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Langmuir 2008, 24, 6060-6066
Synthesis and Characterization of a New Gemini Surfactant Derived from 3r,12r-Dihydroxy-5β-cholan-24-amine (Steroid Residue) and Ethylenediamintetraacetic Acid (Spacer) Mercedes Alvarez Alcalde,† Aida Jover,† Francisco Meijide,† Luciano Galantini,‡ Nicolae Viorel Pavel,‡ Alvaro Antelo,† and Jose´ Va´zquez Tato*,† Departamento de Quı´mica Fı´sica, Facultad de Ciencias, UniVersidad de Santiago de Compostela, AVenida Alfonso X El Sabio s/n, 27002 Lugo, Spain, and Dipartimento di Chimica, Research Center SOFT-INFM-CNR, UniVersita` di Roma “La Sapienza”, Piazza le A. Moro 5, 00185 Roma, Italy ReceiVed NoVember 13, 2007. ReVised Manuscript ReceiVed March 4, 2008 A new gemini steroid surfactant derived from 3R,12R-dihydroxy-5β-cholan-24-amine (steroid residue) and ethylenediamintetraacetic acid (spacer) was synthesized and characterized in aqueous solution by surface tension, fluorescence intensity of pyrene, and light scattering (static and dynamic) measurements. These techniques evidence the existence of a threshold concentration (cac), below which a three layers film is formed at the air-water interface. Above the cac, two types of aggregates;micelles and vesicle-like aggregates;coexist in a metastable state. Filtration of a solution with a starting concentration of 2.6 mM (buffer 150 mM, pH 10) allows isolation of the micelles, which have an average aggregation number of 12, their density being 0.28 g cm-3. Under conditions where only the vesiclelike aggregates are detected by dynamic light scattering, a value of 5.5 × 104 was obtained for their aggregation number at 30 µM, their density being 6.8 × 10-4 g cm-3. At high concentrations, the intensity ratio of the vibronic peaks of pyrene, I1/I3, () 0.68) is very close to published values for deoxycholate micelles, indicating that the probe is located in a region with a very low polarity and far from water. A hypothesis to explain the observed aggregation behavior (small aggregates are favored with increasing gemini concentration) is outlined.
Introduction During the past few years, an increasing number of papers have been published on the surface and micellar properties of gemini surfactants.1–3 This is mainly because of their better efficiency in decreasing both the surface tension of water and the critical micelle concentration (cmc) in comparison to their corresponding monomeric analogues. Most of them contain two hydrophobic long alkyl chains and two hydrophilic groups that are linked through a flexible or rigid spacer.4,5 Although bile salts are very well-known surfactants6–8 and good solubilizers of hydrophobic compounds (including drugs9 and cholesterol,10), little attention has been paid to their potential use as amphiphile residues to design new gemini surfactants. Only a few examples of gemini surfactants formed by two bile acid residues have been published,11–13 the first one being synthesized by McKenna et al.11 In these examples, the carboxylic acid group is lost because it has been used to link the two steroid residues to the spacer. Here we have designed, synthesized, and characterized a dicarboxylic gemini steroid surfactant derived * Corresponding author. † Universidad de Santiago de Compostela. ‡ Universita` di Roma “La Sapienza”.
(1) Rosen, M. J.; Tracy, D. J. J. Surfactants Deterg. 1998, 1, 547. (2) Zana, R. AdV. Colloid Interface Sci. 2002, 97, 205. (3) Hait, S. K.; Moulik, S. P. Curr. Sci. 2002, 82, 1101. (4) Song, L. D.; Rosen, M. J. Langmuir 1996, 12, 1149. (5) Mathias, J. H.; Rosen, M. J.; Davenport, L. Langmuir 2001, 17, 6148. (6) Carey, M. C.; Small, D. M. Arch. Int. Med. 1972, 130, 506. (7) Coello, A.; Meijide, F.; Rodrı´guez Nu´n´ez, E.; Va´zquez Tato, J. J. Pharm. Sci. 1996, 85, 9. (8) Mukhopadhyay, S.; Maitra, U. Curr. Sci. 2004, 87, 1666. (9) Wiedmann, T. S.; Kamel, L. J. Pharm. Sci. 2002, 91, 1743. (10) Almgren, M. Biochim. Biophys. Acta 2000, 1508, 146. (11) McKenna, J.; McKenna, J. M.; Thornthwaite, D. W. J. Chem. Soc., Chem. Commun. 1977, 809. (12) Li, Y.; Dias, J. R. Chem. ReV. 1997, 97, 283. (13) Ronsin, G.; Kirby, A. J.; Rittenhouse, S.; Woodnutt, G.; Camilleri, P. J. Chem. Soc., Perkin Trans. 2002, 2, 13026.
Figure 1. Structure of the g-2DC24-EDTA gemini, derived from 3R,12Rdihydroxy-5β-cholan-24-amine and EDTA as the spacer.
from 3R,12R-dihydroxy-5β-cholan-24-amine (i.e., a 24-amino derivative of deoxycholic acid) (see Figure 1). The gemini, g-2DC24-EDTA, was prepared by reacting the dianhydride derivative of ethylenediaminetetraacetic acid (EDTA), which constitutes the spacer of the gemini, with the steroid amine. Thus two amide bonds are formed, and two carboxylic groups are released (Figure 1).
Experimental Section Synthesis. Dimethyl ester of EDTA (0.61 g, 1.9 mmol) was dissolved in a mixture of 5 mL of dried dimethylformamide (DMF) and 15 mL of dried tetrahydrofuran (THF). Diethyl cyanophosphate (DEPC, 0.65 mL, 4.28 mmol) was added to this solution. After 30 min, the solution was cooled to 0 °C, and a solution of 3R,12Rdihydroxy-5β-cholan-24-amine14 (1.51 g, 4.00 mmol) and triethylamine (0.6 mL, 4.30 mmol) in 20 mL of dried THF was added dropwise with stirring. After 90 min, the ice bath was removed, and the reaction was maintained for 6 h at room temperature. The solvent was evaporated in vaccum. Then 200 mL of chloroform were added and washed twice with water (50 mL) to remove all DMF. The organic phase was dried (Na2SO4) and partially evaporated under reduced pressure. Finally the product was purified by column (14) Fini, A.; Fazio, G.; Roda, A.; Bellini, A. M.; Mencini, E.; Guarneri, M. J. Pharm. Sci. 1992, 81, 726.
10.1021/la7035218 CCC: $40.75 2008 American Chemical Society Published on Web 05/23/2008
Synthesis and Characterization of g-2DC24-EDTA
Langmuir, Vol. 24, No. 12, 2008 6061
chromatography (silica gel 70-230 mesh; eluent 7:3 ethyl acetate/ methanol). Identity of the compound was confirmed by NMR and fast atom bombardment mass spectrometry (FAB-MS). Overall yield 45%. To remove the methyl groups of the ester in the spacer, the compound was refluxed with KOH 1 M in methanol for one hour at 80 °C. The solvent was evaporated, and the solid was redissolved in water (200 mL) and acidified with HCl (pH ≈ 1). When the solution was cooled, the compound precipitated in its diacid form. The precipitate was filtered and dried in a vaccum oven. The disodium salt was obtained by adding the stoichiometric amount of NaOH. Both the diacid and the disodium salts were repeatedly crystallized to guarantee the purity (checked by thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), FAB-MS, and surface tension measurements; see Supporting Information) of the gemini compound. The melting point was 129.56 °C. Instrumental Techniques. Light scattering measurements were carried out in a Brookhaven instrument constituted by a BI2030AT digital correlator with 136 channels and a BI200SM goniometer. The light source was a Uniphase solid-state laser system model 4601 operating at λ ) 532 nm. The samples were placed in the cell for at least 30 min prior to the measurement to allow for thermal equilibration. Their temperature was kept constant within 0.5 °C by a circulating water bath. Samples were filtered through filters with different pore size (see Results and Discussion). The basic theories of static and dynamic light scattering techniques are very well-known and can be found elsewhere.15,16 For each sample, the excess Rayleigh scattering ratio ∆Rq was estimated as a function of the scattering vector q ) 4πnsin(θ/2)/λ, where n is the solvent refractive index, θ is the scattering angle, and λ is the wavelength. Whenever a q dependence was observed, an apparent gyration radius (Rg) and a q f 0 extrapolated ∆Rq value (∆Ro) were estimated by fitting the experimental data with the equation
Figure 2. Surface tension and fluorescence intensity ratio I1/I3 of pyrene (as a probe) vs log [g-2DC24-EDTA]/M in water. pH (at the highest concentration of gemini) ) 8.33. T ) 25.0 ( 0.5 °C.
the q f 0 limiting value was obtained from the linear Dapp versus q2 plot. This value was used to calculate Rapp. Surface tension measurements were carried out in a Kruss K10ST tensiometer by the Wilhelmy method. Fluorescence measurements were carried out in a Hitachi model F-3010 spectrofluorimeter at an excitation wavelength of 336 nm, and excitation and emission bandwidth of 5 nm. Samples were thermostatted at 25 °C.
Results and Discussion
From this value, an apparent hydrodynamic radius Rapp was calculated by the Stokes-Einstein equation, Rapp ) kBT/6πηDapp, where kB is the Boltzmann constant, T is the absolute temperature, and η is the solvent viscosity. Whenever a q dependence of Dapp was observed,
In Figure 2, surface tension data, γ, are plotted against log C. Below the cac, γ varies linearly with log C, as for many classical and gemini surfactants, while, above this threshold concentration (4.6 µM, in water), γ smoothly decreases with log C. As usual, the cac can be accepted as a critical aggregation concentration (cac) above which aggregates are formed. This value is 3 orders of magnitude lower than the cmc () 4.4 ( 2.9 mM, calculated from compiled values by Coello et al.)7 for sodium deoxycholate, indicating that the new compound is a much better surfactant than the natural bile salt. Such a difference is typical of a gemini surfactant in comparison to its monomeric analogue.1 Surface tension measurements were also carried out at different pH values (9.33-11.2 range) and different buffer concentrations (15-150 mM, carbonate/bicarbonate). All plots are similar to the one observed in water (Figure 2) and the average value for the cac obtained from these experiments is 2.7 ( 0.5 µM. Figure 2 also shows the dependence of the intensity ratio, I1/I3, of the vibronic peaks of pyrene with log C. At very low surfactant concentrations, I1/I3 is almost constant and then decreases gradually with increasing concentration of g-2DC24EDTA, reaching a plateau at 0.68. This value is close to the one measured for pyrene included in sodium deoxycholate aggregates () 0.70 ( 0.01)17 and not far from its value in the very apolar cyclohexane () 0.61).18 The value obtained for I1/I3 at very diluted concentrations is close to the observed value in water () 1.96).18 At these limiting gemini concentrations, the concentration of pyrene () 2 µM) is comparable to the surfactant concentration, meaning that not all pyrene is incorporated into the aggregates. So, the I1/I3 ratio corresponds to the sum on the I1/I3 ratios for pyrene in water and in the aggregates, taking into account the respective fraction of pyrene in both media. This can partially explain the gradual decrease of I1/I3 over the broad range of concentration observed in Figure 2 (much wider than
(15) Wyatt, P. J. Anal. Chim. Acta 1993, 272, 1–40. (16) Schmitz, K. S. An Introduction to Dynamic Light Scattering by Macromolecules; Academic Press: Boston, 1990.
(17) Jover, A.; Meijide, F.; Rodrı´guez Nu´n´ez, E.; Va´zquez Tato, J.; Mosquera, M.; Rodrı´guez Prieto, F. Langmuir 1996, 12, 1789. (18) Hashimoto, S.; Thomas, J. K. J. Colloid Interface Sci. 1984, 102, 152.
(
q2Rg 1 1 ) 1∆Rq ∆Ro 3
)
(1)
at qRg , 1. Hence an apparent molecular weight was estimated as
Mapp )
∆R0 KC
(2)
where C is the solute concentration (g mL-1) and K is a constant given by
K)
2π2 dn n λ4NA dC
2
( )
(3)
where NA is Avogadro’s constant, and dn/dC is the differential refractive index increment of the solution. The differential refractive index was measured in an Atago model DD7 instrument. When no angular dependence was shown by the measured ∆Rq, the value at θ ) 90° was used in eq 2. In the DLS experiments, the intensity-intensity autocorrelation function was measured at a particular q value, and related to the normalized electric field autocorrelation function g1(q,τ) by the Siegert relation. The autocorrelation function was analyzed either by CONTIN or the cumulant expansion.16 In the latter case, the socalled apparent diffusion coefficient Dapp was obtained from the first cumulant by the relation
Dapp ) -
1 dlng1(q, τ) dτ q2
|
(4) τ)0
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the typical sharp drop found for surfactants with cmc values in the millimolar range),5 since, at the lowest concentrations, the pyrene in water increases the measured value of the I1/I3 ratio. Under these experimental conditions, steady-state fluorescence is not a suitable technique to determine some parameters of the aggregates such as their aggregation number, but this qualitative analysis supports the formation of aggregates. Surface tension results suggest that the gemini forms aggregates with increasing concentration, while fluorescence results indicate that the probe is finally located in a region of the aggregate with a polarity similar to that of sodium deoxycholate aggregates and far from water. Vethamuthu et al.19 have determined that, in sodium deoxycholate aggregates, only a small fraction of pyrene () 4%) is in contact with water, evidencing a closed packing of the surfactant molecules around the probe. Below the cac, the values for the slope (∂γ/∂ log C) of the straight line do not show any clear dependency on pH (9.3-11.2) or ionic strength, and, consequently, only the average value () -15 ( 4 mN/m) will be discussed. The low influence of ionic strength on this critical concentration and on slope (or, equivalently, on the molecular area at the interface, As) is in agreement with observations for several zwitterionic surfactants.20–22 Prosser and Franses23 have reviewed the application of the Gibbs adsorption isotherm to the surface tension of ionic surfactants at the air-water interface. For a strong ionic surfactant of ν+ free positive ions and ν- free negative ions of charges z+ j , is given and z-, respectively, the surfactant surface density, Γ by _
Γ)-
dγ 1 RTm(c, cs) dlnC
(
)
cs
(5)
where m(c,cs) is a function of ν+, ν-, the surfactant concentration, C, the concentration of added inert salt, Cs, and the stoichiometry coefficient of the counterion of the surfactant in the supporting s electrolyte, ν+ . m(c,cs) is given by
m(c, cs) ) ν- +
ν2+ ν+ + νs+
Cs C
(6)
It can be noticed that m(c,cs) is not a function of the coion valence s of the supporting electrolyte ν. In the absence of inorganic electrolyte, m ) (ν- + ν+), and the surface excess density is inversely proportional to the total number of free ions in solution. However, when the electrolyte concentration is high, the term involving ν+ becomes negligible and m(c,cs) ) ν-. For the present gemini, ν) 1. Therefore, at pH ) 10.1 and concentrations of Cs(Na+) much higher than C (45 mM and below 3 µM, respectively), m(c,cs) ≈ 1. For highly surface-active surfactants in dilute solutions, the surface excess density may be approximated by the adsorbed surface density, j ≈ Γ. Since Γ ) 1/(AsNA) (NA is Avogadro’s number), a value Γ of As) 27 ( 6 Å2/molecule is calculated. The low influence on the surface properties of ionic strength mentioned above (a behavior shared with zwitterionic surfactants) also supports the use of ν- ) 1, as for zwitterionic surfactants.24,25 (19) Vethamuthu, M. S.; Almgren, M.; Mukhtar, E.; Bahadur, P. Langmuir 1992, 8, 2396. (20) Chorro, M.; Kamenka, N.; Faucompre, B.; Partyka, S.; Lindheimer, M.; Zana, R. Colloids Surf., A 1996, 110, 249. (21) Zajac, J.; Chorro, C.; Lindheimer, M.; Partyka, S. Langmuir 1997, 13, 1486. (22) Aydogan, N.; Abbott, N. L. Langmuir 2002, 18, 7826. (23) Prosser, A. J.; Franses, E. I. Colloids Surf., A 2001, 178, 1. (24) Sesta, B.; La Mesa, C. Colloid Polym. Sci. 1989, 267, 748. (25) Chevalier, Y.; Storet, Y.; Pourchet, S.; Le Perchec, P. Langmuir 1991, 7, 848.
A simple theoretical calculation (performed with the program CHEM3D of ChemOffice) gives a value of 90 Å2 for a single deoxycholic acid molecule lying flat. This value is in close agreement with the experimental one for lithocholic acid lying flat on the air-water interface.26 The calculated length of a fully extended molecule of g-2DC24-EDTA is 37 Å. This value is comparable to the experimental one (34.662 Å)27 obtained from X-ray diffraction of the crystal of a methyl diester of a 3β-cholic amine dimer in which the two steroid residues are linked by amide bonds to a succinyl group. The area occupied for the fully extended g-2DC24-EDTA molecule with the two steroid residues lying flat on the surface is 220 Å2 (Figure 3). For an upright orientation of the gemini (ionic carboxylic groups oriented toward the water and steroid moieties oriented toward the aerial phase), the area occupied by a molecule depends on the angle formed by the two branches of the gemini. For a maximum packing of the steroids (minimum angle), the projected area on the surface is 100 Å2/molecule. None of these theoretical values is close to the experimental values mentioned above. Previous interpretations for bile salts will help in understanding such a difference. Small26 has reviewed the compression isotherms of natural bile acids. Particularly interesting is the behavior of lithocholic acid. At pH 10 and 3 M NaCl, there is only one collapse point at about 44 Å2/molecule which corresponds to the molecule in an upright position. (It can be noticed that our calculated value for the upright orientation of the gemini is not far from twice the experimental value for lithocholic acid). Similar values (between 40 and 46 Å2/molecule) were obtained for other bile acids or salts.26,28 At pH ) 2, the isotherm is completely different. At about 119 Å2/molecule, there is a rise in pressure to a first collapse point at 81 Å2/molecule. This range (119-81 Å2/molecule) of areas corresponds to a monolayer of molecules lying flat on the surface. At about 27 Å2/molecule a second increase in pressure originates a decrease in area of 5 Å2/molecule. Since this last value for area is one-third of the minimum area (81 Å2/molecule) of lithocholic acid lying flat in a monolayer, Ekwall29 suggested that the bile acid forms a single bulk phase made up of a trilayer of bile acid. Rosen et al.30 have also proposed the formation of multilayer structures to explain the aberrant behavior of some surfactants (having a large number of carbon atoms in the alkyl chain) belonging to a family of bis(quaternary ammonium halide) gemini surfactants. A similar aberrant behavior is observed by Tsubone et al.31 who have also accepted the formation of multilayer films. Therefore, the comparison between theoretical and experimental results suggests that g-2DC24-EDTA forms a multilayer film at the air-water interface. The length of the steroid side chain plus the EDTA bridge (∼11.7 Å), which is almost twice the length of the steroid nucleus, would allow the formation of the multilayer without preventing the interaction of the ionic groups of upper layers with water. Comparison between experimental surface area and theoretical calculations suggests that three layers are the most probable structure of the film at the air-water interface in the presence of added salt. At high pH values, where only the carboxylate groups are charged, the presence of salt would allow the unfolding of carboxylate (26) Small, D. M. In The Bile Acids, Chemistry, Physiology, and Metabolism; Nair, P. P., Kritchevski, D., Eds.; Plenum Press: New York, 1971; Chapter 8, p 249. (27) Soto Tellini, V. H. Doctoral Thesis, University of Santiago de Compostela, 2006. (28) Swanson-Vethamuthu, M.; Almgren, M.; Hansson, P.; Zhao, J. Langmuir 1996, 12, 2186. (29) Ekwall, P.; Ekholm, R. Proc. Int. Congr. Surf. Act., 2nd, London 1957, 1957, 23. (30) Rosen, M. J.; Mathias, J. H.; Davenport, L. Langmuir 1999, 15, 7340. (31) Tsubone, K.; Ogawa, T.; Mimura, K. J. Surfactants Deterg. 2003, 6, 39.
Synthesis and Characterization of g-2DC24-EDTA
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Figure 3. Representation of the surface configuration of the g-2DC24-EDTA molecule lying in flat (above) and upright (below) positions. The theoretical value of 37 Å in the extended structure is very close to the one () 34.662 Å)27 obtained from the resolution of a crystal of a diester of a 3β-cholic amine dimer (in which the two steroid residues are linked through a succinyl group).
Figure 4. Histogram size distributions of particles calculated from a CONTIN analysis of the autocorrelation function for a sample of 3.00 mM of the gemini g-2DC24-EDTA in bicarbonate/carbonate buffer: (a) 15 mM, (b) 150 mM at pH 10. Temperature 25.0 ( 0.5 °C. Scattering angle θ ) 90°.
chains into water, their mutual interaction being screened by added salt ions. Static (SLS) and dynamic (DLS) light scattering measurements were performed to characterize the nature of the aggregates. The measurements were carried out in water and at high pH values in carbonate/bicarbonate buffer, at concentrations of the gemini above the cac. Figure 4 shows the results obtained from a CONTIN analysis of the autocorrelation function g1(q,τ) (measured at 90°) of a 3 mM gemini sample at pH 10 and two buffer concentrations
(15 and 150 mM). In both cases, bimodal distributions of aggregates are observed with two maxima at hydrodynamic radii values of 2.0 and 77 nm (buffer 15 mM) and 2.5 and 82 nm (buffer 150 mM). These results evidence that (i) two types of aggregates are present in solution and (ii) the ionic strength has a low influence on either of the two maxima. At NaCl 150 mM, Mazer et al.32 have obtained the hydrodynamic radius for taurocholate, taurodeoxycholate, tauroursodeoxycholate, and taurochenodeoxycholate, ranging from 1.0 to1.5 nm for taurocholate and from 1.5 to 2.2 nm for the other three bile salts. These results were analyzed in terms of the formation of micelles with low aggregation numbers (ranging from 3 to 22, depending on bile salt and temperature). As we discuss below, from SLS measurements we have obtained a value of 12 for the aggregation number of the aggregate of smaller size. The agreement between size and aggregation number of this aggregate and those results for natural bile salts suggests a micellar structure for it. Reviews on micelle structure for bile salts have been published elsewhere.7,33–35 Table 1 shows that the size of this aggregate is invariant with the total surfactant concentration and that it slightly grows with the addition of inert salts. Table 1 and Figure 5 show that the hydrodynamic radius of the aggregate of greater size (obtained from a CONTIN analysis of the autocorrelation funtion) does not significantly change with the gemini surfactant in the range of concentration 0.10-3.0 mM. The average values are 77 ( 5 nm and 78 ( 3 nm at 15 mM and 150 mM of buffer concentration, respectively, evidencing that the ionic strength does not affect the size of the aggregate. As we show below, this is a vesicle-like aggregate. Table 1 evidences that the aggregate of greater size is always present in the whole range of concentrations above 10 µM and that the (32) Mazer, N. A.; Carey, M. C.; Kwasnick, R. F.; Benedek, G. B. Biochemistry 1979, 18, 3064. (33) Small, D. M. AdV. Chem. Ser. 1968, 84, 31. (34) Kawamura, H.; Murata, Y.; Yamaguchi, T.; Igimi, H.; Tanaka, M.; Sugihara, G.; Kratohvil, J. P. J. Phys. Chem. 1989, 93, 3321. (35) Campanelli, A. R.; Candeloro de Sanctis, S.; Giglio, E.; Pavel, N. V.; Quagliata, C. J. Inclusion Phenom. Macrocyclic Chem. 1989, 7, 391.
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Table 1. Hydrodynamic Radii of the Two Populations Observed for the Gemini g-2DC24-EDTA Determined from DLS at a Scattering Angle of θ ) 90°, at Two Buffer Concentrations and pH 10a buffer 15 mM (pH 10) C/mM 9.00 5.00 3.00 2.50 1.50 1.00 0.750 0.650 0.500 0.320 0.250 0.150 0.100 0.060 0.050 0.040 0.030 0.020 0.010
Rh90(1)/nm
Rh90(2)/nm
2.0 2.0 2.0 2.0 2.0 2.0
77.5 79.5 75.0 72.0 79.0 72.5
2.0 2.0 2.0 2.0
71.7 76.6 76.5 89.0
2.0
75.0
buffer 150 mM (pH 10) Rh90(1)/nm
Rh90(2)/nm
2.5 3.0 2.5 2.0 2.5 2.5 2.5
81.5 82.0 82.0 79.5 80.5 74.0 75.0
2.5
79.3
2.5 2.5 2.5
74.3 76.0 78.5 58.2 62.3 60.9 61.1 62.4 63.0
Figure 6. Apparent molecular weight (scattering angles of 40° and 90°) vs log [g-2DC24-EDTA]/M. Bicarbonate/carbonate buffer 150 mM at pH 10. Temperature 25.0 ( 0.5 °C. Samples were filtered through filters with a diameter of 0.45 µm.
a Temperature 25.0 ( 0.5°C. All samples were filtered through filters with a diameter of 0.45 µm. Typical standard error of the hydrodynamic radius for the larger aggregate is lower than 7%, while the one for the smaller aggregate is lower than 1 nm.
Figure 7. Rayleigh scattering ratio (θ ) 90°) vs log [g-2DC24-EDT]/M. Bicarbonate/carbonate buffer 150 mM at pH 10. Temperature 25.0 ( 0.5 °C. (b) Sample prepared in the scattering cell by dilution of a starting solution of higher concentration. (g) Samples prepared individually by weighing the right amount of sample and solvent. Samples were filtered through filters with a diameter of 0.45 µm.
Figure 5. Histogram size distributions of particles calculated from a CONTIN analysis of the autocorrelation function of g-2DC24-EDTA samples of different concentrations in 150 mM bicarbonate/carbonate buffer at pH 10. Temperature 25.0 ( 0.5 °C. Scattering angle θ ) 90°. Samples were filtered through filters with a diameter of 0.45 µm.
distribution is monomodal with a low polydispersity (σ < 0.2) in the range 30-60 µM (buffer 150 mM). In this interval of concentrations, the aggregate of greater size has an average hydrodynamic radius of R90 ) 61 ( 2 nm (determined at a scattering angle of 90°). Although micelles are not detected, it does not mean that they are not present in the solution since they can be completely masked by the contribution of the vesicle-like aggregates because of their much higher molar mass (see below), 36 i.e., micelles and vesicle-like aggregates coexist on the whole range of concentration above the cac. Under these conditions, a fit of the correlation functions of the scattering intensity with a cumulant expansion was performed, and a realistic apparent translational diffusion coefficient, Dapp, at q f 0 was estimated. From these data, an average value of Rapp ) 114 ( 5 nm is obtained for the apparent hydrodynamic radius of the particle.
Figure 6 shows the apparent molecular weight versus log C (buffer 150 mM and pH 10). Above 10 µM, a sudden increase of Mapp is observed, suggesting the formation of aggregates above the cac. From the initial value determined at 20 µM, Mapp smoothly decreases with concentration, reaching a plateau at relatively high gemini concentration (∼1 mM). Identical results are obtained independently of the method used to prepare the samples, as evidenced in Figure 7. Samples were prepared either by direct weighing of the gemini and its solubilization in the right amount of solvent or by dilution from a more concentrated sample. The second method allows us to discard possible bias errors due to loss of mass because of the filtration since it is not required. SLS measurements were carried out at experimental conditions where only the vesicle-like aggregates have been detected by DLS experiments. Values of 5.8, 4.6, and 3.6 have been obtained for Mapp/107g · mol-1 at surfactant concentrations of 30, 45, and 60 µM, respectively (buffer 150 mM, pH 10). For the calculation of the molar mass, a value of 0.168 mL · g-1 was used for the increment of the solution refraction index with gemini concentration, dn/dc. As we have already noticed, micelles are probably present in this interval of concentration, thus the observed tendency of Mapp would be in agreement with an increasing percentage of micelles with increasing surfactant concentration. It also suggests that the average molar mass of these vesicle-like
Synthesis and Characterization of g-2DC24-EDTA
Figure 8. TEM image of the aggregates formed by g-2DC24-AEDT in D2O. Starting gemini concentration was 50 µM.
aggregates is probably higher than the maximum value for Mapp measured at 30 µM since Mapp is a weigth average molar mass of all species in the solution. In this interval of concentrations, an average value of Rg ) 132 ( 15 nm was obtained for the radius of gyration of aggregates. The influence of the temperature was studied at the interval 25-60 °C at a gemini concentration of 30 µM, and no significant changes were observed either in the intensity of solution or in the size of aggregates. No changes were observed after sonication of the solution for 1 h at 25 °C and different ionic strengths (NaCl 25-350 mM). Transmission electron microscopy (TEM) measurements were performed to obtain information about the morphology of the aggregates. Figure 8 shows a typical TEM image obtained for a sample with an initial gemini concentration of 50 µM. Circular structures (vesicle-like) are evident, having an average radius of 227 ( 45 nm, in acceptable agreement with the Rg and Rh values from light scattering measurements, if effects on the sample preparation for TEM measurements are considered. On the other hand, the comparison of the hydrodynamic radius with the radius of gyration is usually done by the parameter defined by F ) Rg/Rh,37 which has distinctly different values depending on the architecture of the particle. For instance, for a homogeneous impenetrable sphere, F ) 0.788, while a value of F close to unity suggests the presence of spherical shells, i.e., vesicles.38,39 From the average value of Rg and the value of Rapp, it results in F)1.1 ( 0.2, supporting the formation of vesiclelike aggregates. Although a gradual decrease of the vesicle-like aggregate contribution to the CONTIN distribution is observed by increasing the gemini concentration, it does not disappear completely, even at the highest surfactant concentrations. However, we observed that, at these concentrations, it is possible to completely remove these aggregates by filtering the samples. This is probably due to fact that the large aggregates are in a metastable state, as is observed in several vesicle systems. The aggregation number of the small aggregate was estimated as follows. A sample of g-2DC24-AEDT 2.6 mM (buffer 150 mM, pH 10) was prepared (36) Pedersen, J. S.; Egelhaaf, S. U.; Schurtenberger, P. J. Phys. Chem. 1995, 99, 1299. (37) Burchard, W. EnVironmental Particles; Buffle, J., Van Leeuvwn, H. P.; Environmental Analytical and Physical Chemistry Series; Lewis Publishers: Boca Raton, FL, 1992; Vol. 2, Chapter 4. (38) Zhou, S.; Burger, C.; Chu, B.; Sawamura, M.; Nagahama, N.; Toganoh, M.; Hackler, U. E.; Isobe, H.; Nakamura, E. Science 2001, 291, 1944. (39) Savariar, E. N.; Aathimanikandan, S. V.; Thayumanavan, S. J. Am. Chem. Soc. 2006, 128, 16224.
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and filtered through a filter with a porous diameter of 0.03 µm, which should retain the vesicle-like aggregates. The concentration of the filtered sample was determined by measuring the amount of sample retained by the filter (∼19%) of the initial mass. Under these conditions, a monomodal distribution of aggregates in solution is observed, their hydrodynamic radius being 2.6 nm (polydispersity 0.14). Similar results are obtained after 24 h. That value is close to those values obtained from unfiltered solutions of similar initial concentration resolved by CONTIN (see above). Under these conditions, ∆R90 ) 1.35 × 10-5, and by accepting a negligible angle dependence (an approximation supported by the low value of Rh) we have calculated a value of 1.3 × 104 g · mol-1 for the molar mass of the micelle. This gives a value of 12 for the aggregation number, which is close to those values for natural bile salts determined from DLS.32 The isolation of vesicles by filtering the solution is reminiscent of the observations by Schurtenberger et al.40 for a bile salt/lecithin solution. These authors have observed that is possible to almost completely remove by dialysis the bile salt molecules from mixed vesicles prepared by dilution, without changing the vesicle size by more than 10%. They presume that such systems are in a metastable state. From previous parameters and Mapp ) 5.8 × 107g · mol-1 for the large aggregate, densities of 0.28 g cm-3 and 6.8 × 10-4 g cm-3 are estimated for micelles and vesicle-like aggregates, respectively. This large difference confirms the different nature of both aggregates, although the vesicle-like aggregate value is probably underestimated. The last value is comparable to published values (1.3-1.8 × 10-3 g cm-3) for homopolymers forming large spheres with a hollow cavity.41 Surface tension, fluorescence intensity of pyrene, and light scattering (static and dynamic) measurements evidence the existence of a threshold cac. Below the cac, a multilayer film is formed at the air-water interface, while above it vesicle-like aggregates in equilibrium with micelles are present, i.e., transitions from a multilayer to micelles or vesicle-like aggregates are involved. Kumaran42 has discussed the formation of vesicles by weakly charged membranes due to spontaneous curvature, as a consequence of an asymmetry in the charge density on the two layers. The decrease in the electrostatic energy when the membrane forms a vesicle would compensate for the increase in the bending energy of the membrane. This could be the driven force for the spontaneous formation of vesicle-like aggregates in this system. Furthermore, above the cac, vesicle-like aggregates and micelles are simultaneously present in solution, although micelles are favored at high gemini concentrations, suggesting a vesicle-micelle transition. Micelle-vesicle transitions have often been observed for mixtures of cationic and anionic surfactants43,44 by the addition of inert salts or by varying the anionic/cationic surfactant ratio. In some cases, the transition can be induced by the addition of apolar hydrocarbons45 or the addition of divalent metal cations in a gemini surfactant.46 The ratio of vesicles to micelles can be actively controlled by the addition of polar (40) Schurtenberger, P.; Mazer, N. A.; Ka¨nzig, W. J. Phys. Chem. 1985, 89, 1042. (41) Duan, H.; Chen, D.; Jiang, M.; Gan, W.; Li, S.; Wang, M.; Gong, J. J. Am. Chem. Soc. 2001, 123, 12097. (42) Kumaran, V. J. Chem. Phys. 1993, 99, 5490. (43) Yatcilla, M. T.; Herrington, K. L.; Brasher, L. L.; Kaler, E. W.; Chiruvolu, S.; Zasadzinski, J. A. J. Phys. Chem. B 1996, 100, 5874. (44) Renoncourt, A.; Vlachy, N.; Bauduin, P.; Drechsler, M.; Touraud, D.; Verbavatz, J.-M.; Dubois, M.; Kunz, W.; Ninham, B. W. Langmuir 2007, 23, 2376. (45) Yin, H.; Lei, S.; Zhu, S.; Huang, J.; Ye, J. Chem. Eur. J. 2006, 12, 2825. (46) Huang, X.; Cao, M.; Wang, J.; Wang, Y. J. Phys. Chem. B 2006, 110, 19479.
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additives.45 In our case, none of the above-mentioned variables were modified since only the total concentration of the gemini have been changed. This phenomenon is reminiscent of those occurring in systems of mixed micelles of species with different solubilities in water. In these systems, the dilution beyond a limit concentration gives rise to a micellar growth because of a progressive loss of the more soluble surfactant from micelles and a consequent increase of micellar low soluble surfactant percentage. Schurtenberger et al.40 observed a micelle-to-vesicle transition in aqueous solutions of bile salts and lecithin upon dilution. According to these authors, the phase limit between these species is reached when, upon dilution, the remaining bile salt molecules are too few to solubilize the lecithin in diskshaped mixed micelles with a finite size. This is due to the fact that the amount of bile salt in the mixed micelles (lecithin/bile salt) decreases by diluting the system. For the present case, the mechanism of the growth process is unclear, and only some considerations can be outlined. pKa values in the ranges 3.5-4.4 and 6.7-7.3 have been published for the dissociation of the tertiary amino groups of EDTA diamides.47 It is expected that g-2DC24-AEDT has pKa values in those intervals.48–50 Thus, in buffer solution (pH∼10), only a very small amount of monoanionic form is present in equilibrium with the most abundant dianionic form. The calculation of concentration of all species in the bulk solution is straightforward, as the system can be simplified because of the large difference (3 pKa units) between the basicity of the two nitrogen atoms in the EDTA bridge. Thus the ratio [dianion]/[anion] is equal to Ka/[H+]. For instance for pKa ) 7.3 and pH ) 9.3, the ratio is 100:1. However, hydrogen ions have the tendency to be adsorbed onto bile salt micelles as they form (micellization tends to increase the pH of the solution), 28 and acidic species in micelles and at the interface are present in a more appreciable extent than in the bulk aqueous solution.51 Thus micelles of natural bile salts are mixed micelles formed by species with a different degree of ionization in which anionic bile salt solubilizes neutral forms.51,52 Furthermore, protons are adsorbed more weakly onto smaller aggregates than onto larger aggregates, 28 meaning that neutral forms (with a much lower solubility) are better accommodated in larger aggregates than in smaller ones. In analogy with previous phenomena observed for natural bile salts, we propose that g-2DC24-AEDT aggregates are formed by a mixture of dianionic and monoanionic species; and that the proportion of dianionic
species/monoanionic species is probably higher for micelles than for vesicle-like aggregates. The previous hypothesis means that species with a lower proportion of dianionic species are favored by dilution since large aggregates are mainly formed at low surfactant concentrations. Lowering the concentration of surfactant reduces the total number of aggregates that will be surrounded by the same concentration of protons, provided that pH is kept constant. This will increase the probability of proton adsorption for a given aggregate. Additional dianionic species have to join the aggregate to maintain the ratio monoanionic species/dianionic species under an acceptable (unknown) limit in the aggregate. This would accrue the size of the aggregate, facilitating the adsorption of additional protons, leading to a further increment of the aggregate size. It is important to remark, however, that other processes such as those involving the binding equilibrium of the counterion to the micelles can affect the growth. These equilibriums could slightly change by dilution and sensitively bias the structure of the EDTA derivative dianionic polar head with relevant consequences on the gemini aggregation. Further work is in progress to outline a detailed mechanism. However, the high puritiy of our gemini surfactant allow us to rule out hydrophobic impurities as being responsible for the light scattering results.
(47) Danil de Namor, A. F.; Pacheco Tanaka, D. A. J. Chem. Soc., Faraday Trans. 1998, 94, 3105. (48) Wang, Y.-M.; Wang, Y.-J.; Wu, Y.-L. Polyhedron 1998, 18, 109. (49) Fisher, A. E. O.; Naughton, D. P. Transition Met. Chem. 2004, 29, 315. (50) Platas-Iglesias, C.; Corsi, D. M.; Vander Elst, L.; Muller, R. N.; Imbert, D.; Buenzli, J.-C. G.; Toth, E.; Maschmeyer, T.; Peters, J. A. Dalton Trans. 2003, 727. (51) Carey, M. C. Hepatology 1984, 4, 66S (52) Igimi, H.; Carey, M. C. J. Lipid Res. 1980, 21, 72.
Supporting Information Available: Synthesis of the dimethyl ester of EDTA; synthesis of 24-deoxycholate amine; synthesis scheme of g-2DC24-EDTA; 1H and 13C NMR spectra of previous compounds; FAB-MS spectra of the dimethyl ester and diacid form of g-2DC24EDTA. This material is available free of charge via the Internet at http://pubs.acs.org
Conclusion A new gemini steroid surfactant, g-2DC24-AEDT, derived from 3R,12R-dihydroxy-5β-cholan-24-amine (steroid residue) and EDTA (spacer) was synthesized and characterized in aqueous solution. Surface tension measurements evidence the existence of a threshold concentration, or cac, that is 3 orders of magnitude lower than the cmc for sodium deoxycholate. Below the cac, a three-layer film is formed at the air-water interface, and above the cac two types of aggregates, small micelles and vesicle-like aggregates, coexist in a metastable state since they can be separated by filtration. Micelles have a very apolar region with a polarity similar to that of cyclohexane. Acknowledgment. The authors from USC thank the Ministerio de Ciencia y Tecnologı´a (Project MAT2004-04606) and Xunta de Galicia (PGIDIT05PXIC26201PN) for financial support. The authors from “La Sapienza” are thankful for the MUR financial support (PRIN 2006039789.00). We also acknowledge the Spain-Italy Integrated Action grant.
LA7035218
