Association of amphiphilic cyclodextrins with dipalmitoylphosphatidylcholine in mixed insoluble monolayers at the air-water interface

Association of Amphiphilic Cyclodextrins with Dipalmitoylphosphatidylcholine in Mixed Insoluble Monolayers at the Air-Water Interface SVETLA TANEVA, *'1 KATSUHIKO ARIGA,* WAICHIRO TAGAKI,t AND YOSHIO OKAHATA *'2 * Department of Polymer Chemistry, Tokyo Institute of Technology, Ookayama, Meguro-ku, Tokyo 152, Japan, and t Department of Applied Chemistry, Osaka City University, Sugimoto, Sumiyoshi-ku, Osaka 558, Japan Received September 12, 1988; accepted November 22, 1988 Surface pressure vs molecular area curves of binary spread monolayers of amphiphilic cyclodextrin (~-, {~-,or "{-C16CD) and dipalmitoylphosphatidylcholine (DPPC) were measured at various compositions. Reduction in the mean molecular area was observed in three C~rCD/DPPC monolayer systems with a maximal effect corresponding to monolayer mixture where the DPPC mole fraction was 0.78 _+ 0.02. A model of molecular packing of C16CD and DPPC in the two-dimensional layer was employed and showed that at the above composition the contact between molecules of phospholipid and amphiphilic cyclodextrin is realized to the greatest extent. Thus, the reduction in the mean molecular area in the mixed CI6CD/DPPC monolayers could be attributed to enhanced molecular attraction between two components rather than to inclusion complex formation. © 1989AcademicPress,Inc. 1. INTRODUCTION

miristoylphosphatidylcholine (DMPC) and decrease the surface pressure in the order aRecently, the interaction between cyclo>/3> y-CD (4). The same authors isolated dextrins (CDs) and biological membrane the inclusion complexes when aqueous solucomponents such as steroids and phospholiptions of aor 7-cyclodextrin and small uniids has been studied in relation to the hemolamellar vesicles from diacylphosphatidylcholysis of human erythrocytes ( 1). For example, a high concentration of CDs has been reported line had been mixed (5). On the contrary, no to hemolyze human erythrocytes in the order interactions have been observed between water /3- > a- > y-CD and this effect has been at- dispersions or liposomes of cell-membrane tributed to the removal of membrane com- lipids and cyclodextrins (3). In the present work, the monolayer method ponents by CDs (2, 3). has been employed to investigate the associThe interaction between monolayers of phospholipid and cholesterol with cyclodex- ation between cyclodextrins and phospholipids trins dissolved in the aqueous subphase has by using mixed monolayer systems of newly been studied: the surface pressure of the cho- synthesized amphiphilic cyclodextrins (a-, lesterol monolayer decreases when CDs are fl-, and T - C I 6 C D , see Fig. 1) and dipalmipresent in the subphase in the order ~3- > y- toylphosphatidylcholine (DPPC). We have > a-CD (4). Cyclodextrins in the aqueous already reported the interaction between amphase can interact with the monolayer of di- phiphilic C16CDs with cholesterol in the mixed monolayer and the results have shown that the cholesterol molecule is completely inl On leave from the Department of Physical Chemistry, cluded in a 3,-cyclodextrin cavity, whereas it Faculty of Chemistry, University of Sofia, Anton Ivanov is accommodated with alkyl chain regions of 1, 1126 Sofia, Bulgaria. 2 To whom correspondence should be addressed. a-CI6CD and f l - C l 6 C D ( 6 ) . Formation of sta561 0021-9797/89 $3.00 Journal of Colloid and Interface Science, Vol. 131, No. 2, September 1989

Copyright © 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

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C154H301028N7 " 5H20: C, 66.38; H, 11.14; N, 3.52. Found: C, 65.83; H, 11,15; N, 3.49. Five moles of water was determined by Karl Fisher analysis, o~-C16CD and "y-C16CD were also prepared by the same procedures from a- and 3,-cyclodextrins, respectively: a-CI6CD , mp 205°C (dec.), Anal. Calcd for C148H291023N7 n = 6 : (x-C16CD × 4H20: C, 68.18; H, 11.48; N, 3.76. Found: n = 7 : ~-q6CD C, 68.02; H, 11.40; N, 3.80. 'y-C16CD, mp n = 8 : %'-C16CD 195°C (dec.), Anal. Calcd for C160H311033N7 FIG. 1. Structuralformulaofamphiphiliccyclodextrins × 5H20: C, 65.13; H, 10.92; N, 3.32. Found: (CI6CDs). C, 65.22; H, 11.02; N, 3.31. Commercially available L-a-DPPC (Nihon Yushi Co. Ltd., Tokyo) was used. Chloroform ble monolayers from amphiphilic /3-cyclo- of a spectrophotometric grade was employed dextrins (fl-C12CD) and deposition of Lang- as a spreading solvent. The binary monolayers muir-Blodgett films of host-guest complex were obtained by spreading of a premixed between/3-C~2CD and cis- or trans-azobenzene chloroform solution ( 1.0 mg m1-1 ) of C]6CDs derivatives have been studied (7). and DPPC. The temperature of the subphase (Milli-Q water, pH 5.8 ) was kept constant at 2. EXPERIMENTAL 20 + 0.5°C. Ten minutes after spreading, the Amphiphilic cyclodextrins, hexakis(6- monolayer in gaseous state was continuously hexadecylamino-6-deoxy)-eyclodextrin ( a- compressed. A compressional velocity was C16CD), heptakis (6-hexadecylamino-6-de- 0.88 cm 2 s-L Below this value the effect of oxy)-cyclodextrin (fl-C16CD), and octakis(6- compression rates on the molecular area was hexadecylamino - 6 - deoxy) - cyclodextrin within the experimental error. Wilhelmy's (3'-CI6CD) were prepared as follows (8). As plate method (filter paper plate) and a Teflona typical example, preparations of/3-C16CD coated trough with a microprocessor-conwere described below. Heptakis(6-bromo-6- trolled film balance (San-Esu Keisoku Ltd., deoxy)-/3-cyclodextrin was synthesized from Fukuoka, Japan) with a precision of 0.01 mN /3-cyclodextrin and methanesulfonyl bromide m -1 were used for surface pressure measurein dimethylformamide according to the liter- ments. ature (9). The bromo-derivative (5 g, 2.8 3. RESULTS mmole) was allowed to react with hexadecylamine (12 g, 49 mmole) in 50 ml of dimeth3.1. Surface Pressure (~r)-Area (A) ylformamide at 70°C for 72 h under stirring. Isotherms The reaction mixture was evaporated and crystallized by the addition of CH3CN (400 Figures 2-4 show surface pressure (Tr) vs ml). The powder obtained was neutralized area (A) curves for single-component monowith an aqueous solution of NaOH and pu- layers of the amphiphilic cyclodextrin (a-, rified with silica gel column chromatography /3-, and T-C16CD) or DPPC and their binary (solvent, CH3Cl:CH3OH = 5:1); yield 62%, mixtures at 20 + 0.5°C, where XDPpc denotes mp 195°C (dec.), ~H NMR (CDC13, ppm), the mole fraction of DPPC in the mixed 0.8 (21 H, CH3 of alkyl groups), 1.2 ( 196 H, monolayer. Isotherms of additional mixtures CH2 of alkyl groups minus CH2 NH), 2.0-3.1 were measured but not shown for the sake of (28 H, CH2 adjacent to NH), 3.1-3.6 clarity in the figures. The inspection of 7r-:~ (7 H, 7 H; CD C2-H, Ca-H), 3.6-4.2 (7 H, isotherms showed that the main phase tran7 H; CD C r H , NH). Anal. Calcd for sition of DPPC was completely eliminated at Journal of Colloid and Interface Science, Vol. 131, NO, 2, September 1989

563

MONOLAYER OF AMPHIPHILIC CYCLODEXTRIN

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FIG. 4. Surface pressure (Tr) vs area (¢]) curves of mixed "y-CI6CD/DPPC monolayers.

in the mixed monolayer.

the DPPC mole fraction XDpVC< 0.90. The collapse pressures (Trc) of each pure component and C~6CD/DPPC mixtures were determined from the lateral compressibility (k =-(6A/67r)/Ao)T vs area (A) curves. The collapse pressures were defined as pressures corresponding to the minima in k-A relations in the region of high pressures. In Fig. 5, collapse pressures (Try) are plotted as a function of the monolayer composition (XDPeC). Full lines in the figure were calculated using the concentration dependence of collapse pressure in the case of ideal surface mixtures ( 10, 11 ). Good correlations between the experimental and the theoretical values of collapse pressures were observed for c~-C]6CD/DPPC,/3-C16CD/ DPPC, and 3,-C16CD/DPPC mixed monolayers.

pressures of 2 and 10 m N m -1 and plotted against the DPPC mole fraction (XDPPC) in the mixed monolayer in Figs. 6a and 6b. Dotted lines in these figures represent the additivity rule of zJ = XCI6CDAC16CD q- XDPPCADPPC '

[1 ]

where AC,6CD and ADVPCstand for the molecular area in the single-component monolayer of Cl6CD and DPPC, respectively. Xc16cD and XDPPCare the respective mole fractions in the mixed monolayer. Negative deviations of the experimental areas from the additivity rule were observed at surface pressures below the main phase transition of DPPC monolayer, i.e., in the liquid expanded state (Fig. 6a). In three o~-,/3-, and "y-CI6CD/DPPCsystems the maximal condensation was reached at XDvpc = 0.78 + 0.02. When the plots -~-Xuvvc were

3.2. Mean Molecular Areas (A) in Mixed Monolayers The m e a n area per molecule (A) was determined from the 7r-A isotherms at surface

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FIG. 5. Collapse pressure (7rc) as a function of the composition in mixed a-CI6CD/DPPC (O), fl-Ct6CD/DPPC (©), and 3,-C~6CD/DPPC (A) monolayers. Full lines represent the theoretical values in the case of ideal surface mixtures (Refs. ( 10, 11 )). Journal of Colloid and lnteoCace Science, Vol. 131,No. 2, September1989

564

TANEVA ET AL. (OA 2 raN rn"1

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normalized with respect to the alkyl chain concentration, the maximally condensed mixture corresponded to an approximately 1:1 ratio between the hydrocarbon chains of Cl 6CD and DPPC. At higher surface pressures (>5 m N m -1) mixed monolayers displayed ideal behavior (Fig. 6b). 4. DISCUSSION The areas of the cavities of a-, ~-, and 3'cyclodextrins are estimated to be 0.19 +_ 0.02, 0.30 _+ 0.02, and 0.49 __ 0.05 n m 2 from their internal diameters (0.50 _+ 0.02, 0.62 +_ 0.02, and 0.79 + 0.04 nm, respectively) (12). A cross-sectional area of DPPC was obtained to be 0.41 n m 2 from the 7r-A isotherm. These data showed that a DPPC molecule was sizecompatible only with the largest ~,-cyclodextrin cavity which could include one lipid molecule. Different cavity sizes of a-,/3-, and 3/-C16CDs, however, could not be distinguished by the monolayer data indicating that the maximum condensation was reached at the average molecular ratio of 1:3.5 (XDPPC = 0.78 ___ 0.02) in the three mixed CI6CD: DPPC systems. On the other hand, assuming that DPPC is completely inserted into the "y-C16CD cavity and the inclusion complex of a 1:1 molecular ratio is formed, then the phospholipid molecules will not occupy any area in the mixed Journal of Colloid and Interface Science, Vol. 131, No. 2, September 1989

monolayers up to XDPPC = 0.50, i.e., ADPPC = 0 in Eq. [ 1 ]. In this case, the mean molecular area at a given surface pressure will be determined by .,~ = XC,6cDAc16CD,

[2 ]

which is represented by dashed lines in Fig. 6. The experimental area did not coincide with the thus calculated .4 value. This suggests that DPPC is not inserted into any cyclodextrin cavity. This is not surprising because in the above estimation the dimension of a DPPC molecule was used as obtained from the monolayer experiment. This value corresponds to a highly ordered and oriented monolayer structure and it is comparable with the area ofa phospholipid molecule in the lipid bilayer crystalline phase. In the chloroform solution, however, the effective diameter of DPPC molecule is likely to be larger and hence different steric requirements for the inclusion process would exist. It is worth noting that in the mixed monolayers of amphiphilic cyclodextrins and cholesterol the latter (molecular area of 0.399 nm 2) was confirmed to be completely included into the ~'-CI6CD cavity from both calculations and monolayer experiments (6). Considering the rigid planar structure of the steroid molecule, the effective dimension of cholesterol in the chloroform solution could be expected to be close to that obtained from the monolayer data. To visualize the condensing effect seen in the mixed C16CD/DPPC monolayers, the model of molecular packing of nonspecifically interacting steroid and phospholipid molecules in a two-dimensional layer was employed ( 13, 14). Using the external cross-sectional areas of ot-CI6CD, /~-Ct6CD, "y-Ct6CD, and DPPC as obtained from the monolayer experiments (6) (1.618, 2.153, 2.870, and 0.410 n m 2, respectively), we found that maximum 9, 10, and 11 individual peripheral ph0spholipid molecules could be accommodated in direct contact with the a-, ~3-, and T-C16CD molecules, respectively. These molecular structures correspond to DPPC mole fractions (XDppc) of 0.90, 0.91, and 0.92, respectively.

MONOLAYER OF AMPHIPHILIC CYCLODEXTRIN The molecular packing model, however, showed that even at a lower phospholipid concentration XDPVC = 0.77, 0.78, and 0.79 for o~-C16CD/DPPC,~3-C16CD/DPPC, and ~C16CD / DPPC systems, respectively, there are just enough DPPC molecules to surround each particular CI6CD molecule with a layer consisting of 9, 10, and 11 molecules. In this case, each DPPC molecule is not individual for a given C16CD molecule but shared by the neighboring amphiphilic cyclodextrins. Thus, from the molecular packing model it follows that the contacts between the molecules of amphiphilic cyclodextrin and DPPC are realized to the largest extent at this composition. Using the model, the compositions XDpec = 0.95, 0.96, and 0.97 were found for a-, fi-, and "y-C16CD/DPPC systems, respectively, where all DPPC molecules could be grouped within two layers around C~6CD molecules; i.e., phospholipid molecules which are not directly in contact with CI6CD molecules would be present in the mixtures at these compositions. The model results are supported by the monolayer data which show that for the three C~6CD/DPPC systems maximal condensation is reached at XDPPC = 0.78 + 0.02 (Fig. 6a) and therefore it could be associated with a maximal contact between the cyclodextrin and phospholipid molecules. At a monolayer composition corresponding to XDepc = 0.90 the 7r-.~ curves of three C16CD/DPPC monolayers (curves 2 in Figs. 2-4) do not clearly display phase transition but it was detectable from the respective compressibility vs molecular area plots. In other words, at XDpec = 0.90 in the mixed C16CD/DPPC monolayers the enhanced attraction between C16CD and DPPC molecules exists (as indicated by the reduction in the mean molecular areas) and therefore the cyclodextrin/phospholipid contacts are preferred; an excess of DPPC molecules with respect to the maximally condensed mixture (XDPeC = 0.78 _+ 0.02) that are not directly associated with CI6CDs is present. These phospholipid molecules which are more weakly influenced by C16CDs could be ex-

565

pected to give rise to the phase transition observed in the k-A curves. The phase transition was readily seen at XDppc = 0.95 on the 7r-.~ isotherms of three C16CD/DPPC monolayers (not shown). As discussed above, at this composition DPPC molecules which are not in direct contact with C~6CDs are available in the C16CD/DPPC mixtures. 5. SUMMARY The results of the present work show that the amphiphilic cyclodextrins (a-,/3-, and 3,_ C16CDs) and DPPC are miscible in the binary spread monolayers over the entire range of concentrations and pressures studied. Negative deviations of the experimental molecular areas from the additivity rule were observed at lower pressures Or < 5 m N m -1 ). Maximal condensation occurred at the monolayer composition XDePC= 0.78 + 0.02. From a molecular packing model of CtrCD and DPPC in the twodimensional layer the above composition was found to correspond to a maximum contact between Ct6CD and DPPC molecules. Considering that the condensing effect in the mixed monolayers of C~6CD and DPPC was detected only at surface pressures below the main phase transition of DPPC, it could be regarded as resulting from enhanced interactions between their hydrocarbon chains which are of identical length and in fluid state. At higher pressures the C16CD/DPPC monolayers display ideal behavior as judged from the concentration dependence of their collapse pressures (Fig. 5) and the additivity of the mean molecular areas (Fig. 6b). Therefore, a conclusion could be drawn that in the mixed insoluble monolayers of amphiphilic cyclodextrin (~-, {]-, or "y-C16CD) and DPPC, the phospholipid molecules are not included into the cyclodextrin cavities. REFERENCES 1. Szejtli,J., "Cyclodextrinsand Their Inclusion Complexes." Akademia Kiado, Budapest, 1982. Journal of Colloid and Interface Science, Vol. 131, No. 2, September 1989

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2. Irie, T., Otagiri, M., Sunada, M., Uekama, K., Ohtani, Y., Yamada, Y., and Sugiyama, Y., J. Pharmacobio-Dyn. 5, 741 (1982). 3. Szejtli, J., Czerhati, T., and Szogyi, M., Carbohydr. Polym. 6, 35 (1986). 4. Miyajima, K., Saito, H., and Nakagaki, M., J. Chem. Soc. Japan, 306 (1987). 5. Miyajima, K., Tomita, K., and Nakagaki, M., Chem. Pharm. Bull. 33, 2587 (1985). 6. Taneva, S., Ariga, K., Okahata, Y., and Tagaki W., Langmuir 5, 111 (1989). 7. Kawabata, Y., Matsumoto, M., Tanaka, M., Takahashi, H., Irinatsu, Y., Tamura, S., Tagaki, W., Nakahara, H., and Fukuda, K., Chem. Lett., 1933 (1986); 1307 (1987); 1 (1988).

JournalofColloidand InterfaceScience,Vol.13t, No. 2, September1989

8. Takahashi, H., Irinatsu, Y., Kozuka, S., and Tagaki, W., Mere. Fac. Eng. Osaka City Univ. 26, 93 (1985). 9. Takeo, K., Sumitomo, T., and Kuge, T., Straerke 26, i l i (1974). 10. Joos, P., and Demel, R, Biochim. Biophys. Acta 183, 447 (1969). 11. Tomoaia-Cotisel, M., and Chifu, E., J. Colloid Interface Sci. 95, 355 (1983). 12. Saenger, W., Angew. Chem. Int. Ed. Engl. 19, 344 (1980). 13. Engelman, D., and Rothman, J., J. Biol. Chem. 247, 3694 (1972). 14. Muller-Landau, F., and Cadenhead, D., Chem. Phys. Lipids 25, 315 (1979).