Langmuir 1989,5,111-113
further improvements in amphiphile design can be expected to significantly increase the number of monolayers which can be successfully adsorbed. We note also that new reactions useful for the self-assembly of multilavers can be expected to be discovered, of which the recerk report of a seven-layer film linked by zirconium-phosphonate bonds is a recent and creative example.19 A still unclear issue is to what degree multilayer formation will remain compatible with incorporation of diverse functionalities into-the structure of the film, and we are continuing to explore this problem. From the results presented here it
111
appears, however, that self-assembly represents a viable alternative to the Langmuir-Blodgett technique for the construction of ordered, multilayer films of thicknesses amroaching -- 0.1 um. Acknowledgment. We acknowledge the contributions of vita DePalma, for obtaining optical microscopy results, and of Ravi Sharma, for stimulating discussions, both of EivemifiedTecholot$es Research Group, h t m m Kodak Lo-
Registry No. 1,103946-41-4; L m ,16853-85-3; Si, 7440-21-3.
Association between Amphiphilic Cyclodextrins and Cholesterol in Mixed Insoluble Monolayers at the Air-Water Interface Svetla Taneva,? Katsuhiko Ariga, and Yoshio Okahata* Department of Polymer Chemistry, Tokyo Institute of Technology, Ookayama, Meguro-ku, Tokyo 152, Japan
Waichiro Tagaki Department of Applied Chemistry, Osaka City University, Sugimoto, Sumiyoshi-ku, Osaka 558, Japan Received July 19, 1988
Two-component insoluble monolayers of amphiphilic cyclodextrins (a-, 0-, r-CleCDs) and cholesterol (CH) were studied at the air-water interface. Surface pressure vs area measurements were carried out at various compositionsof the mixtures. Negative deviations of the mean molecular area from the additivity rule are observed in the three binary monolayers (a-C16CD/CH,P-C16CD/CH,and r-C16CD/CH). Both calculations and monolayer data showed that cholesterol can be included into only the r-CleCD cavity. In the cases of mixed a-or @-Cl6CD/CHmonolayers, cholesterol seems to be accommodated in the alkyl chain region of amphiphilic cyclodextrins.
Introduction Cyclodextrins (CDs) are unique molecules capable of forming inclusion complexes with molecules of an appropriate size in their apolar cavities. Because CDs are able to encapsulate guest molecules and thus modify their physicochemical properties, the practical application of CDs in the pharmaceutical and food industries has gained in importance.'I2 Recently, the interaction between cyclodextrins and biological membrane components such as steroids and phospholipids has been studied in connection with the hemolysis of human erythrocytes. For example, a high concentration of CDs has been reported to hemolyze human erythrocytes in the order 0- > a- > y-CD,s*4and this effect has been attributed to the removal of membrane components by CDs. Inconsistent data, however, have been reported by other workers concerning the interaction of CDs with steroids in aqueous solutions: formations of a stable inclusion complex between a-CD and cholesterol with a molar ratio of almost 1:16 and no interactions of a-CD with steroid molecules.6 p- and y-CDs have been proposed to include a part of steroid molecules in their cavities.w A qualitative monolayer study has showed that 'On leave from the Department of Physical Chemistry, Faculty of Chemistry, University of Sofia, Anton Ivanov 1,1126 Sofia, Bul; garia.
the surface pressure of a cholesterol monolayer decreases when CDs are dissolved in the subphase in the order 8> y- > a!-CDmg In the present work, the monolayer method has been employed to investigate the association between cyclodextrins and cholesterol by using mixed monolayer system of amphiphilic cyclodextrins (a-, b-, and r-C,,CDs) and cholesterol (CH). Formation of stable insoluble monolayers by amphiphilic @-cyclodextrinshaving seven long alkyl chains'O and deposition of Langmuir-Blodgett films (1) Saenger, W. Angew. Chem., Int. Ed. Engl. 1980,19, 344. ( 2 ) Szejtly,J. Cyclodextn'na and Their Inclusion Complexes; Akademia Kiado: Budapest, 1982; Chapter 5. (3) hie, T.;Otagiri, M.; Sunada, M.; Uekama, K.; Ohtani, Y.; Yamada, Y.; Sugiyama, Y. J. Pharmacobio-Dyn. 1982,5, 741, (4) Szejtli, J.; Czerhati, T.; Szogyi, M. Carbohydr. Polym. 1986,6,35. ( 5 ) Hammami, M.; Maume, G.; Maume, B. Cell. Biol. Toxicol. 1986, 2, 41. (6) Kempfle, M.; Mueller, R.; Palluk, R. Freaeniwr. 2.Anal. Chem. 1984,317, 700. (7) Kempfle, M.; Mueller, R.; Palluk, R.; Winkler, H. Biochim. Biophys. Acta 1987,923,83. (8) Lach, J.; Pauli, W. J . Pharm. Sci. 1966,55, 32. (9) Miyajima, K.; Saitq H.; Nakagaki, M. J. Chem. SOC.Jpn. 1987, 306.
(10)Kawabata, Y.; Matsumoto, M.; Tanaka, M.; Takahaehi, H.; Irinatau, Y.; Tamura, s.; Tagaki, W.; Nakahara, H.;Fukuda, K. Chem. Lett. 1986, 1933.
0743-7463/89/2405-0111$01,50/00 1989 American Chemical Society
Taneva
112 Langmuir, Vol. 5,No. 1, 1989
l
A I nmholec-1 Figure 1. Surface pressure ( T ) vs molecular area (A) isotherms of (a) cholesterol and amphiphilic cyclodextrins (b) a-C16CD, (c) fi-C,&D, and (d) y C & D a t 20 "c.
Table I. Molecular Area of Amphiphilic Cyclodextrins cyclodextrin a-C&D @-C&D r-C&D
diameter/ nma cavity external 0.47-0.52 1.46 0.04 0.60-0.64 1.54 f 0.04 0.75-0.83 1.75 0.04
* *
Ao/nm2 A'/nm2 per alkyl molecule-' exptlb calcd' chaind 0.204 1.618 1.58-1.77 0.218 2,153 1.77-1.96 0.223 2.870 2.29-2.52
-b
05
X1
Figure 2. Mean molecular area ( A )vs cholesterol mole fraction (X,)in the mixed monolayers of ( 0 ) a-C16CD/CH, (0)fiC16CD/CH,and (A)y-C16CD[CH at the constant surface pressure of (a) 10 and (b) 42 mN m- .
the cholesterol monolayer is in good agreement with that obtained in the literat~re,'~ and the area per molecule of cholesterol extrapolated at zero surface pressure (A,) was As measured on unsubstituted ,CDs from CPK molecular modfound to be 0.399 nm2 molecule-l. els (ref 1). bThe area per molecule extrapolated at zero surface pressure. e Calculated from external diameters of unsubstituted The amphiphilic cyclodextrins formed expanded monCDs. dThe area per alkyl chain at 45-50 mN m-l, calculated by olayers at 20 "C. The obtained areas per molecule at zero dividing experimental values by number of alkyl chains per CD pressure (A,) for a-,p-, and -y-C16CDsare summarized in molecule. Table I. The A, value for the P-C16CDmonolayer (2.153 nm2) was very consistent with that reported for the pof host-guest complex between P-Cl2CDand azobenzene C12CDmonolayer (2.17 nm2).l0 The areas (A,) for a-,pderivatives" have been reported. and Y-C&DS corresponded well to the respective cross sectional areas calculated from the external diameters of unsubstituted cyclodextrins (see Table I). When the lateral compressibility (k = -(6A/6r)/A,JT was plotted against the molecular area for three C&D monolayers, the minima were observed at pressures between 45 and 50 mN m-l. The area occupied per alkyl chain (A? L H OH _In at these pressures was obtained by dividing molecular areas of C16CDscorresponding to the minimum compressibility by the number of alkyl chains per cyclodextrin molecule. n = 6 : O(-C16CD The values thus obtained (A'in Table I) were consistent n = 7 : (3-CI6CD with the hydrocarbon chain cross sectional area (0.20 nm2). n = 8 : Y-C~GCD This suggests that at the high pressure the alkyl chains of C&Ds are closely packed in the two-dimensional layer Experimental Section and further compression would result in the monolayer Preparations of amphiphilic cyclcdextrins (a-, j3-,and Y-C~~CDS) collapse. have been reported elsewhere.12 High-purity cholesterol was Mean Molecular Areas in Mixed Monolayers. Recommercially available (Nakarai Chemicals, Ltd., Tokyo). The lations between the mean molecular area (A)at a given binary monolayers were obtained by spreading of a premixed surface pressure and the cholesterol mole fraction (XI) in chloroform solution (1.0 mg mL-') of two components. The the mixed monolayers are shown in Figure 2. Dotted lines temperature of the subphase (Milli-Q water, pH 5.8) was kept in these figures represent the additivity rule of eq 1: constant at 20 f 0.5 OC. Ten minutes after spreading, the monolayer in gaseous state was continuously compressed. A A = XIA1 + X2A2 (1) Compressionalvelocity w a 0.88 ~ em2s-'. Below this value the effect of compression rates on the molecular area was within the experimental error. Wilhelmy's plate method (filter paper plate) and a Teflon-coated trough with a microprocessor-controlled film balance (San-&u Keisoku Ltd., Fukuoka, Japan) with a precision of 0.01 m N rn-l were used for surface pressure measurements.
Results r-A Isotherms in Single-Component Monolayers. Figure 1 shows surface pressure (a)- molecular area (A)
curves of the pure monolayer of cholesterol and a-,@-, and r-C16CDs,respectively. The compressional isotherm for
(11)Tanaka, M.; Ishizuka, Y.; Mataumoto, M.; Nakamura, T.; Yabe, A.; Nakaniahi, H.; Kawabata,Y.; Takahashi, H.; Tamura, S.; Tagaki, W.; Nakahara, H.; Fukuda, K. Chem. Lett. 1987,1307. (19)Takahashi, H.;Irinatau, Y.; Kozuka, S.; Tagaki, N. Mem. Fac. Eng., Osaka City Uniu. 1985,26,93.
where Al and A2 stand for the molecular area in the single-component monolayer of cholesterol and amphiphilic cyclodextrin, respectively. X1 and X 2 are the respective mole fraction in the mixed monolayers. If the cholesterol is completely inserted into a cyclodextrin cavity and the inclusion complex of 1:1molecular ratio is assumed to be formed, then cholesterol will not occupy any area in the mixed monolayers up to composition of X1 = 0.50, Le., Al = 0, and eq 1 is reduced to A = X2A2 (2) The mean molecular areas (A)thus calculated are shown by broken lines in Figure 2. At the surface pressure of 10 (13) Muller-Landau, F.; Cadenhead,D. Chem. Phys. Lipids 1979,25, 299.
Langmuir, Vol. 5, NO.1, 1989 113
Association between Cyclodextrins and Cholesterol At low pressures
At high pressures
0.21 I
00
I
05
x;
1.0
Figure 3. Mean molecular area per alkyl chain (23 va cholesterol in the mixed monolayers of ( 0 )a-ClsCD/CH, mole fraction (Xi) (0) @Cl&D/CH, and (A) yC&D/CH.
mN m-l (Figure 2a), negative deviations of experimental areas from the additivity line were observed in the three binary systems composed of cholesterol and a-,8-, and y-C16CDs,respectively. The maximal condensations correspond to the cholesterol mole fraction of 0.66,0.70,and 0.50 in the mixed monolayers of a-C16CD/CH, 8C16CD/CH,and y-C16CD/CH, respectively. In the cases of a-C16CD/CH and fl-C16CD/CHmonolayers, the condensing effect decreased with increasing surface pressure and disappeared at the high pressure (342 mN m-l), where the alkyl chains of C16CDswere closely packed (see Figure 2b). In the mixed y-C16CD/CH monolayers, the condensation was independent of the surface pressure and the experimental area coincided with the value calculated by eq 2. The latter did not hold for the a-and fi-Cl&DS/CH systerns, For a better understanding of the condensing effect observed in three mixed C16CD/CH monolayers, the results were examined on a “per alkyl chain” basis.13 The mean area per alkyl chain (A? in the binary monolayers were calculated in and the cholesterol mole fraction (Xl’) reference to the hydrocarbon chain concentration and plotted in Figure 3. The maximal condensing effect occurred a t X1’= 0.25, i.e., a t the alkyl chain/cholesterol ratio of 3:l in the a-and ,8-C16CD/CHmonolayers. For the y-C16CD/CH monolayers, the maximal condensation was observed at X1’= 0.11 (i.e., the 8:l alkyl chain/ cholesterol ratio), which corresponds to the 1:l molar ratio of y-Cl6CD/cho1esterol.
Discussion In general, two mechanisms of host-guest, complex formation are possible for the cyclodextrin molecules.218 Molecules which are size-compatible with the CD cavity could be completely inserted. In the case of the larger molecule, only a certain part or a side chain of the molecule may penetrate into the cyclodextrin cavity. In the present study, amphiphilic cyclodextrins (C16CDs)have an additional hydrophobic moiety of six to eight long alkyl chains. Hence, one more inclusion mechanism is possible in the monolayer system; namely, hydrophobic guest molecules could be accommodated by the region of alkyl chains attached to CD rings. If the cross sectional area of the cholesterol molecule (0.399nm2) is represented as a circle, its diameter is estimated to be 0.71 nm. The comparison of this value with the cyclodextrin cavity dimension (Table I) shows that cholesterol is size-compatible only with the internal cavity of y-cyclodextrin. This is supported by the monolayer data
Y-C&D Figure 4. Schematic illustration of the mixed monolayers of C16CDs and cholesterol.
that the complete inclusion occurs in the mixtures composed of y-C16CDand cholesterol. The condensing effect observed in the mixed monolayers of cholesterol and a- or /3-Cl&D could be interpreted in terms of geometrical accommodation of cholesterol by the intermolecular cavity formed by the cyclodextrin alkyl chains in fluid state. This “intermolecular cavity effect”14 has been observed in binary monolayers of cholesterol and or fatty acids16exhibiting an expanded phosph01ipids~~J~ state, and it always is accompanied by altered molecular interactions.16 A preferred packing ratio of 3:l alkyl chain/cholesterol has been reported, independent of the polar head group of the host lipid.13 Similar results have been obtained in the present work for the mixed monolayers of cholesterol with a-or 8-C16CDwhen the number of alkyl chains per C16CD molecule is taken into account (Figure 3). On the basis of the obtained monolayer data, a certain mechanism of the interaction between amphiphilic cyclodextrins and cholesterol in the mixed monolayers is assumed and shown in Figure 4. In the case of a- and /3-C16CDs, the cyclodextrin cavities are not large enough to completely incorporate cholesterol molecules, and cholesterol is accommodated into the intermolecular cavity formed by the alkyl chains at the lower pressure (T < 42 mN m-l). At the higher pressure, where the hydrocarbon chains are closely packed, each molecule of both species occupies its own area in the mixed monolayer. In the binary y-C&D/CH monolayers, the condensing effect can be attributed to the cholesterol accomodation into the intramoleclar cyclodextrin channels both at low and high pressures. In conclusion, both the calculations and the monolayer experiments performed with mixed monolayers of amphiphilic cyclodextrins and cholesterol indicate that the complete inclusion of the steroid molecule is possible only into the y-cyclodextrin cavity. This result is in accord with data reported about the association of cholesterol with unsubstituted cyclodextrins in an aqueous solution: no interactions with a a-CD6 and partial inclusions into a The data concerning y-cyclodextrin are 8-CD rather scarce. Our results may be related to the cyclodextrin-induced hemolysis of human erythrocyte^.^ Registry No. a-C16CD,116399-45-2;@-CleCD,117183-39-8; y-C&D, 116399-46-3; CH, 57-88-5. (14)Shah, D.; Schulman, J. Adu. Chem. Ser. 1968, &, 189. (15) Motomura, K.; Terazono, T.;Matuo, H.; Matuura, R. J. Colloid Interface Sci. 1976,57, 52. (16)Cadenhead, D.; Muller-Landau,F. J. Colloid Interface Sci. 1980, 78, 269.
