality. The magnitude of the attractive interaction of CyA molecules is remarkably similar to that seen with medium chain alcohols.23 Specifically, the attractive forces among CyA molecules are comparable to decanol, although the surface balance was not sufficiently sensitive to characterize the shape of the curve a t very low surface pressures. Other investigators have used the limiting intercept as a means of determining the molecular weight and degree of aggregation of proteins.24 Although extreme care must be used in such an approach, the observation that the (23) Adam, N. K. In The Physics and Chemistry of Surfaces, 3rd ed.; Oxford University Press: London, 1941. (24) Mishuck, E.; Eirich, F. In Monomolecular Layers; American Association for the Advancement of Science: Washington, DC, 1954; pp 14-32.
Wiedmann and Jordan
320 Langmuir, Vol, 7, No, 2, 1991
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Figure 3. Hysteresis loops of a spread monolayer of cyclosporin A. The arrows indicate the direction of the barrier and the associated numbers indicate the order in which the loops were obtained. bonds which are important in the crystalline structure and also in nonpolar solvents. At the surface, CyA could 0 1 2 3 assume one of the above or even a different conformation. To a first approximation, CyA may be considered to lay Surface Pressure (Dydcm) with its ring of amino acids parallel to the surface. The Figure 2. Product of the surface pressure and surface area ((dyn/ nitrogen and oxygen atoms in the amide groups would be cm)(A2/molecule))given as a function of surface pressure (dyn/ expected to orient in a manner to maximize hydrogen cm) for a monolayer of cyclosporin A. The dotted line represents bonding to the aqueous subphase whereas the hydrophobic the results from a least-squaresbest fit,and the solid line indicates side chains would orient away from the subphase. The the behavior of an ideal monolayer. side group of MeBmt has greater flexibility due to its greater length and also has greater hydrogen bonding limiting intercept is much lower than the ideal limiting capability due to the presence of the hydroxy group. The intercept suggests that association is occurring among the remaining amino acids are small and hydrophobic inCyA molecules. The number in the aggregates would cluding four methylleucines, two alanines (one of the D appear to consist of trimers or quaternions, since the ratio conformer), and one each of valine, alanine, methylalaof the ideal intercept to the observed intercept is 3.6. nine, and sarcosine (N-methylglycine). Estimates of the In addition to the characterization of the attractive area/molecule for CyA in this conformation based on forces, the slope of the curve provides an indication of the molecular models are about 250 A2 depending on the repulsive forces encountered a t higher surface pressures.22 orientation of Bmt-1. For an ideal isotherm, the product of the surface pressure and surface area is independent of surface pressure; thus There are several other dimensions of CyA that have the resulting plot would have a slope of zero. From the been obtained experimentally. From X-ray measureleast-squares fit of CyA, a slope of 135 A2/molecule was ments, the probable dimensions are 13 X 14 X 24 A, with found. This corresponds to an estimate of the excluded a projected area of 312 A2.9For use in dynamic studies, area effect at large surface pressures. CyA has been modeled as a prolate ellipsoid of evolution A great deal of information is known concerning the with major and minor axes of 10 and 23 A; the projected actual molecular conformation of CyA, which has been area would be 230 A2.28 The point of measurable surface shown to depend on the solvent and physical form.25126 pressure occurs a t the lower end of these estimates, which X-ray crystallography indicates that amino acids two indicates that close packing must occur before appreciable through six form a @-sheetconformation conveying conreduction in surface tension is seen. siderable rigidity to the structure. The amino acid in the Measurements of CyA at higher surface pressures also one position is unusual and consists of a 2-(S)-(methyldisplay interesting features. Since, the equilibrium spreadamino)-3-(R)-hydroxy-4-(R)-methyl-oct-6-en-l-oic acid. ing pressure was found to be about 30 dyn/cm2,the results This side chain is believed to fold into the ply of the obtained with the continuous compression above 15 dyn/ @-pleatedsheet. The remaining amino acids serve as a cm2 require additional consideration. The fact that the loop, but a hydrogen bond exists between the sixth and surface pressure decreases when the barrier is no longer eighth amino acids. A similar structure is seen in noncompressing the film indicates that a nonequilibrium state polar solvents; however, the MeBmt-1 side chain appears exists at surface pressures greater than 15 dyn/cm. Under to form a intramolecular hydrogen bond with the carcontinuous compression either the film is elastically overbonyl 0 atom of MeBmt itself to replace the intermoleccompressed or CyA is lost to the subphase. Both would ular hydrogen bond found in the crystal structure. This cause a reduction in the area/molecule. Talc spread causes the MeBmt to form a proboscis-like structure which behind the barrier failed to show any leakage occurring is known to be important for its immunosuppressent underneath the barrier. activity.27 In Figure 3, the results obtained from recycling the CyA As opposed to the solid-state or nonpolar solvents, many film at the interface are given. The monolayer was different conformations are present in polar ~ o l v e n t s , ~ ~compressed ~~~ to 30 dyn/cm and then reexpanded a t the no doubt in part due to the disruption of the hydrogen same rate of compression. After complete expansion, the film was again compressed and expanded. With both (25) Kessler, H.; Loosli, H. R.; Oschkinat, H. Helu. Chim. Acta 1985, cycles, large hysteresis loops are observed. In addition, 68,661-681. (26) Loosli, H.R.;Kessler, H.; Oschkinat, H.; Weber, H. P.; Petcher, T.J.; Widmer, A. Helu. Chim. Acta 1985,68,682-703. (27) Hess, A. D.; Colombani, P. M. h o g . Allergy 1986, 38, 198-221.
(28) Dellwo, M.J.; Wand, A. J. J.Am. Chem. SOC.1989, 111, 46714578.
Interaction of C y A with DPPC the second loop is shifted to smaller molecular areas relative to the first loop. The latter observation suggests that compression to high surface pressures causes an irreversible loss of CyA molecules to the subphase. The irreversible loss of molecules, however, is insufficient to account for the length of the plateau region seen with pressures between 15 and 30 dyn/cm.29 One possibility in light of the above information concerning the structure of CyA is that it undergoes a change in orientation a t the surface. That is at 15 dyn/ cm, which corresponds to an area of 125 A2/molecule,CyA undergoes a transition from an orientation where the ring of the peptide chain lies parallel to the surface to one where it lifts off to a perpendicular orientation. By use of the above estimates of the dimensions from the X-ray and dynamic studies, the cross sectional area for CyA would be from 100 to 182 A2for its ring positioned perpendicular to the plane of the surface. This proposed change in orientation would reduce the area/molecule by about 100 A*/molecule. One the other hand, from the shape of the hysteresis loops, there is an indication that other processes may be more important. For example, CyA may be forced from the surface at surface pressures above 15 dyn/cm but retains sufficient attraction to the other surface molecules that it remains in the subphase. This would be consistent with the continuous increase in the pressure above 15dyn/ cm with compression. Above, CyA was shown to have significant intermolecular attractive interactions at the air/water interface. In addition, CyA is known to dimerize in the bulk solution4 and also appears to associate at the surface. Upon expansion of the area, CyA molecules that were “squeezed out” could return to the surface, thereby giving rise to a reversible loss of molecules at the surface with compression. Finally, given the strong association among CyA molecules, elastic overcompression may also occur. Comparison of CyA with other proteins is facilitated if the areas are corrected for differences in the number of amino acids. Therefore, on an aminoacid basis, the surface pressure of pure CyA becomes measurable a t about 18 A2/aminoacid. The transition occurs at 125 A2/molecule or equivalently a t 11.4 A2/amino acid. Other proteins such as albumin and trypsin typically have area/amino acids of 10-15 A2/amino acid.1° Malcomb’O suggested that proteins in an a-helix or &sheet would have areas of 15 A2/amino acid. Since CyA is small as well as cyclic, the conformational freedom is considerably smaller in comparison to these large, linear proteins, which would cause adecrease in the area/molecule. However, overlap of peptide chain is not possible, which would lead to an increase in the observed area/molecule. In addition, the high value of the equilibrium spreading pressure (ESP) is also noteworthy. Graham and Phillips15 found the ESP to lie in the range of 15-22 dyn/cm for six different proteins. The higher ESP seen with CyA may be a result of the relatively larger number of hydrophobic amino acids and the more restricted conformations available. Overall, CyA appears to have a slightly smaller area/ amino acid as found with other proteins, which may be a result of CyA undergoing considerable association at the surface as indicated above. The other aspect of consideration in this study is the interaction of CyA with DPPC. Returning to Figure 1, the a-A isotherms of a range of mixtures of DPPC with CyA are given. With increasing mole fractions of CyA, (29) Taneva, S.;Panaiotov, I.; Ter-Minassian-Saraga,L. Colloids Surf. 1984, 10, 101-111.
Langmuir, Vol. 7, No. 2, 1991 321
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Figure 4. Observed area/molecule (mean f standard deviation (n 2 3), AZ/molecule) given as a function of mole fraction of cyclosporin A with dipalmitoylphosphatidylcholine. The solid line represents the theoretic result for simple additivity of the
area/molecule.
the area per molecule a t which the surface pressure is detectable increases, reflecting the larger size of CyA relative to DPPC. There are no noticeable discontinuities or “kinks” in the a-A isotherms for any of the lipidpeptide mixtures below 15 dyn/cm2. From the T-A isotherms, the total area/total number of molecules at the surface for a surface pressure of 5 dyn/ cm was calculated. The resulting area/molecule as a function of mole fraction of CyA in DPPC is given in Figure 4. The results are given as the mean f standard deviation ( n L 3) with a line connecting the average area/molecule of pure DPPC and CyA. Thus, the line represents the expected area/molecules for the two extreme cases of ideal mixing or complete phase separation.I6 That is, the theoretical line of the area/molecule (A) is simply the sum of the areas occupied by each species at the surface A = x1A1 + xzA, where x is the mole fraction a t each component present a t the surface. At low and at high CyA mole fractions, the observed area/molecule exhibits significant positive deviations from ideal behavior. However, at the intermediate mole fractions, there does not appear to be any difference between the observed behavior and the simple additive relationship. One possible interpretation is that the aggregates of DPPC and CyA are disrupted by the presence of the other component. This induced disruption in turn increases the area/molecule occupied by the phospholipid molecule leading to the positive deviations. It is also interesting that the maximum deviation from the additive relationship occurs at a lipid to protein ratio where in principle each lipid could be a nearest neighbor to the protein. In the intermediate range, the extent of positive deviations is negligible. The additivity relationship would be fulfilled by ideal behavior or by phase separation. In the case of phase separation, the phase transitions seen with pure DPPC at the higher pressures should again be visible in the isotherm. This apparently does not occur, although there may be a pressure dependence on the extent of miscibility. The more likely explanation is that a t the intermediate mole fraction mixtures there is a compensation mechanism that leads to the additive relationship.
322 Langmuir, Vol, 7, No. 2, 1991 Clearly more studies examining the lipid to protein ratio are needed to fully characterize the interaction of CyA with phospholipids.
Conclusions CyclosporinA has been shown to form a kinetically stable monolayer at the air/water interface at pressures less than 15 dyn/cm. Analysis of the isotherm indicates that appreciable association may be occurring. Relatively little loss of CyA occurs from the surface when compressed to 30 dyn/cm, although substantial hysteresis of the monolayer is seen. This may be a result of a combination of molecular rearrangement. association. and /or elastic overcompression. MLtures of CyA with DPPC display an
Wiedmann and Jordan additive relationship a t intermediate protein to lipid ratios indicating a compensatory mechanism is in effect, whereas at low and high ratios, positive deviations are seen which may arise from disruption of the association of aggregates of CyA.
Acknowledgment. We thank the Center of Interfacial Engineering, University of Minnesota, for the use of the Langmuir-Blodgett instrument. The financial support of the American Lung Association is gratefully acknowledged. Discussionswith Dr. Y .-L. Chen during preparation of this manuscript are also appreciated as well as comments received from the reviewers. Registry No. CyA, 59865-13-3; DPPC, 2644-64-6.
