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Micellization of bile salts in aqueous medium: A fluorescence study
Colloids and Surfaces B: Biointerfaces 57 (2007) 102–107
Micellization of bile salts in aqueous medium: A fluorescence study Usharani Subuddhi, Ashok K. Mishra ∗ Department of Chemistry, Indian Institute of Technology-Madras, Chennai 36, India Received 4 January 2007; received in revised form 23 January 2007; accepted 23 January 2007 Available online 30 January 2007
Abstract Owing to the physiological importance of the micellization process of bile salts, the critical micelle concentration (CMC) becomes a fundamental parameter in the evaluation of their biological activities. The present study suggests fluorescence probing, using 1,6-diphenylhexatriene (DPH), as a simple, convenient, sensitive and economic method for monitoring the micellization process of bile salts in aqueous medium. Three independent parameters: fluorescence intensity, anisotropy and lifetime of DPH have been employed successfully for determining the CMC of two bile salts, sodium deoxycholate (NaDC) and sodium cholate (NaC), in aqueous medium. The CMC values reported by all the above three parameters of DPH are found to be same and it is 16 mM for NaC and 6 mM for NaDC at 25 ◦ C in unbuffered solution. The effect of temperature and ionic strength on the micellization process has also been investigated employing DPH as a fluorescent probe. Increasing temperature leads to the formation of fluffier micelles with less rigid interior for both NaC and NaDC. The micelle core of NaC is less perturbed by the presence of NaCl whereas in case of NaDC, the aggregates provide DPH a more nonpolar and rigid environment in presence of NaCl than that in absence of salt. © 2007 Elsevier B.V. All rights reserved. Keywords: Bile salt; Sodium cholate; Sodium deoxycholate; Micelles; Fluorescence; 1,6-Diphenylhexatriene
1. Introduction The physiological importance of bile salts lies in their ability to solubilize and emulsify cholesterol, dietary lipids and fatsoluble vitamins in the gastrointestinal tract [1]. Apart from these biological applications, bile salts also have got much recognition as delivery systems for medicines, cosmetics and several other chemicals because of their unusual solubilizing and emulsifying capacity. The detergent action of bile salts is mainly due to their micelle-forming capacity and these are known as “biosurfactants”. In this context, the critical micelle concentration (CMC) becomes a fundamental parameter in the evaluation of their biological activity. The decisive factor for any molecule to form micelle is its amphiphilicity, i.e. the presence of both polar and nonpolar groups in the same molecule. Bile salts have a large, rigid, and hydrophobic steroid nucleus with hydrophilic moieties of two or three hydroxyl groups and an ionic head of a carboxyl group, which provide the molecule a planar polarity with hydrophilic and hydrophobic domains (Scheme 1). Because of their unique molecular structure, bile salts do not behave anal-
∗
Corresponding author. Tel.: +91 44 22574207; fax: +91 44 22574202. E-mail address: [email protected] (A.K. Mishra).
0927-7765/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2007.01.009
ogous to conventional surfactants that contain clear-cut polarity gradient between the hydrophilic and hydrophobic parts. They show distinct behaviour with respect to self-association and molecular solubilization, the CMC for a given bile salt being largely determined by its hydrophilic:hydrophobic balance. Interplay of the effects of numbers and orientations of various ring hydroxyls, length and polarity of the side chain contributes to the hydrophilic:hydrophobic balance of bile salt molecules and play an important role in its micellization behaviour [2]. The process of micellization in a surfactant solution brings about several changes, which can be related to appreciable alterations in phenomena, such as light scattering, surface tension, viscosity, solubilization of other organic molecules etc. These changes are the basis of several analytical techniques that can be employed for the investigation of micelles formation [3–13]. Because of the absence of a well-defined critical point in most of the experimental curves obtained from different techniques the values of CMC reported in the literature vary drastically depending on the technique used [11]. The reported values range from 3 to 19 mM for NaC and 2–10 mM for NaDC. Fluorescence technique has long been used in the structural and dynamics study of microheterogeneous media [14]. The sensitivity of a fluorescent molecule to its immediate environment and the instantaneous response of it to the changes in the
U. Subuddhi, A.K. Mishra / Colloids and Surfaces B: Biointerfaces 57 (2007) 102–107
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Scheme 1. Molecular structures of sodium cholate (NaC), sodium deoxycholate (NaDC) and 1,6-diphenylhexatriene (DPH).
microenvironment make fluorescence a versatile technique to be used for acquiring important information about these systems. The four independent fluorescence parameters namely, fluorescence peak maximum, intensity, anisotropy and lifetime provide valuable information about different aspects of these constrained media. The measured changes in these fluorescence parameters of a fluorescent probe can be easily related to various molecular properties of the environment, such as polarity, viscosity, diffusion coefficients, distance between groups, structural changes due to existence of different phases, formation of microstructures and microdomains. In this study, the fluorescent molecule, 1,6-diphenylhexatriene (DPH) (Scheme 1), has been used for monitoring the micellization process of two bile salts, sodium cholate (NaC) and sodium deoxycholate (NaDC), in aqueous medium. DPH offers efficient tools in terms of its three independent parameters, fluorescence intensity, anisotropy and lifetime for monitoring the process of micellization. Using fluorescence intensity at the peak maximum and anisotropy of DPH the effects of temperature and ionic strength on the micellization process of bile salts have also been investigated.
experiments at different ionic strengths, a fixed volume of NaCl stock solution was added to the experimental solution. Except for the temperature dependence experiments the temperature was always maintained at 25 ◦ C for all experiments. 3.1. Fluorescence measurements
2. Experimental
Fluorescence measurements were carried out with JobinYvon-Spex Fluorolog II spectrofluorimeter. Temperature was controlled by circulating water through a jacketed cuvette holder from a refrigerated bath (INSREF Ultra Cryostat, India). In fluorescence spectrum, the intensity is often expressed in arbitrary units, but in the present case the absolute intensities are given in order to make a comparison between the environments of DPH in NaDC and NaC micelles, where DPH concentration is kept constant at 5 × 10−7 M. The steady state fluorescence anisotropy (rss ) values were obtained using the expression rss = (I − GI⊥ )/(I + 2GI⊥ ), where I and I⊥ are fluorescence intensities when the emission polarizer is parallel and perpendicular, respectively, to the direction of polarization of the excitation beam, and G is the factor that corrects for unequal transmission by the diffraction gratings of the instrument for vertically and horizontally polarized light [15].
2.1. Materials
3.2. Fluorescence lifetime measurements
1,6-Diphenylhexatriene (DPH) was purchased from Sigma Chemical Company (USA) and used as such. The bile salts, sodium deoxycholate (NaDC) and sodium cholate (NaC) were obtained from S.D. Fine Chemical Company, India and were recrystallized from hot ethanol. Water distilled twice from alkaline permanganate solution, was used for all the experiments.
Lifetime measurements were carried out using the IBH single-photon counting fluorimeter in a time-correlated singlephoton counting arrangement consisting of ps/fs Ti–Sapphire Laser system (Tsunami Spectra Physics, Bangalore, India). The pulse repetition rate was 82 MHz and the full width half maximum is less than 2 ps. The emission was collected at magic angle polarization (54.7) to avoid any polarization in the emission decay. The instrument response time is approximately 50 ps. The decay data were further analysed using IBH software. A value of χ2 , 0.99 ≤ χ2 ≤ 1.4 was considered as a good fit.
3. Preparation of bile salt solutions and labeling The stock solutions of NaDC and NaC were prepared in water and the experimental solutions were prepared by subsequent dilutions from the stock. Fresh solutions of bile salt were prepared every time to avoid the problem of aging. The pH of the stock solution was found to be ∼8, which did not change significantly after dilution. Solutions of DPH in bile salt were prepared by passing a gentle stream of nitrogen gas over the appropriate volume of the DPH stock solution to evaporate THF and by redissolving it in the bile salt solution by sonication. The solutions were left for 2 h to achieve equilibrium. The DPH concentration was maintained at 5 × 10−7 M for all the experiments. For the
4. Results and discussion Fig. 1A and B represents the emission spectra of DPH in NaC and NaDC solutions with increasing concentration of the two bile salts. DPH, which is practically nonfluorescent in aqueous medium show a remarkable increase in intensity in both NaC and NaDC. Initially, when the bile salt concentration is low, the intensity is almost close to zero and with increase in bile salt concentration it shows a steep increase and then it attains saturation.
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Fig. 1. Emission spectra of DPH with increasing concentration of (A) NaC and (B) NaDC. Temperature = 25 ◦ C, [DPH] = 5 × 10−7 M, [NaC] = 0–30 mM, [NaDC] = 0–20 mM, λex = 360 nm.
4.1. Fluorescence intensity as the parameter to determine CMC
4.2. Fluorescence anisotropy as the parameter to determine CMC
The variation of fluorescence intensity of DPH with concentration of the two bile salts is shown in Fig. 2A and B. The fluorescence intensity in both cases shows a sigmoid variation with the concentration of bile salt. The point of intersection of the horizontal line and the line of inflation represents the CMC and it is found to be 16 mM for NaC and 6 mM for NaDC (shown as dashed lines in Fig. 2A and B). The remarkable increase in the fluorescence intensity of DPH suggests that bile salt micelles provide a solubilizing hydrophobic environment to DPH molecules. This experimental finding is in agreement with Small’s model of micelle formation, which says the aggregation of bile salts in aqueous medium takes place through the back-to-back stacking of the hydrophobic planes of the molecules [16]. For the same concentration of probe (5 × 10−7 M), the increase in intensity is almost four times higher in NaDC solution as compared to that in NaC solution under similar experimental conditions. This implies that the dihydroxy bile salt micelles provide more hydrophobic microenvironment to DPH as compared to that of trihydroxy bile salts. Trihydroxy bile salts being more hydrophilic form looser and smaller micelles than dihydroxy bile salts [17].
DPH is one of the most widely used fluorescence anisotropy probes, which because of its rod-shaped structure shows high retention of polarization. In an organized media, the anisotropy of DPH reflects the resistance offered by the microenvironment to its rotational movement; hence, the change in the polarization of DPH fluorescence acts as an index for the change in microviscosity of the immediate environment [18]. Fig. 3A and B shows the variation of steady state fluorescence anisotropy of DPH as a function of respective bile salt concentrations. Initially, when the concentration of bile salt is low, the anisotropy is found to be high, which can be due to the hydrophobic aggregation of DPH in aqueous medium. As the bile salt concentration increases there is a pronounced solubilization of DPH and a corresponding break down of DPH aggregates resulting in the decrease in the anisotropy value. After the CMC, when bile salt exists as micelles, DPH is completely solubilized in the hydrophobic core that has lower microviscosity; hence, anisotropy is low and remains independent of concentration. The surfactant concentration at which the anisotropy value becomes independent of concentration is considered as the CMC of the surfactant [18]. Thus, the CMCs for NaC and NaDC are found to be 16 and 6 mM, respectively. Thus, two totally independent parameters of DPH, i.e. fluorescence intensity and anisotropy,
Fig. 2. Plots showing variation of fluorescence intensity of DPH at emission maximum (428 nm) as a function of concentration of (A) NaC and (B) NaDC. Temperature = 25 ◦ C, [DPH] = 5 × 10−7 M, λex = 360 nm. The error in the data as calculated from three independent sets of experiments is within ±5%.
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Fig. 3. Variation of steady state fluorescence anisotropy of DPH as a function of (A) NaC and (B) NaDC concentration. Temperature = 25 ◦ C, [DPH] = 5 × 10−7 M, λex = 360 nm, λem = 428 nm. The error in the data as calculated from three independent sets of experiments is within ±5%.
report the same value of CMC. The higher anisotropy value in NaDC as compared to that in NaC indicates a higher microviscosity in the interior of the deoxycholate micelle. This observation also suggests that the micelles of dihydroxy bile salts are larger and more rigid than that of the trihydroxy bile salts. 4.3. Fluorescence lifetime studies on NaC and NaDC in water
with the micelles formation DPH experiences a nonpolar environment. The slightly longer lifetime in NaDC as compared to that in NaC indicates that NaDC micelles provide relatively more nonpolar and rigid microenvironment to DPH. The ability of DPH fluorescence to truthfully reflect the CMC of bile salt micelles can be tested further by changing the experimental conditions that affect the micellization process. 4.4. Effect of temperature on CMC of bile salts
Tables 1 and 2 give the fluorescence lifetime data for DPH at different concentrations of sodium cholate and sodium deoxycholate solutions. Below 16 mM of NaC, the fluorescence shows biexponential decay with a short lifetime and a long lifetime component and above 16 mM of NaC, only the longer component exists. Similarly, in case of NaDC below 6 mM, the decay is biexponential and above this only the longer component exists. Thus, 16 mM for NaC and 6 mM for NaDC are the critical concentrations above which there is a change in the DPH environment in the solutions. These values are same as found earlier from the intensity and anisotropy studies. Thus, this indicates that fluorescence lifetime of DPH is as sensitive as its intensity and anisotropy towards the process of micellization and support the earlier findings. Below the CMC the biexponential nature of DPH decay suggests the heterogeneity in its distribution, but Table 1 Fluorescence lifetime data for DPH in sodium cholate solutions at 25 ◦ C (λex = 370 nm, λem = 428 nm) NaC concentration (mM)
τ 1 (ns)
α1 (%)
τ 2 (ns)
α2 (%)
χ2
8 10 12 14 15 16 20 24 28 32
1.03 1.45 1.60 2.41 3.24
28.59 22.44 19.04 17.52 17.28
4.64 5.86 6.09 6.85 6.90 7.01 7.11 7.21 7.23 7.32
71.41 77.56 80.96 82.48 82.72
1.26 1.27 1.01 1.20 1.01 1.25 1.24 1.17 1.15 1.17
Bile salt micelles are known to be sensitive to temperature. The CMC is known to increase with increase in temperature [19,20]. The effect of temperature on the process of micellization of NaDC and NaC was monitored by using fluorescence intensity and fluorescence anisotropy of DPH. Fig. 4A and B shows the response of fluorescence intensity and fluorescence anisotropy of DPH to temperature in sodium deoxycholate solution. As is clear from the plots both fluorescence intensity as well as anisotropy shows good response to the change in CMC of NaDC with temperature. The CMC value shifts from 5 mM at 15 ◦ C to 6 mM at 25 ◦ C, 8 mM at 35 ◦ C and to 10 mM at 45 ◦ C. Similar effect is also seen in case of NaC micellization and the CMC shifts from 15 mM at 15 ◦ C, 16 mM at 25 ◦ C and 17 mM at 35 ◦ C and 45 ◦ C. Table 2 Fluorescence lifetime data for DPH in sodium deoxycholate solutions at 25 ◦ C (λex = 370 nm, λem = 428 nm) NaDC concentration (Mm)
τ 1 (ns)
α1 (%)
τ 2 (ns)
α2 (%)
χ2
1 2 3 4 5 6 7 8 10 14 18 20
2.48 2.46 2.38 2.33 1.48
34.55 31.18 31.33 31.83 20.46
5.37 5.64 5.74 5.95 7.68 8.54 8.59 8.64 9.25 9.40 9.41 9.79
65.45 68.82 68.67 68.17 79.54
1.16 1.07 1.11 1.07 0.99 1.11 1.06 1.05 1.26 1.27 1.01 1.23
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Fig. 4. Plot of fluorescence (A) intensity and (B) anisotropy of DPH vs. NaDC concentration at various temperatures. Temperature = 15–45 ◦ C.
Fig. 5. Variation of fluorescence (A) intensity and (B) anisotropy of DPH as a function of sodium cholate concentration at different ionic strengths.
As is clearly seen in the figures, with increase in temperature the fluorescence intensity at the saturation decreases and the anisotropy value after the micellization also decreases. This suggests that the micelles’ interior becomes less hydrophobic and provides more rotational freedom for DPH at high temperature. Thus, it can be concluded that increasing temperature leads to the formation of fluffier micelles with less rigid interior.
4.5. Effect of ionic strength on CMC of bile salts The increase in ionic strength of the aqueous medium is known to promote the micellar aggregates of bile salts. The effect of counter-ion on the CMC of bile salt micelles is mainly mediated by progressive neutralization of the ionic charges on the carboxylate group. As the CMC is an index of hydrophobic interaction and ionic repulsion, reduction of the latter allows
Fig. 6. Variation of fluorescence (A) intensity and (B) anisotropy of DPH as a function of sodium deoxycholate concentration at different ionic strengths.
U. Subuddhi, A.K. Mishra / Colloids and Surfaces B: Biointerfaces 57 (2007) 102–107
micelle formation to occur at lower concentration thus resulting in a decrease in the CMC [14]. Fig. 5A and B represents the variation of fluorescence intensity and fluorescence anisotropy of DPH in NaC solution at different ionic strengths. With increase in the ionic strength, the CMC shifts to lower value. It decreased from 16 mM in absence of salt, to 12 mM at 0.1 M NaCl and 10 mM at 0.2 M NaCl. However, there is not much of change in overall intensity or in anisotropy on addition of salt, which indicates that the micellar core or more specifically the solubilizing site for DPH in NaC micelles is less affected with the addition of NaCl. The micellization process of dihydroxy bile salt NaDC shows strong dependence on ionic strength (Fig. 6A and B). The CMC drops from 6 mM in absence of salt to 3.5 mM at 0.05 M and 2.5 mM at 0.1 M of NaCl. There is almost a two-fold increase in the intensity in presence of 0.1 M NaCl as compared to that in absence of salt. The anisotropy values in the micelles also show slight increase in presence of salt. This indicates that in NaDC micelles not only the CMC is affected on addition of salt but also the nature of aggregation. The aggregates formed in presence of NaCl provide DPH a more nonpolar and rigid environment. There is literature report that says the aggregations of dihydroxy bile salts are much greater than that of trihydroxy bile salts in presence of NaCl [21]. 5. Conclusion Fluorescence properties of DPH are found to be very sensitive to the process of micellization of bile salts in aqueous medium. DPH offers efficient tools in terms of its three independent parameters: fluorescence intensity, anisotropy and lifetime for monitoring the process. The CMC thus obtained are 16 mM for NaC and 6 mM for NaDC at 25 ◦ C in an unbuffered solution. Using DPH as a probe, it is possible to investigate the effect of temperature and ionic strength on the micellization process. Increasing temperature leads to the formation of fluffier micelles with less rigid interior. The micelle core of NaC is less perturbed by the presence of NaCl whereas in case of NaDC, the aggregates provide DPH a more nonpolar and rigid environment in presence of NaCl than that in absence of salt. In summary fluorescence probing, using DPH as a molecular probe provides a simple,
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convenient, sensitive and economic method for monitoring the process of micellization of bile salts in aqueous medium. Acknowledgements Usharani thanks IIT Madras for financial assistance. The authors thank SAIF, IIT Madras for the access to the fluorimeter and Dr. P. Ramamurthy and Mr. Kumaran of NCUFP, University of Madras, India for the lifetime measurements. References [1] R. Coleman, Biochem. Soc. Trans. 15 (1987) S68. [2] D.G. Oakenfull, in: E. Wyn-Jones, J. Gormally (Eds.), Aggregations Processes in Solutions, Elsevier Scientific Publishing Company, Amsterdam, 1983, p. 118. [3] M.C. Carey, D.M. Small, Arch. Intern. Med. 130 (1972) 506. [4] E. Pramauro, E. Pelizzetti, Surfactants in Analytical Chemistry. Applications of Organized Amphiphilic Media, Elsevier, New York, 1996. [5] S. Gouin, X.X. Zhu, Langmuir 14 (1998) 4025. [6] B.R. Simonovic, M. Momirovic, Mikrochim. Acta 127 (1997) 101. [7] H. Matsuoka, J.P. Kratohvil, N. Ise, J. Colloid Interface Sci. 118 (1987) 387. [8] T. Nakashima, T. Anno, H. Kanda, Y. Sato, T. Kuroi, H. Fujji, S. Nagadome, G. Sugihara, Colloids Surf. B 24 (2002) 103. [9] P. Mukerjee, J. Cardinal, J. Pharm. Sci. 65 (1976) 882. [10] L. Antonian, S. Deb, W. Spivak, J. Lipid Res. 31 (1990) 947. [11] S. Reis, C.G. Moutinho, C. Matos, B. de Castro, P. Gameiro, J.L.F.C. Lima, Anal. Biochem. 334 (2004) 117. [12] P. Ranajit, M.K. Mathew, R. Narayanan, P. Balaram, Chem. Phys. Lipids 25 (1979) 345. [13] C. Ju, C. Bohne, Photochem. Photobiol. 63 (1996) 60. [14] K. Kalyanasundaram, in: V. Ramamurthy (Ed.), Photochemistry in Organized and Constrained Media, VCH Publishers, New York, 1991, p. 39. [15] J.R. Lakowicz, Principles of Fluorescence Spectroscopy, Kluwer Academic, Plenum Publishers, New York, 1999. [16] D.M. Small, Advances in Chemistry Series, Molecular Association in Biological and Related Systems, vol. 84, American Chemical Society, 1968, p. 31. [17] G. Li, L.B. McGown, J. Phys. Chem. 98 (1994) 13711. [18] X. Zhang, J.K. Jackson, H.M. Burt, J. Biochem. Biophys. Methods 31 (1996) 150. [19] K. Matsuoka, Y. Moroi, Biochim. Biophys. Acta 1580 (2002) 189. [20] H. Sugioka, K. Matsuoka, Y. Moroi, J. Colloid Interface Sci. 259 (2003) 156. [21] S.Z. Zhang, J.W. Xie, C.S. Liu, Anal. Chem. 75 (2003) 91.
Micellization of bile salts in aqueous medium: A fluorescence study Usharani Subuddhi, Ashok K. Mishra ∗ Department of Chemistry, Indian Institute of Technology-Madras, Chennai 36, India Received 4 January 2007; received in revised form 23 January 2007; accepted 23 January 2007 Available online 30 January 2007
Abstract Owing to the physiological importance of the micellization process of bile salts, the critical micelle concentration (CMC) becomes a fundamental parameter in the evaluation of their biological activities. The present study suggests fluorescence probing, using 1,6-diphenylhexatriene (DPH), as a simple, convenient, sensitive and economic method for monitoring the micellization process of bile salts in aqueous medium. Three independent parameters: fluorescence intensity, anisotropy and lifetime of DPH have been employed successfully for determining the CMC of two bile salts, sodium deoxycholate (NaDC) and sodium cholate (NaC), in aqueous medium. The CMC values reported by all the above three parameters of DPH are found to be same and it is 16 mM for NaC and 6 mM for NaDC at 25 ◦ C in unbuffered solution. The effect of temperature and ionic strength on the micellization process has also been investigated employing DPH as a fluorescent probe. Increasing temperature leads to the formation of fluffier micelles with less rigid interior for both NaC and NaDC. The micelle core of NaC is less perturbed by the presence of NaCl whereas in case of NaDC, the aggregates provide DPH a more nonpolar and rigid environment in presence of NaCl than that in absence of salt. © 2007 Elsevier B.V. All rights reserved. Keywords: Bile salt; Sodium cholate; Sodium deoxycholate; Micelles; Fluorescence; 1,6-Diphenylhexatriene
1. Introduction The physiological importance of bile salts lies in their ability to solubilize and emulsify cholesterol, dietary lipids and fatsoluble vitamins in the gastrointestinal tract [1]. Apart from these biological applications, bile salts also have got much recognition as delivery systems for medicines, cosmetics and several other chemicals because of their unusual solubilizing and emulsifying capacity. The detergent action of bile salts is mainly due to their micelle-forming capacity and these are known as “biosurfactants”. In this context, the critical micelle concentration (CMC) becomes a fundamental parameter in the evaluation of their biological activity. The decisive factor for any molecule to form micelle is its amphiphilicity, i.e. the presence of both polar and nonpolar groups in the same molecule. Bile salts have a large, rigid, and hydrophobic steroid nucleus with hydrophilic moieties of two or three hydroxyl groups and an ionic head of a carboxyl group, which provide the molecule a planar polarity with hydrophilic and hydrophobic domains (Scheme 1). Because of their unique molecular structure, bile salts do not behave anal-
∗
Corresponding author. Tel.: +91 44 22574207; fax: +91 44 22574202. E-mail address: [email protected] (A.K. Mishra).
0927-7765/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2007.01.009
ogous to conventional surfactants that contain clear-cut polarity gradient between the hydrophilic and hydrophobic parts. They show distinct behaviour with respect to self-association and molecular solubilization, the CMC for a given bile salt being largely determined by its hydrophilic:hydrophobic balance. Interplay of the effects of numbers and orientations of various ring hydroxyls, length and polarity of the side chain contributes to the hydrophilic:hydrophobic balance of bile salt molecules and play an important role in its micellization behaviour [2]. The process of micellization in a surfactant solution brings about several changes, which can be related to appreciable alterations in phenomena, such as light scattering, surface tension, viscosity, solubilization of other organic molecules etc. These changes are the basis of several analytical techniques that can be employed for the investigation of micelles formation [3–13]. Because of the absence of a well-defined critical point in most of the experimental curves obtained from different techniques the values of CMC reported in the literature vary drastically depending on the technique used [11]. The reported values range from 3 to 19 mM for NaC and 2–10 mM for NaDC. Fluorescence technique has long been used in the structural and dynamics study of microheterogeneous media [14]. The sensitivity of a fluorescent molecule to its immediate environment and the instantaneous response of it to the changes in the
U. Subuddhi, A.K. Mishra / Colloids and Surfaces B: Biointerfaces 57 (2007) 102–107
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Scheme 1. Molecular structures of sodium cholate (NaC), sodium deoxycholate (NaDC) and 1,6-diphenylhexatriene (DPH).
microenvironment make fluorescence a versatile technique to be used for acquiring important information about these systems. The four independent fluorescence parameters namely, fluorescence peak maximum, intensity, anisotropy and lifetime provide valuable information about different aspects of these constrained media. The measured changes in these fluorescence parameters of a fluorescent probe can be easily related to various molecular properties of the environment, such as polarity, viscosity, diffusion coefficients, distance between groups, structural changes due to existence of different phases, formation of microstructures and microdomains. In this study, the fluorescent molecule, 1,6-diphenylhexatriene (DPH) (Scheme 1), has been used for monitoring the micellization process of two bile salts, sodium cholate (NaC) and sodium deoxycholate (NaDC), in aqueous medium. DPH offers efficient tools in terms of its three independent parameters, fluorescence intensity, anisotropy and lifetime for monitoring the process of micellization. Using fluorescence intensity at the peak maximum and anisotropy of DPH the effects of temperature and ionic strength on the micellization process of bile salts have also been investigated.
experiments at different ionic strengths, a fixed volume of NaCl stock solution was added to the experimental solution. Except for the temperature dependence experiments the temperature was always maintained at 25 ◦ C for all experiments. 3.1. Fluorescence measurements
2. Experimental
Fluorescence measurements were carried out with JobinYvon-Spex Fluorolog II spectrofluorimeter. Temperature was controlled by circulating water through a jacketed cuvette holder from a refrigerated bath (INSREF Ultra Cryostat, India). In fluorescence spectrum, the intensity is often expressed in arbitrary units, but in the present case the absolute intensities are given in order to make a comparison between the environments of DPH in NaDC and NaC micelles, where DPH concentration is kept constant at 5 × 10−7 M. The steady state fluorescence anisotropy (rss ) values were obtained using the expression rss = (I − GI⊥ )/(I + 2GI⊥ ), where I and I⊥ are fluorescence intensities when the emission polarizer is parallel and perpendicular, respectively, to the direction of polarization of the excitation beam, and G is the factor that corrects for unequal transmission by the diffraction gratings of the instrument for vertically and horizontally polarized light [15].
2.1. Materials
3.2. Fluorescence lifetime measurements
1,6-Diphenylhexatriene (DPH) was purchased from Sigma Chemical Company (USA) and used as such. The bile salts, sodium deoxycholate (NaDC) and sodium cholate (NaC) were obtained from S.D. Fine Chemical Company, India and were recrystallized from hot ethanol. Water distilled twice from alkaline permanganate solution, was used for all the experiments.
Lifetime measurements were carried out using the IBH single-photon counting fluorimeter in a time-correlated singlephoton counting arrangement consisting of ps/fs Ti–Sapphire Laser system (Tsunami Spectra Physics, Bangalore, India). The pulse repetition rate was 82 MHz and the full width half maximum is less than 2 ps. The emission was collected at magic angle polarization (54.7) to avoid any polarization in the emission decay. The instrument response time is approximately 50 ps. The decay data were further analysed using IBH software. A value of χ2 , 0.99 ≤ χ2 ≤ 1.4 was considered as a good fit.
3. Preparation of bile salt solutions and labeling The stock solutions of NaDC and NaC were prepared in water and the experimental solutions were prepared by subsequent dilutions from the stock. Fresh solutions of bile salt were prepared every time to avoid the problem of aging. The pH of the stock solution was found to be ∼8, which did not change significantly after dilution. Solutions of DPH in bile salt were prepared by passing a gentle stream of nitrogen gas over the appropriate volume of the DPH stock solution to evaporate THF and by redissolving it in the bile salt solution by sonication. The solutions were left for 2 h to achieve equilibrium. The DPH concentration was maintained at 5 × 10−7 M for all the experiments. For the
4. Results and discussion Fig. 1A and B represents the emission spectra of DPH in NaC and NaDC solutions with increasing concentration of the two bile salts. DPH, which is practically nonfluorescent in aqueous medium show a remarkable increase in intensity in both NaC and NaDC. Initially, when the bile salt concentration is low, the intensity is almost close to zero and with increase in bile salt concentration it shows a steep increase and then it attains saturation.
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Fig. 1. Emission spectra of DPH with increasing concentration of (A) NaC and (B) NaDC. Temperature = 25 ◦ C, [DPH] = 5 × 10−7 M, [NaC] = 0–30 mM, [NaDC] = 0–20 mM, λex = 360 nm.
4.1. Fluorescence intensity as the parameter to determine CMC
4.2. Fluorescence anisotropy as the parameter to determine CMC
The variation of fluorescence intensity of DPH with concentration of the two bile salts is shown in Fig. 2A and B. The fluorescence intensity in both cases shows a sigmoid variation with the concentration of bile salt. The point of intersection of the horizontal line and the line of inflation represents the CMC and it is found to be 16 mM for NaC and 6 mM for NaDC (shown as dashed lines in Fig. 2A and B). The remarkable increase in the fluorescence intensity of DPH suggests that bile salt micelles provide a solubilizing hydrophobic environment to DPH molecules. This experimental finding is in agreement with Small’s model of micelle formation, which says the aggregation of bile salts in aqueous medium takes place through the back-to-back stacking of the hydrophobic planes of the molecules [16]. For the same concentration of probe (5 × 10−7 M), the increase in intensity is almost four times higher in NaDC solution as compared to that in NaC solution under similar experimental conditions. This implies that the dihydroxy bile salt micelles provide more hydrophobic microenvironment to DPH as compared to that of trihydroxy bile salts. Trihydroxy bile salts being more hydrophilic form looser and smaller micelles than dihydroxy bile salts [17].
DPH is one of the most widely used fluorescence anisotropy probes, which because of its rod-shaped structure shows high retention of polarization. In an organized media, the anisotropy of DPH reflects the resistance offered by the microenvironment to its rotational movement; hence, the change in the polarization of DPH fluorescence acts as an index for the change in microviscosity of the immediate environment [18]. Fig. 3A and B shows the variation of steady state fluorescence anisotropy of DPH as a function of respective bile salt concentrations. Initially, when the concentration of bile salt is low, the anisotropy is found to be high, which can be due to the hydrophobic aggregation of DPH in aqueous medium. As the bile salt concentration increases there is a pronounced solubilization of DPH and a corresponding break down of DPH aggregates resulting in the decrease in the anisotropy value. After the CMC, when bile salt exists as micelles, DPH is completely solubilized in the hydrophobic core that has lower microviscosity; hence, anisotropy is low and remains independent of concentration. The surfactant concentration at which the anisotropy value becomes independent of concentration is considered as the CMC of the surfactant [18]. Thus, the CMCs for NaC and NaDC are found to be 16 and 6 mM, respectively. Thus, two totally independent parameters of DPH, i.e. fluorescence intensity and anisotropy,
Fig. 2. Plots showing variation of fluorescence intensity of DPH at emission maximum (428 nm) as a function of concentration of (A) NaC and (B) NaDC. Temperature = 25 ◦ C, [DPH] = 5 × 10−7 M, λex = 360 nm. The error in the data as calculated from three independent sets of experiments is within ±5%.
U. Subuddhi, A.K. Mishra / Colloids and Surfaces B: Biointerfaces 57 (2007) 102–107
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Fig. 3. Variation of steady state fluorescence anisotropy of DPH as a function of (A) NaC and (B) NaDC concentration. Temperature = 25 ◦ C, [DPH] = 5 × 10−7 M, λex = 360 nm, λem = 428 nm. The error in the data as calculated from three independent sets of experiments is within ±5%.
report the same value of CMC. The higher anisotropy value in NaDC as compared to that in NaC indicates a higher microviscosity in the interior of the deoxycholate micelle. This observation also suggests that the micelles of dihydroxy bile salts are larger and more rigid than that of the trihydroxy bile salts. 4.3. Fluorescence lifetime studies on NaC and NaDC in water
with the micelles formation DPH experiences a nonpolar environment. The slightly longer lifetime in NaDC as compared to that in NaC indicates that NaDC micelles provide relatively more nonpolar and rigid microenvironment to DPH. The ability of DPH fluorescence to truthfully reflect the CMC of bile salt micelles can be tested further by changing the experimental conditions that affect the micellization process. 4.4. Effect of temperature on CMC of bile salts
Tables 1 and 2 give the fluorescence lifetime data for DPH at different concentrations of sodium cholate and sodium deoxycholate solutions. Below 16 mM of NaC, the fluorescence shows biexponential decay with a short lifetime and a long lifetime component and above 16 mM of NaC, only the longer component exists. Similarly, in case of NaDC below 6 mM, the decay is biexponential and above this only the longer component exists. Thus, 16 mM for NaC and 6 mM for NaDC are the critical concentrations above which there is a change in the DPH environment in the solutions. These values are same as found earlier from the intensity and anisotropy studies. Thus, this indicates that fluorescence lifetime of DPH is as sensitive as its intensity and anisotropy towards the process of micellization and support the earlier findings. Below the CMC the biexponential nature of DPH decay suggests the heterogeneity in its distribution, but Table 1 Fluorescence lifetime data for DPH in sodium cholate solutions at 25 ◦ C (λex = 370 nm, λem = 428 nm) NaC concentration (mM)
τ 1 (ns)
α1 (%)
τ 2 (ns)
α2 (%)
χ2
8 10 12 14 15 16 20 24 28 32
1.03 1.45 1.60 2.41 3.24
28.59 22.44 19.04 17.52 17.28
4.64 5.86 6.09 6.85 6.90 7.01 7.11 7.21 7.23 7.32
71.41 77.56 80.96 82.48 82.72
1.26 1.27 1.01 1.20 1.01 1.25 1.24 1.17 1.15 1.17
Bile salt micelles are known to be sensitive to temperature. The CMC is known to increase with increase in temperature [19,20]. The effect of temperature on the process of micellization of NaDC and NaC was monitored by using fluorescence intensity and fluorescence anisotropy of DPH. Fig. 4A and B shows the response of fluorescence intensity and fluorescence anisotropy of DPH to temperature in sodium deoxycholate solution. As is clear from the plots both fluorescence intensity as well as anisotropy shows good response to the change in CMC of NaDC with temperature. The CMC value shifts from 5 mM at 15 ◦ C to 6 mM at 25 ◦ C, 8 mM at 35 ◦ C and to 10 mM at 45 ◦ C. Similar effect is also seen in case of NaC micellization and the CMC shifts from 15 mM at 15 ◦ C, 16 mM at 25 ◦ C and 17 mM at 35 ◦ C and 45 ◦ C. Table 2 Fluorescence lifetime data for DPH in sodium deoxycholate solutions at 25 ◦ C (λex = 370 nm, λem = 428 nm) NaDC concentration (Mm)
τ 1 (ns)
α1 (%)
τ 2 (ns)
α2 (%)
χ2
1 2 3 4 5 6 7 8 10 14 18 20
2.48 2.46 2.38 2.33 1.48
34.55 31.18 31.33 31.83 20.46
5.37 5.64 5.74 5.95 7.68 8.54 8.59 8.64 9.25 9.40 9.41 9.79
65.45 68.82 68.67 68.17 79.54
1.16 1.07 1.11 1.07 0.99 1.11 1.06 1.05 1.26 1.27 1.01 1.23
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Fig. 4. Plot of fluorescence (A) intensity and (B) anisotropy of DPH vs. NaDC concentration at various temperatures. Temperature = 15–45 ◦ C.
Fig. 5. Variation of fluorescence (A) intensity and (B) anisotropy of DPH as a function of sodium cholate concentration at different ionic strengths.
As is clearly seen in the figures, with increase in temperature the fluorescence intensity at the saturation decreases and the anisotropy value after the micellization also decreases. This suggests that the micelles’ interior becomes less hydrophobic and provides more rotational freedom for DPH at high temperature. Thus, it can be concluded that increasing temperature leads to the formation of fluffier micelles with less rigid interior.
4.5. Effect of ionic strength on CMC of bile salts The increase in ionic strength of the aqueous medium is known to promote the micellar aggregates of bile salts. The effect of counter-ion on the CMC of bile salt micelles is mainly mediated by progressive neutralization of the ionic charges on the carboxylate group. As the CMC is an index of hydrophobic interaction and ionic repulsion, reduction of the latter allows
Fig. 6. Variation of fluorescence (A) intensity and (B) anisotropy of DPH as a function of sodium deoxycholate concentration at different ionic strengths.
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micelle formation to occur at lower concentration thus resulting in a decrease in the CMC [14]. Fig. 5A and B represents the variation of fluorescence intensity and fluorescence anisotropy of DPH in NaC solution at different ionic strengths. With increase in the ionic strength, the CMC shifts to lower value. It decreased from 16 mM in absence of salt, to 12 mM at 0.1 M NaCl and 10 mM at 0.2 M NaCl. However, there is not much of change in overall intensity or in anisotropy on addition of salt, which indicates that the micellar core or more specifically the solubilizing site for DPH in NaC micelles is less affected with the addition of NaCl. The micellization process of dihydroxy bile salt NaDC shows strong dependence on ionic strength (Fig. 6A and B). The CMC drops from 6 mM in absence of salt to 3.5 mM at 0.05 M and 2.5 mM at 0.1 M of NaCl. There is almost a two-fold increase in the intensity in presence of 0.1 M NaCl as compared to that in absence of salt. The anisotropy values in the micelles also show slight increase in presence of salt. This indicates that in NaDC micelles not only the CMC is affected on addition of salt but also the nature of aggregation. The aggregates formed in presence of NaCl provide DPH a more nonpolar and rigid environment. There is literature report that says the aggregations of dihydroxy bile salts are much greater than that of trihydroxy bile salts in presence of NaCl [21]. 5. Conclusion Fluorescence properties of DPH are found to be very sensitive to the process of micellization of bile salts in aqueous medium. DPH offers efficient tools in terms of its three independent parameters: fluorescence intensity, anisotropy and lifetime for monitoring the process. The CMC thus obtained are 16 mM for NaC and 6 mM for NaDC at 25 ◦ C in an unbuffered solution. Using DPH as a probe, it is possible to investigate the effect of temperature and ionic strength on the micellization process. Increasing temperature leads to the formation of fluffier micelles with less rigid interior. The micelle core of NaC is less perturbed by the presence of NaCl whereas in case of NaDC, the aggregates provide DPH a more nonpolar and rigid environment in presence of NaCl than that in absence of salt. In summary fluorescence probing, using DPH as a molecular probe provides a simple,
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convenient, sensitive and economic method for monitoring the process of micellization of bile salts in aqueous medium. Acknowledgements Usharani thanks IIT Madras for financial assistance. The authors thank SAIF, IIT Madras for the access to the fluorimeter and Dr. P. Ramamurthy and Mr. Kumaran of NCUFP, University of Madras, India for the lifetime measurements. References [1] R. Coleman, Biochem. Soc. Trans. 15 (1987) S68. [2] D.G. Oakenfull, in: E. Wyn-Jones, J. Gormally (Eds.), Aggregations Processes in Solutions, Elsevier Scientific Publishing Company, Amsterdam, 1983, p. 118. [3] M.C. Carey, D.M. Small, Arch. Intern. Med. 130 (1972) 506. [4] E. Pramauro, E. Pelizzetti, Surfactants in Analytical Chemistry. Applications of Organized Amphiphilic Media, Elsevier, New York, 1996. [5] S. Gouin, X.X. Zhu, Langmuir 14 (1998) 4025. [6] B.R. Simonovic, M. Momirovic, Mikrochim. Acta 127 (1997) 101. [7] H. Matsuoka, J.P. Kratohvil, N. Ise, J. Colloid Interface Sci. 118 (1987) 387. [8] T. Nakashima, T. Anno, H. Kanda, Y. Sato, T. Kuroi, H. Fujji, S. Nagadome, G. Sugihara, Colloids Surf. B 24 (2002) 103. [9] P. Mukerjee, J. Cardinal, J. Pharm. Sci. 65 (1976) 882. [10] L. Antonian, S. Deb, W. Spivak, J. Lipid Res. 31 (1990) 947. [11] S. Reis, C.G. Moutinho, C. Matos, B. de Castro, P. Gameiro, J.L.F.C. Lima, Anal. Biochem. 334 (2004) 117. [12] P. Ranajit, M.K. Mathew, R. Narayanan, P. Balaram, Chem. Phys. Lipids 25 (1979) 345. [13] C. Ju, C. Bohne, Photochem. Photobiol. 63 (1996) 60. [14] K. Kalyanasundaram, in: V. Ramamurthy (Ed.), Photochemistry in Organized and Constrained Media, VCH Publishers, New York, 1991, p. 39. [15] J.R. Lakowicz, Principles of Fluorescence Spectroscopy, Kluwer Academic, Plenum Publishers, New York, 1999. [16] D.M. Small, Advances in Chemistry Series, Molecular Association in Biological and Related Systems, vol. 84, American Chemical Society, 1968, p. 31. [17] G. Li, L.B. McGown, J. Phys. Chem. 98 (1994) 13711. [18] X. Zhang, J.K. Jackson, H.M. Burt, J. Biochem. Biophys. Methods 31 (1996) 150. [19] K. Matsuoka, Y. Moroi, Biochim. Biophys. Acta 1580 (2002) 189. [20] H. Sugioka, K. Matsuoka, Y. Moroi, J. Colloid Interface Sci. 259 (2003) 156. [21] S.Z. Zhang, J.W. Xie, C.S. Liu, Anal. Chem. 75 (2003) 91.