Structural Study of the Micellar Aggregates of Sodium

Thus, the stronger Coulombic interactions of the carboxylate groups with the cations give rise to a more .... The carboxylate and, to a less extent, t...
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Langmuir 1997, 13, 5811-5815

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Articles Structural Study of the Micellar Aggregates of Sodium Chenodeoxycholate and Sodium Deoxycholate M. D’Alagni,†,‡ A. A. D’Archivio,§ L. Galantini,§ and E. Giglio*,‡ Centro di Studio per la Chimica dei Recettori e delle Molecole Biologicamente Attive del C. N. R., c/o Istituto di Chimica, Universita` Cattolica, Largo F. Vito 1, 00168 Roma, Italy, Dipartimento di Chimica, Universita` di Roma “La Sapienza”, P.le A. Moro 5, 00185 Roma, Italy, and Dipartimento di Chimica, Ingegneria Chimica e Materiali, Universita` di L’Aquila, 67010 L’Aquila, Italy Received March 29, 1997. In Final Form: July 23, 1997X Quasi-elastic light-scattering data of sodium chenodeoxycholate (NaCDC), sodium deoxycholate (NaDC), and sodium taurodeoxycholate (NaTDC) show that the micellar size increases upon increasing ionic strength (NaCDC, NaDC, and NaTDC) and upon decreasing temperature and pH (NaCDC and NaDC). Their mean hydrodynamic radii (Rh) are in the following order: NaDC > NaTDC > NaCDC. The difference between the Rh values of NaDC and NaCDC is negligible without NaCl and increases upon increasing the NaCl concentration. The average intensities scattered by 0.01-0.10 M aqueous solutions are in the sequence NaTDC > NaDC > NaCDC. Thus, the growth of NaCDC is less than those of NaDC and NaTDC. The plots of the average intensity vs the square root of the mole fraction show that the experimental data are fitted by one straight line for NaCDC and two straight lines for NaDC and NaTDC, suggesting that the NaCDC or NaDC and NaTDC micellar aggregates can be represented by one or two structures, respectively. Since the Rh and the average intensity values of NaCDC and NaDC with added NaCl decrease by increasing the temperature within the range 15-85 °C, their micellar aggregates are stabilized mainly by polar interactions as well as the helical micellar aggregates previously proposed for NaDC and NaTDC. The decrease of pH causes a slight increase of the Rh values of NaDC and NaCDC up to a certain pH value, where a sudden increase is observed. Circular dichroism spectra of NaDC and NaCDC aqueous solutions above and below the critical micellar concentration indicate that the two salts give rise to different types of aggregation. Those of the interaction complexes between bilirubin-IXR and NaCDC or NaDC or NaTDC suggest that the micellar structures are chiral and helical. The enantioselective abilities of NaDC and NaTDC are somewhat similar, but they are different from that of NaCDC. The X-ray fiber patterns of NaCDC, NaDC, and NaTDC can be interpreted by means of different helices and support the quasi-elastic light-scattering and circular dichroism results.

Introduction Micellar aggregates of sodium and rubidium deoxycholate (NaDC and RbDC, respectively) and their interaction complexes with probe molecules were studied by means of several techniques.1-11 Similar helices stabilized by polar interactions were observed in crystals of NaDC * To whom correspondence should be addressed. † Universita ` Cattolica. ‡ Universita ` di Roma “La Sapienza”. § Universita ` di L’Aquila. X Abstract published in Advance ACS Abstracts, September 15, 1997. (1) Esposito, G.; Giglio, E.; Pavel, N. V.; Zanobi, A. J. Phys. Chem. 1987, 91, 356. (2) Giglio, E.; Loreti, S.; Pavel, N. V. J. Phys. Chem. 1988, 92, 2858. (3) Burattini, E.; D’Angelo, P.; Giglio, E.; Pavel, N. V. J. Phys. Chem. 1991, 95, 7880. (4) D’Angelo, P.; Di Nola, A.; Giglio, E.; Mangoni, M.; Pavel, N. V. J. Phys. Chem. 1995, 99, 5471. (5) Campanelli, A. R.; Candeloro De Sanctis, S.; Chiessi, E.; D’Alagni, M.; Giglio, E.; Scaramuzza, L. J. Phys. Chem. 1989, 93, 1536. (6) D’Alagni, M.; Forcellese, M. L.; Giglio, E. Colloid Polym. Sci. 1985, 263, 160. (7) D’Alagni, M.; Delfini, M.; Galantini, L.; Giglio, E. J. Phys. Chem. 1992, 96, 10520. (8) Conte, G.; Di Blasi, R.; Giglio, E.; Parretta, A.; Pavel, N. V. J. Phys. Chem. 1984, 88, 5720. (9) Esposito, G.; Zanobi, A.; Giglio, E.; Pavel, N. V.; Campbell, I. D. J. Phys. Chem. 1987, 91, 83. (10) Chiessi, E.; D’Alagni, M.; Esposito, G.; Giglio, E. J. Inclusion Phenom. Mol. Recognit. Chem. 1991, 10, 453. (11) Bottari, E.; Festa, M. R.; Jasionowska, R. J. Inclusion Phenom. Mol. Recognit. Chem. 1989, 7, 443.

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and RbDC8,12 and were assumed as approximate models of the NaDC and RbDC micellar aggregates in aqueous solutions.13 Apolar groups covered the helical lateral surface, which is in contact with the aqueous medium in solution. Electromotive force (emf) measurements, carried out on NaDC as a function of pH and ionic strength,11 applying the method of the constant ionic medium,14 provided the distribution of the micellar aggregation numbers. The micellar size increased as a function of ionic strength and concentration. A 7/1 helix was proposed as a model of the micellar aggregates of sodium taurodeoxycholate (NaTDC) on the basis of X-ray fiber patterns15,16 and emf measurements.17 The 7/1 helix was formed by trimers,15,16 and most of the aggregates had aggregation numbers which are multiples of three.17 Mean hydrodynamic radii were calculated by means of the distribution of the micellar aggregation numbers. A two-structures model characterized by oblate and cylindrical aggregates (7/1 helices) was used. The agreement with quasi-elastic light-scattering (12) Campanelli, A. R.; Candeloro De Sanctis, S.; Giglio, E.; Petriconi, S. Acta Crystallogr., Sect. C 1984, C40, 631. (13) Campanelli, A. R.; Candeloro De Sanctis, S.; Giglio, E.; Pavel, N. V.; Quagliata, C. J. Inclusion Phenom. Mol. Recognit. Chem. 1989, 7, 391. (14) Biedermann, G.; Sille´n, L. G. Ark. Kemi 1953, 5, 425. (15) D’Alagni, M.; D’Archivio, A. A.; Giglio, E.; Scaramuzza, L. J. Phys. Chem. 1994, 98, 343. (16) Briganti, G.; D’Archivio, A. A.; Galantini, L.; Giglio, E. Langmuir 1996, 12, 1180. (17) Bottari, E.; Festa, M. R. Langmuir 1996, 12, 1777.

© 1997 American Chemical Society

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(QELS) data at low NaTDC concentration was satisfactory.16 As for NaDC the micellar size increased as a function of ionic strength and concentration.16,17 QELS, circular dichroism (CD), and dielectric measurements supported the two-structures model.18 Sodium chenodeoxycholate (NaCDC) provoked scientific and medical interest, since it induces dissolution of cholesterol gallstones after oral administration,19,20 decreases the secretion rate of cholesterol into bile, and often desaturates bile with cholesterol.21 Valuable studies deal with the interfacial and micellar properties of NaCDC, observed by means of several complementary techniques, and with its cholesterol-solubilizing capacity,22 as well as with surface tension and QELS measurements.23 This paper deals with the measurements and the comparison of some NaCDC, NaDC, and NaTDC properties. The comparison between NaCDC and NaDC is particularly interesting, since they have the same molecular formula and very similar molecular structures.

D’Alagni et al.

Figure 1. Rh values of 0.10 M NaCDC, NaDC, and NaTDC aqueous solutions as a function of NaCl concentration at 25 °C. The pH values are 8.3, 8.1, and 6.4 for NaCDC, NaDC, and NaTDC, respectively. The average standard deviation is (0.4 Å for NaCDC and NaDC and (0.3 Å for NaTDC.

Experimental Section Materials. NaCDC (Sigma), NaDC (Calbiochem), and NaTDC (Sigma) were twice crystallized from a mixture of water and acetone. NaCDC fibers were drawn from a viscous gel obtained in an aqueous solution of NaCDC with or without NaCl by adding HCl. The solution was turbid and contained a suspension of insoluble material.24 NaDC fibers were prepared by adding 0.015 M HI to a 0.1 M NaDC aqueous solution. The solution was drawn out into glassy and brittle fibers. I- was oxidized to I2 and removed from the solution by means of oxygen to prevent the presence of NaI in the fibers. NaCl (Merck, Suprapur) and bilirubin-IXR (BR) puriss. (about 99%, Fluka) were used. HPLC measurements showed that the commercial sample contains at least 96% BR. BR was added to the aqueous solutions by using a 0.01 M aqueous NaOH vehicle. Octaethylene glycol monododecyl ether (C12E8) was from Fluka (purity > 98%). Trizma base 0.025 M, reagent grade, was from Sigma. X-ray Measurements. Fibers of NaCDC and NaDC were preferentially oriented microcrystalline specimens. Their X-ray diffraction photographs were recorded on flat and cylindrical films by means of Weissenberg, Debye-Scherrer, and Buerger precession cameras, using Ni-filtered Cu KR radiation (λ ) 1.5418 Å). The density of NaDC fibers was measured by flotation in a mixture of cyclohexane-carbon tetrachloride, the density of which was determined by means of an Anton Paar DMA 02C densimeter. Thermogravimetric analyses were accomplished by means of a DuPont 990 thermal analyzer and 951 thermobalance. Melting points were measured at atmospheric pressure by means of a Leitz 350 heating plate. QELS Measurements. A Brookhaven instrument constituted of a BI-2030AT digital correlator with 136 channels and a BI-200SM goniometer was used. The light source was an argon ion laser model 85 from Lexel Corporation operating at 514.5 nm. Dust was eliminated by means of a Brookhaven ultrafiltration unit (BIUU1) for flow-through cells, the volume of the flow cell being about 1.0 cm3. Nuclepore filters with a pore size of 0.1 µm were used. The samples were placed in the cell for at least 30 min prior to measurement to allow for thermal equilibration. Their temperature was kept constant within 0.5 °C by a circulating water bath. The scattered intensity and the time-dependent light-scattering correlation function were analyzed only at the 90° scattering angle. The observed intensity (18) Bonincontro, A.; Briganti, G.; D’Archivio, A. A.; Galantini, L.; Giglio, E. Paper submitted for publication to J. Phys. Chem. (19) Danzinger, R. G.; Hofmann, A. F.; Schoenfield, L. J.; Thistle, J. L. N. Engl. J. Med. 1972, 286, 1. (20) Makino, I.; Shinozaki, K.; Yoshino, K.; Nakagawa, S. Nippon Shokakibyo Gakkai Zasshi 1975, 72, 690. (21) Stiehl, A.; Czygan, P.; Kommerell, B.; Weiss, H. J.; Holtermu¨ller, K. H. Gastroenterology 1978, 75, 1016. (22) Carey, M. C.; Montet, J.-C.; Phillips, M. C.; Armstrong, M.; Mazer, N. A. Biochemistry 1981, 20, 3637. (23) Roe, J. M.; Barry, B. W. J. Colloid Interface Sci. 1985, 107, 398. (24) van Berge-Henegouwen, G. P.; Hofmann, A. F.; Gaginella, T. S. Gastroenterology 1977, 73, 291.

and the apparent diffusion coefficients did not depend on the exchanged wave vector in the range 30-150° under our experimental conditions. The scattering decays were analyzed by means of cumulant expansion up to second order, because higher order contributions did not improve the statistics. The results were reported in terms of the apparent hydrodynamic radius obtained by the Stokes-Einstein relationship. The scattered intensity was calibrated by using C12E8 as standard25 and is relative to that scattered by pure water at 25 °C. CD Measurements. CD spectra were recorded on a JASCO J-500A spectropolarimeter by using quartz cells with path lengths of 0.1, 0.2, 0.5, 1.0, and 2.0 cm and by flushing with dry ultrapurified nitrogen before and during the experiments. A slit program that gives a wavelength accuracy better than 0.5 nm was used. The instrument was calibrated with androsterone (1.69 × 10-3 M in dioxane) on the basis of a molar ellipticity [θ]304 ) 11 180 deg cm2 dmol-1. All the CD spectra were recorded starting from 3 min of solution preparation and refer to a 1.0 cm path length.

Results and Discussion QELS Study of NaCDC, NaDC, and NaTDC Aqueous Solutions. The apparent hydrodynamic radius (Rh) of 0.10 M NaCDC, NaDC, and NaTDC16 aqueous solutions vs NaCl concentration is reported in Figure 1.The distribution and the composition of the micellar aggregates depend on pH for NaDC.11 Therefore, the pH values of the NaDC and NaCDC solutions must be approximately equal (8.1 and 8.3, respectively) for a correct comparison. The Rh values are practically equal up to 0.20 M NaCl. NaDC shows a faster increase of Rh than NaCDC, whereas NaTDC falls in between. The Rh values of NaDC are very similar to those of sodium glycodeoxycholate and greater than those of NaTDC.16 The charge density of the carboxylate group is higher than that of the sulfonate group, because the charge is distributed within a smaller volume. Thus, the stronger Coulombic interactions of the carboxylate groups with the cations give rise to a more stable micellar structure. On the other hand, NaCDC has the lowest Rh values in spite of its carboxylate group. These results show that the NaCDC micellar aggregates grow less and are less stable than those of NaDC, although their molecules differ only for the axial hydroxyl group at C7 or C12. The increase of the temperature in the range 15-85 °C causes a continuous decrease of Rh with a slope greater for NaDC than for NaCDC. As an example, the data of 0.05 M NaCDC and NaDC aqueous solutions with 0.50 M (25) Degiorgio, V.; Corti, M.; Minero, C. Nuovo Cimento, Sect. D 1984, 3, 44.

Sodium Chenodeoxycholate and Sodium Deoxycholate

Figure 2. Rh values of 0.05 M NaCDC and NaDC aqueous solutions with 0.50 M NaCl as a function of temperature. The pH values are 8.3 and 8.1 for NaCDC and NaDC, respectively. The average standard deviation is (0.3 Å.

Figure 3. Rh values of 0.05 M NaCDC and NaDC aqueous solutions, containing 0.025 M Tris-HCl, as a function of pH at 25 °C. The average standard deviation is (0.4 Å.

NaCl are reported in Figure 2. The temperature range investigated includes the values that generally give rise to association by hydrophobic interactions. Since a decrease of Rh is observed by increasing the temperature, the polar forces rather than the apolar ones could be the driving forces in the growth of the micellar aggregates, in agreement with the NaDC helical model that is stabilized by polar forces.1-13 The pH influences the composition and the aggregation numbers of the NaDC micellar aggregates.11 Rh values as a function of pH are shown in Figure 3 for 0.05 M NaCDC and NaDC aqueous solutions. No difference is observed without NaCl, since comparable small aggregates are present. When NaCl is added, the difference increases by decreasing the pH (as an example, the data with 0.30 M NaCl are shown in Figure 3), since the micellar size of NaDC increases more quickly than that of NaCDC. When the pH is lowered below a certain value, a dramatic increase of Rh occurs, owing to a solution-gel transition. The more the ionic strength is high, the more the pH value corresponding to the gelation increases. The plot of the average intensity scattered by NaTDC aqueous solutions vs the square root of the NaTDC mole fraction indicates that no less than two structures are in equilibrium.18 Since the light-scattering form factor is in the limit where it approximates unity,26,27 the experimental points are expected to lie on a straight line,28 (26) Mazer, N. A.; Carey, M. C.; Kwasnick, R. F.; Benedek, G. B. Biochemistry 1979, 18, 3064. (27) Schurtenberger, P.; Mazer, N.; Ka˚nzig, W. J. Phys. Chem. 1983, 87, 308. (28) Missel, P. J.; Mazer, N. A.; Benedek, G. B.; Young, C. Y.; Carey, M. C. J. Phys. Chem. 1980, 84, 1044.

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Figure 4. Average intensity scattered by NaCDC, NaDC, and NaTDC aqueous solutions as a function of the square root of their mole fraction at 25 °C. I and Is are the average intensities scattered by the solution and the water at 25 °C, respectively.

Figure 5. Average intensity scattered by 0.05 M NaCDC and NaDC aqueous solutions with 0.50 M NaCl as a function of temperature. The pH values are 8.3 and 8.1 for NaCDC and NaDC, respectively. I and Is are the average intensities scattered by the solution and the water at the different temperatures, respectively, and I0 is the average intensity scattered by the water at 25 °C.

assuming that the micellar aggregates have only one type of structure and grow along one direction, as in the case of the helix. On the contrary, the NaTDC data are fitted by two straight lines, which intersect at a concentration of about 0.04 M, suggesting the presence of two structures. CD and dielectric data support this hypothesis.18 Similar intensity data, collected for solutions without NaCl, are shown in Figure 4 for NaDC and NaCDC, together with those of NaTDC for comparison. NaDC follows the trend of NaTDC within the concentration range 0.01-0.10 M, whereas the NaCDC experimental points lie on one straight line. This suggests that the NaCDC micellar aggregates grow, keeping the same structure. The remarkable difference between the NaCDC and NaDC micellar sizes is confirmed by the average intensity scattered by the samples of Figure 2 as a function of temperature (Figure 5). The NaDC micellar aggregates seem to be more stable and grow more than those of NaCDC under the same conditions. CD Study of NaCDC, NaDC, and NaTDC Aqueous Solutions. CD spectra of NaCDC and NaDC above and below their critical micellar concentrations are shown in Figure 6. In this connection it must be stressed that the concept of critical micellar concentration is questionable for bile salts, which very probably give rise to a continuous self-aggregation as a function of concentration, pH, ionic strength, and temperature. The NaCDC molar ellipticity is greater than that of NaDC. Passing from the premicellar to the micellar state, NaCDC, NaDC, and sodium

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Figure 6. CD spectra at 20 °C of aqueous solutions of NaCDC [(a) 7.9 × 10-4 M, pH ) 6.79; (b) 1.012 × 10-2 M, pH ) 7.88] and NaDC [(c) 8.0 × 10-4 M, pH ) 6.72; (d) 1.015 × 10-2 M, pH ) 7.84].

cholate29 decrease their rotational strength because of aggregation, in contrast with NaTDC.30 The most interesting feature of the CD spectra is the position of the minima centered at about 202 and 209 nm for NaCDC and NaDC, respectively. The carboxylate and, to a less extent, the carboxylic groups are the chromophores mainly responsible for the absorption and, therefore, are involved in different interactions in the two salts. Since the role of the carboxylate groups is crucial in the NaDC selfaggregation,1,2,8,12 the structures of the NaCDC and NaDC micellar aggregates must be different. CD spectra of BR in aqueous micellar solutions of NaDC7 and NaTDC15 showed a preferential complexation of the left-handed BR enantiomer on the basis of the exciton coupling theory,31 which expects a (-) longer wavelength Cotton effect followed by a (+) shorter wavelength Cotton effect. Moreover, the chiral recognition of BR was sensitive to the structure of the micellar aggregates, which could be helical, since a helix is a chiral object. Hence, CD spectra of NaCDC with BR were recorded as a function of ionic strength in order to compare them with those of NaDC and NaTDC. The effects of the BR self-association were made negligible by using a concentration less than 5.0 × 10-6 M. Some CD spectra of NaCDC and NaDC with BR are shown in Figure 7. Plainly, the NaCDC and NaDC enantioselective abilities increase by increasing the micellar size, and the enantioselective complexation of NaCDC is greater than that of NaDC. A more clear picture emerges from the ellipticity values at 465 nm (wavelength of the minimum in the CD spectrum) observed in the spectra of NaCDC, NaDC, and NaTDC with BR (Figure 8). The enantioselective abilities are in the following order: NaCDC > NaDC > NaTDC. Those of NaDC and NaTDC are somewhat similar, in accordance with their strictly related 8/1 and 7/1 helices observed in the fibers (see later). Since that of NaCDC is very different from those of NaDC and NaTDC, the formation of NaCDC interaction complexes dissimilar from those of NaDC and NaTDC is supported. X-ray Study of NaDC and NaCDC Fibers. The NaCDC fiber, obtained by adding HCl, could be formed by molecules of chenodeoxycholic acid. Its melting, which started at 561 K (566 K for NaDC), reasonably ensured that the fiber was composed of the sodium salt, since the (29) D’Alagni, M.; D’Archivio, A. A.; Giglio, E. Biopolymers 1993, 33, 1553. (30) D’Alagni, M.; Giglio, E.; Petriconi, S. Colloid Polym. Sci. 1987, 265, 517. (31) Blauer, G.; Wagnie´re, G. J. Am. Chem. Soc. 1975, 97, 1949.

Figure 7. CD spectra at 25 °C of 4.7 × 10-6 M BR in 0.05 M aqueous solutions of (A) NaCDC, pH ) 8.7 [(a) without NaCl; (b) with 0.10 M NaCl; (c) with 0.20 M NaCl; (d) with 0.40 M NaCl] and (B) NaDC, pH ) 8.3 [(a) without NaCl; (b) with 0.10 M NaCl; (c) with 0.20 M NaCl; (d) with 0.40 M NaCl].

Figure 8. Ellipticity values at 465 nm of 0.02 M NaCDC, NaDC, and NaTDC aqueous solutions, containing 4.6 × 10-6 M BR and 0.025 M Tris-HCl, as a function of added NaCl. The temperature is 25 °C, and the pH is 8.1.

crystal of the high-melting polymorph of chenodeoxycholic acid melts at 438-439 K.32 X-ray diffraction photographs were taken on NaDC and NaCDC air-dried fibers. The most intense NaCl interplanar spacings, which are known with great precision, were used to determine those of NaDC and NaCDC. The NaDC equatorial and meridional reflections can be indexed in the smallest unit cell with a ) 31.8 Å, b ) 18.4 Å, c ) 52.0 Å, and R ) β ) γ ) 90°. The equatorial rectangular lattice of NaDC has a ) b/tan 30° and could be derived from a trigonal or hexagonal lattice (a ) b ) 18.4 Å, γ ) 120°) as in the case of sodium and rubidium glycodeoxycholate16 and sodium, rubidium,15,16 and calcium33 taurodeoxycholate. The fibers of these salts are formed by similar 7/1 helices with a radius of about 10 Å, seven points (seven trimers) in the identity period along the helical axis (45 Å), and a helical axis projection of the distance between two consecutive equivalent points on the helix, Z, of 6.4-6.5 Å. On the basis of the helical (32) Lindley, P. F.; Mahmoud, M. M.; Watson, F. E.; Jones, W. A. Acta Crystallogr. 1980, B36, 1893. (33) D’Archivio, A. A.; Galantini, L.; Giglio, E. Langmuir 1997, 13, 4197.

Sodium Chenodeoxycholate and Sodium Deoxycholate

transform theory,34 the fiber of NaDC is formed by 8/1 helices. They have a radius of about 10 Å, eight points (eight trimers) in the identity period along the helical axis (52 Å), and a Z of 6.5 Å. The unit cell contains two 8/1 helices, which are composed each one of 24 NaDC molecules according to density and thermogravimetric measurements. The 8/1 helix is generated by rotating 360°/8 ) 45° and translating 6.5 Å the repetitive unit (the trimer) and is strictly related to that of NaTDC. The X-ray patterns of NaCDC are characteristic of poorly oriented fibers. They show few diffuse reflections and a low degree of ordering. The intensity distribution of NaCDC is very different from that of NaDC. The main feature of the NaCDC X-ray pattern is a true meridional reflection with a spacing of 7.6 Å belonging to the most intense layer line. This value represents the translation of the asymmetric unit along the fiber axis and differs from the value of 6.5 Å of NaDC. Similar values (7.547.94 Å) were found in several crystal structures of (34) Cochran, W.; Crick, F. H. C.; Vand, V. Acta Crystallogr. 1952, 5, 581.

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dihydroxy and trihydroxy salts of bile acids.15,35-37 They refer to identity periods of “binary units”, which are units with a twofold screw axis, with a twofold rotation axis, or without any symmetry axis. They were proposed as models of the micellar aggregates of the trihydroxy bile salts.37 Probably, the structure of the NaCDC micellar aggregates is similar to that of a binary unit. Further work is in progress to clarify this point. Acknowledgment. This work was sponsored by the Italian Ministero per l’Universita` e per la Ricerca Scientifica e Tecnologica. LA970337N (35) Campanelli, A. R.; Candeloro De Sanctis, S.; Galantini, L.; Giglio, E.; Scaramuzza, L. J. Inclusion Phenom. Mol. Recognit. Chem. 1991, 10, 367. (36) D’Alagni, M.; Galantini, L.; Giglio, E.; Gavuzzo, E.; Scaramuzza, L. Trans. Faraday Soc. 1994, 90, 1523. (37) D’Archivio, A. A.; Galantini, L.; Gavuzzo, E.; Giglio, E.; Scaramuzza, L. Langmuir 1996, 12, 4660.