5880
J. Phys. Chem. 1996, 100, 5880-5887
Toward Building a Better Microreactor: Increasing Microenvironmental Diversity in AOT Reversed Micelles Using a Bile Salt Cosurfactant Scott S. Lee, Alecia M. Rideau, and Linda B. McGown* Department of Chemistry, P.M. Gross Chemical Laboratory, Duke UniVersity, Box 90346, Durham, North Carolina 27708-0346 ReceiVed: September 12, 1995; In Final Form: January 19, 1996X
Fluorescence probe studies show that sodium taurocholate (NaTC), a trihydroxy bile salt, can be used not only to increase the water capacity of AOT reversed micelles but also to modify the micellar organization in order to alter molecular mobility, permeability, and microenvironmental polarity at the interfacial regions. Measurements of fluorescence intensity, spectra, anisotropy, and lifetimes were used in combination with absorption spectroscopy and classical and dynamic light scattering to provide insight into the effects of NaTC on microenvironmental heterogeneity experienced by the probes, which included the hydrophilic fluorescein and lipophilic ANS and NBD-hexanoic acid (NBD-HA), in the novel, four-component system of heptane/ (AOT, NaTC)/water. Small amounts of NaTC were found to disrupt the AOT micellar organization, particularly at the AOT/water interface. At higher NaTC, there is an increase in micellar size as well as structural organization that leads to higher probe anisotropy. At the AOT/water interface, NaTC creates a more polar, water-like environment for fluorescein and decreases the efficiency with which fluorescein is quenched by Tb3+, while at the heptane/AOT interface, NaTC creates more apolar environments for ANS and NBD-HA and increases the efficiency with which ANS is quenched by CCl4. At no or low NaTC, heating to 45 °C has little effect on the micelles; at higher NaTC, heating expands the micelles and disrupts their organization.
Introduction Reversed micelles have been the subject of extensive basic and applied research because of their inherently interesting chemistry as well as their diverse applications in such fields as petroleum, agriculture, and pharmaceuticals. Recently, reversed micelles have excited interest as microreactors for processes such as enzyme catalysis and drug delivery.1,2 A typical reversed micellar system includes the amphiphilic (detergent) compound, water, and the bulk organic solvent. The addition of a cosurfactant can be used to further stabilize, modify, and control the reversed micellar structure. The cosurfactant is a compound that would not be used as the primary amphiphile because of its poor solvency but is blended with other molecules to form the surfactant system.2 Among the amphiphiles capable of forming reversed micelles, the anionic surfactant AOT (sodium bis(2-ethylhexyl) sulfosuccinate) has received particular attention because of its ability to solubilize relatively large amounts of water in a variety of hydrophobic organic solvents. The water is accommodated in the polar centers of the aggregates in spherical pools, the sizes of which can be controlled by the AOT/water ratio. The addition of an alcohol of appropriate chain length, such as butanol or pentanol, can extend the stable, microemulsion phase toward increased water capacity in the AOT system.3-6 The hydrophilic, hydroxyl head groups of the alkanols assist in the formation of microemulsions, acting as both cosurfactant and oil. We have been studying the formation of reversed micelles in a novel, four-component system of heptane/(AOT, bile salt)/ water in which bile salt serves as a cosurfactant. Bile salts are amphiphilic detergents that comprise the most important class of biological detergents,7-9 participating in numerous processes such as intestinal hydrolysis, dispersion, and digestion of lipids, * Corresponding author. X Abstract published in AdVance ACS Abstracts, March 1, 1996.
0022-3654/96/20100-5880$12.00/0
cholesterol solubilization, and drug absorption. Since bile salts are essentially insoluble in the organic solvents, they will be associated with AOT in the organized micellar interface and may also form small aggregates within the interior water pool. The presence of bile salt monomers and aggregates will provide new binding microenvironments within the micellar structure for macromolecules that are contained within the reversed micelle as well as for small molecules that may exist in one or more of the phases. This paper reports on fluorescence probe studies of an AOT reversed micellar system in which sodium taurocholate (NaTC), a trihydroxy bile salt, is the cosurfactant. The fluorescent probes fluorescein, ANS (2-anilinonaphthalene-6-sulfonic acid), and NBD-HA (nitrobenzoxadiazole-hexanoic acid) were used to study microenvironmental heterogeneity in this novel, fourcomponent system of heptane/(AOT, NaTC)/water. Measurements of fluorescence spectra, intensity, anisotropy, and lifetime of the probes, as well as absorption spectroscopy and static and dynamic light scattering, were used to study the effectiveness of NaTC as a cosurfactant and its influence on the molecular structure and organization of the reversed micellar media. The effects of NaTC, AOT, and water concentrations, fluorescence quenchers, electrolytes, and temperature were investigated. Experimental Section Materials. NaTC (ULTROL grade, >98%, Calbiochem), fluorescein (sodium salt, Sigma), phosphate buffer (Sigma), ANS (Molecular Probes), NBD-HA (Molecular Probes), AOT (Fluka), n-heptane (99%, spectrophotometric grade, Aldrich), carbon tetrachloride (Fisher), and terbium(III) nitrate hexahydrate (Aldrich) were all used as received. Absolute ethanol (Aaper Alcohol and Chemical Co., Shelbyville, KY) and deionized, HPLC-grade water were used for solution preparations. Stock solutions of the fluorescent probes were prepared in ethanol. Samples were prepared by evaporat© 1996 American Chemical Society
Building a Better Microreactor ing the ethanol from an appropriate volume of the probe stock solution with a gentle stream of nitrogen, followed by addition of NaTC, then the AOT/heptane solution, and then water or, for the pH studies, buffer. The resulting solution was stirred by a magnetic stir bar at room temperature. Solution pH for the pH studies was adjusted by addition of sodium hydroxide or hydrochloric acid. Solutions were not deoxygenated prior to measurement. Measurements. Steady-state fluorescence and static scattered light were measured using a multifrequency, phase modulation spectrofluorometer (Model 48000S, Spectronic Instruments, Inc., Rochester, NY) with 450 W xenon arc lamp excitation, singlegrating monochromators for excitation and emission wavelength selection, and PMT detection. A reference channel is used for ratiometric correction of intensity fluctuations. The sample compartment was maintained at 25.0 ( 0.1 °C for most measurements with a Haake A81 circulating water bath. For temperature studies, the sample compartment was maintained at the desired temperature and the samples were equilibrated in the sample compartment for at least 15 min prior to measurement. All solutions were contained in quartz cuvettes for measurement. Fluorescence excitation and emission spectra were collected at 1 nm scanning intervals with the monochromator entrance and exit slits set to 2 nm. Scattered light intensity was measured by setting the excitation and emission monochromators to 600 nm and detecting the scattered light at 90° relative to the excitation beam. Fluorescence anisotropy was measured in the L-format. Excitation wavelengths were selected by monochromator, and emission was selected by band-pass filters. Excitation/emission wavelengths (in nm) were 483/520 for fluorescein, 320/404 for ANS, and 465/530 for NBD-HA. Fluorescence lifetimes were measured using a multiharmonic Fourier transform (MHF) phase-modulation fluorometer (Model 4850MHF, Spectronic Instruments, Inc.). For fluorescein and NBD-HA, the 488 nm line of an Ar+ laser (OmniChrome Model 543) was used for excitation and a 520 nm, 10 nm bandwidth band-pass interference filter (Oriel) was used in the emission channel. For ANS, a He-Cd laser (LiCONiX Model 4240NB) provided excitation at 325 nm and a 400 nm, 10 nm bandwidth band-pass interference filter (Oriel) was set in the emission channel. Phase and modulation data were collected simultaneously at 50 modulation frequencies generated from a base frequency of 4.1 MHz and extending up to 205 MHz. The base cross-correlation frequency for detection was 7.000 MHz. The lifetime results were calculated from three replicate measurements in which each measurement was, in most cases, the internal average of 100 samplings taken over a 24 s interval. For all samples, lifetime data were averaged over 15 measurements of sample-reference pairs. The reference solution was either a scattering solution (τref ) 0 ns) or an ethanolic solution of BODIPY493/503 (τref ) 5.88 ns; Molecular Probes) or benz[k]fluoranthene (τref ) 7.63 ns; AccuStandard). Lifetime data were analyzed using two different methods, each in a commercial software package: Non-Linear Least Squares (NLLS) from Globals Unlimited, Urbana, IL and the Maximum Entropy Method (MEM) from Maximum Entropy Data Consultant Ltd., Cambridge, UK. NLLS fits the data to an a priori model that may include one or more discrete components or a continuous distribution such as a Gaussian or Lorentzian distribution. The best model for the data is determined from minimization (toward unity) of a goodness-of-fit parameter, χ2, and randomness of the fitting residuals. MEM is a self-modeling approach in which a unique solution to the data is obtained through simultaneous minimization of the χ2 goodness-of-fit criterion and maximiza-
J. Phys. Chem., Vol. 100, No. 14, 1996 5881 tion of a statistical entropy function.10 In this work, the MEM analysis employed 200 terms that were logarithmically spaced over a lifetime window of 0.1-100 ns. In both MEM and NLLS analyses, the experimentally derived standard deviations of the replicate measurements were used to represent the uncertainties of the phase and modulation data. Quasi-elastic light-scattering measurements were performed using a Malvern Instruments 4700 system with a PCS 100SM autoindexing spectrometer goniometer and a K-7032 automeasure system at room temperature. The 632.8 nm line of a HeNe laser (Spectra-Physics Model 127) or the 488 nm line of an Ar+ laser (OmniChrome Model 543) was used for excitation, and data were collected at angles between 20 and 80° with the aperture size set between 100 and 200 µm. UV-visible absorption spectra were collected by a Perkin Elmer Lambda 6 spectrophotometer. Results and Discussion Selection of Extrinsic Fluorescent Probes. The hydrophilic probe fluorescein and the lipophilic probes ANS and NBDHA were selected in order to explore different locations in the reversed micellar phases. Other fluorescent probes were also investigated, including a series of pyrene derivatives (pyrene, pyrenebutyric acid, and pyrenesulfonic acid) and naphthalene derivatives (2-naphthol, 2-naphthalenesulfonic acid, and 1,5naphthalenedisulfonic acid). Although different hydrophobicities of these derivatives could likely place them in different locations in the AOT reversed micelles, the fluorescence intensities of these probes were largely unaffected by the addition of NaTC. Other xanthene dyes, including rose bengal that differs from fluorescein only by aromatic ring substitution, were also studied. However, among all the fluorescent probes studied, fluorescein and ANS exhibited the largest changes in fluorescence intensity upon NaTC addition. NBD-HA was included because of the high sensitivity of its fluorescence lifetime to solvent polarity. Effects of NaTC. Unless otherwise noted, the reversed micellar system to which NaTC was added is heptane/40 mM AOT/400 mM water, giving a water:AOT ratio (ω) of 10. It should be noted that the ω value refers to the ratio of the total concentrations of water and AOT that are added to the system and does not necessarily describe the actual ratios of water: AOT in each of the individual reversed micelles. NaTC concentration ranged from e1 mM to 20 mM, which is the limit of NaTC solubility in this particular reversed micellar phase. Fluorescein. For fluorescein, the addition of NaTC causes a dramatic increase in fluorescence intensity. Even as little as 1 mM NaTC increases intensity by a full order of magnitude. The increase is due in part to increased absorbance (Figure 1). However, even after the fluorescence intensities have been adjusted to account for increased absorbance by dividing the measured intensity by the absorbance of the solution at that wavelength, there remains a 3-4-fold enhancement between 0 and 1 mM NaTC (Figure 2). Above 1 mM NaTC, the absorbance-adjusted intensity levels off, exhibiting a slight maximum around 7 mM NaTC. As shown in Table 1, which compares the reversed micellar media to simple solvents, the intensity of fluorescein increases with increasing solvent polarity and the addition of NaTC increases the polarity experienced by fluorescein in the reversed micelles. The intensity is still much lower than in bulk water, which indicates that fluorescein is not solubilized in an aqueous continuum in the micellar core and is probably associated with the detergent tail groups at the AOT/water interface; the addition of NaTC creates a more polar, and possibly more restricted and
5882 J. Phys. Chem., Vol. 100, No. 14, 1996
Lee et al. TABLE 1: Fluorescence Intensity of Fluorescein and Fluorescence Maximum of ANS in Simple Solvents and in Reversed Micelles 1 µM fluorescein in water
fluorescence intensity (arbitrary units) 14.64
1 µM ANS in
λEM (max) (nm)
water
457
30% ethanol/70% water
439
heptane/(40 mM AOT, 20 mM NaTC)/water; ω ) 10
4.28
50% ethanol/50% water
438
heptane/(40 mM AOT, 5 mM NaTC)/water; ω ) 10
2.13
ethylene glycol
428
ethanol
1.00
heptane/40 mM AOT/ water; ω ) 10
428
heptane/40 mM AOT/ water; ω ) 10
0.08
heptane/(40 mM AOT, 5 mM NaTC)/water; ω ) 10
421
heptane
0.00
acetonitrile
417
heptane/(40 mM AOT, 413 20 mM NaTC)/water; ω ) 10 ethanol
405
Figure 1. Absorbance spectra of 1 µM fluorescein in water, ethanol, and reversed micelles (heptane/(40 mM AOT, NaTC)/water, ω ) 10) at different NaTC concentrations.
Figure 3. Properties of 1 µM fluorescein vs ω (heptane/40 mM AOT/ water).
Figure 2. Properties of probes vs NaTC concentration (heptane/(40 mM AOT, NaTC)/water, ω ) 10): absorbance-adjusted fluorescence intensity of fluorescein (b); ANS (2); NBD-HA (1); emission maximum of ANS ([); scattered light intensity (9).
protected, binding environment for fluorescein at the interface. In the absence of NaTC, increasing ω causes only modest increases in fluorescein intensity, which passes through a maximum at ω ) 30 (Figure 3). The effects of NaTC are therefore not solely due to increased solubilization of water in the reversed micellar interior. The pH-dependence of fluorescein intensity is probably not responsible, since the pH of the added water had little effect on the intensity of fluorescein in the AOT reversed micellar system with or without NaTC. This is in contrast to the sharp rise in intensity that is observed for fluorescein in water between pH 4.0 and 4.5, which coincides with its pKa. In the absence of information about the actual pH of the aqueous pool within the reversed micellar core, however, further conclusions about the effect of pH on fluorescein cannot be drawn.
The UV-visible absorption spectra of fluorescein (Figure 1) show a red shift in the absorption maximum as NaTC is added and a decrease in the relative contribution from spectral features to the blue of the major peak. Thus, as NaTC is added, the spectrum becomes less like that in ethanol and more like that in water. However, the features at the blue edge persist even at the high NaTC concentrations, supporting the location of fluorescein at the interface rather than in the interior water pool. These blue-edge spectral features were previously observed for fluorescein isothiocyanate in aqueous micellar solutions of NaTC,11 which supports association of fluorescein with NaTC at the reversed micellar interface. The lack of spectral features at 0 NaTC relative to either water or ethanol indicates that fluorescein is located in an environment very unfavorable to fluorescence in the AOT reversed micellar structure in the absence of NaTC. The fluorescence anisotropy of fluorescein initially decreases as NaTC is increased from 0 to 1 mM and then increases as more NaTC is added (Figure 4). The miminum value at 1 mM NaTC is equal to the anisotropy in the absence of NaTC at much higher ω (Figure 3). All of the anisotropy values are higher
Building a Better Microreactor
J. Phys. Chem., Vol. 100, No. 14, 1996 5883
Figure 4. Fluorescence anisotropy of probes (1 µM) vs NaTC concentration (heptane/(40 mM AOT, NaTC)/water, ω ) 10). Limiting anisotropy (r0) is given for each probe in legend.
Figure 5. Fluorescence lifetimes of probes (1 µM) vs NaTC concentration (heptane/(40 mM AOT, NaTC)/water, ω ) 10), recovered from MEM analysis. In cases of multiexponential decay, only the major lifetime component is shown.
than values that have been reported for fluorescein isothiocyanate in aqueous solutions of NaTC up to 20 mM,11 which is further evidence that fluorescein in the reversed micellar system is associated with NaTC among the AOT molecules rather than being free or associated with NaTC in the aqueous core. In the reversed micelles, the anisotropy is determined by the rotational motions of the probe within the micelle and by the overall rotation of the micelle, which tends to become slower as it expands. Since scattered light intensity of the reversed micelles (Figure 2) does not decrease upon addition of low concentrations of NaTC, it is unlikely that the initial decrease in anisotropy is due to a decrease in aggregate size. Instead, there is probably an increase in the local rotational freedom of the probe as the AOT organization is disrupted by the occasional NaTC molecule. As more NaTC is added, it offers sites for fluorescein in an increasingly organized NaTC-AOT structure that expands to accommodate additional water in the core. The anisotropy of fluorescein increases in response to the more rigid, NaTC binding environments and the increasing micellar size. In the absence of NaTC, increasing ω from 2 to 60 decreases the anisotropy, which suggests that fluorescein is freer to rotate as the size of the water pool increases in the absence of tight binding interactions with NaTC at the micellar interface. Fluorescence lifetime analysis of fluorescein consistently yielded two lifetime components, the expected lifetime component of 4-5 ns, which generally provided at least 90% of the total intensity, and a shorter, minor lifetime component of 1-3 ns that may be due to fluorescein aggregation at the micellar interface. No trends were observed in the lifetimes with increasing NaTC concentration. ANS. The properties of ANS (quantum yield, fluorescence emission maximum, and lifetime) are extremely sensitive to microenvironmental polarity. In the AOT reversed micelles, the addition of NaTC causes a dramatic increase in fluorescence intensity of ANS. The absorbance-adjusted intensity (Figure 2) generally increases with increasing NaTC and eventually begins to level off. There is a blue shift in the emission maximum, also shown in Figure 2, which indicates a decreasingly polar microenvironment approaching the polarity of bulk ethanol. These properties were relatively independent of the pH of the added water in the range 2-12. This is not unexpected, since ANS is not in contact with the aqueous
solution or the polar regions of the micellar interface, but it does indicate that changes in pH of the added water do not significantly alter the overall micellar structure. The effects of NaTC on the fluorescence anisotropy of ANS are shown in Figure 4. The curve passes through a minimum that is less pronounced, and at a higher NaTC concentration (around 3-5 mM NaTC), than was observed for fluorescein. The ANS anisotropy is also closer to the limiting anisotropy. The initial decrease reflects the disorganization at the heptane/ AOT interface caused by small amounts of NaTC followed by the increase in organization and size at higher NaTC that was also indicated by fluorescein. ANS lifetime increases with increasing NaTC concentration (Figure 5), exceeding the lifetime in ethanol at 20 mM NaTC, and the widths of the lifetime peaks decrease in the distributions recovered from MEM analysis. This suggests that the ANS microenvironment becomes less polar and more homogeneous with increasing NaTC. Small (
