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Langmuir 2007, 23, 4842-4848
Surfactant Chain-Length-Dependent Modulation of the Prototropic Transformation of a Biological Photosensitizer: Norharmane in Anionic Micelles Alok Chakrabarty,† Arabinda Mallick,† Basudeb Haldar,† Pradipta Purkayastha,‡ Paramita Das,† and Nitin Chattopadhyay*,† Department of Chemistry, JadaVpur UniVersity, Calcutta 700 032, India, and Department of Chemistry, Birla Institute of Technology and Science, Pilani 333031, India ReceiVed January 2, 2007. In Final Form: February 2, 2007 A photophysical study of norharmane (NHM), an efficient cancer cell photosensitizer, has been undertaken in well-characterized biomimetic micellar nanocavities formed by anionic surfactants of varying chain length, namely, sodium decyl sulfate (S10S), sodium dodecyl sulfate (S12S), and sodium tetradecyl sulfate (S14S), using steady-state and time-resolved fluorescence spectroscopy. The effect of the hydrophobic chain length on the structural dynamism of the fluorophore has been reported. Experimental results demonstrate that the equilibrium of this dynamism is sensitive to the environment. Variation in the surfactant chain length plays an important role in promoting a specific prototropic form of the probe molecule. A striking feature of the present study is that an increase in the surfactant chain length (hydrophobicity) favors the cationic species of NHM. This has been rationalized on the basis of changes in the local pH and the aggregation number of the micelles. A fluorescence quenching study of the micelle-bound probe using ionic quencher Cu2+ corroborates this.
1. Introduction Reactants accommodated in molecular assemblies such as micelles, microemulsions, vesicles, and so forth often achieve a greater degree of organization compared to their geometries in homogeneous solution. These organized media can mimic reactions in biosystems and also have the potential for energy storage.1 Surfactants, because of their ability to solubilize membrane proteins, are extremely important in simulating the complex environmental condition present in larger bioaggregates such as biological membranes.2-4 The wide range of functions performed by the biological membranes and membrane proteins have motivated researchers to look for simple model systems. A typical example of such membrane mimetic models is a micelle, which is an organized assembly of surfactants in aqueous media.5,6 Interest in this subject has received remarkable attention in recent years, with explosive growth in research on various bioactive molecules that are promising against a variety of diseases including cancer.7-13 The structure and function of these biologically active molecules have drawn the attention of * Corresponding author. E-mail:
[email protected]. Fax: 91-33-2414 6266. † Jadavpur University. ‡ Birla Institute of Technology and Science. (1) Hashimoto, S.; Thomas, J. K. J. Am. Chem. Soc. 1985, 107, 4655. (2) Tanford, C. The Hydrophobic Effect: Formation of Micelles and Biological Membranes; Wiley: New York, 1973. (3) Rottman, C.; Avnir, D. J. Am. Chem. Soc. 2001, 123, 5730. (4) Chakraborty, H.; Sarkar, M. Biophys. Chem. 2003, 104, 315. (5) Haldar, B.; Chakrabarty, A.; Mallick, A.; Mandal, M. C.; Das, P.; Chattopadhyay, N. Langmuir 2006, 22, 3514. (6) Schille´n, K.; Anghel, D.; Miguel, M. G.; Lindman, B. Langmuir 2000, 16, 10528. (7) Shannigrahi, M.; Bagchi, S. J. Phys. Chem. B 2004, 108, 17703. (8) Deepa, S.; Mishra, A. K. J. Pharm. Biomed. Anal. 2005, 38, 556. (9) Chakraborty, H.; Sarkar, M. J. Colloid Interface Sci. 2005, 292, 265. (10) Mallick, A.; Bera, S. C.; Maiti, S.; Chattopadhyay, N. Biophys. Chem. 2004, 112, 9. (11) Dillon, J.; Spector, A.; Makaniski, K. Nature 1976, 259, 422. (12) Schilitter, E., Bein, H. J., Ed. In Medicinal Chemistry; Academic Press: New York, 1967; Vol. 11. (13) Pardos, J. L.; Leon, A. G.;Olives, A. I.; Martin, M. A.; Del Castillo, B. J. Photochem. Photobiol., A 2005, 173, 287.
researchers because of their ability to achieve specific chemical efficiency as a result of organization in reaction media. For a particular prototropic probe, it is often necessary to opt for one prototopic form or a desired composition of the different prototropic species to achieve better efficiency for a targeted purpose in a specific environment. Norharmane (9H-pyrido[3,4-b]-indole) (NHM) (Scheme 1) belongs to the group of naturally occurring bioactive alkaloids (β-carbolines) with a tricyclic pyrido(3,4-b) indole ring system that are found in about 26 plant families, foods, cells, and animal tissues and human urine. NHM has been shown to act as an efficient photosensitizer in the presence of oxygen to produce both the superoxide radical anion14,15 and singlet oxygen.16 It is also known that these compounds bind selectively with DNA17 and form complexes with flavins.18 Beljansky et al. have found that some β-carbolines can selectively and completely destroy the proliferative capacity of various types of cancer cells, which is enhanced upon excitation with UV radiation.19 Photodynamic therapy (PDT) using photosensitizing chemicals combined with light to produce singlet oxygen is a modality for present day cancer treatments.20 The effectiveness of the photodynamic action depends not only on the yield of singlet oxygen but also on the biodistribution of the probe molecule in the cytoplasmic and mitochondrial membranes, its retention, and the nature of binding inside the target cell. Because NHM is a potential candidate for efficient cancer cell photosensitization,19 it is important to know the location of the probe in a microheterogeneous environment. Studying the binding interaction of NHM with different bio(14) Calle, P.; Fernandez-Arizpe, A.; Siero, C. Appl. Spectrosc. 1996, 50, 1446. (15) Becker, R. S.; Ferreira, L. F. V.; Elisei, F.; Machado, I.; Latterini, L. Photochem. Photobiol. 2005, 81, 1195. (16) Chae, K. H.; Ham, H. S. Bull. Korean Chem. Soc. 1987, 7, 478. (17) Duportail, G. Int. J. Macromol. 1981, 3, 188. (18) Munoz, M. A.; Carmona, C.; Hidaigo, J.; Guaraldo, P.; Balon, M. Bioorg. Med. Chem. 1995, 3, 41. (19) Beljanski, M.; Beljanski, M. S. IRCS Med. Sci. 1984, 50, 587. (20) Henderson, B. W., Dougherty, T. J., Eds. Photodynamic Therapy: Basic Principles and Clinical Applications; Marcel Dekker: New York, 1992.
10.1021/la0700063 CCC: $37.00 © 2007 American Chemical Society Published on Web 03/21/2007
Biological Photosensitizer Prototropic Transformation Scheme 1 . Different Acid-Base Equilibria for Norharmanea
a CN, cation-neutral; NA, neutral-anion; ZA, zwitterion-anion; and CZ, cation-zwitterion. pK values are taken from ref 23. K(S0) and K(S1) refer to the acid dissociation constants in the ground and first excited states, respectively.
systems/organized media thus becomes important when studying the net PDT efficiency. The binding sites and the microenvironment surrounding the probe molecules are indeed complex in nature and influence the photophysical properties. Therefore, we consider the spectroscopic and photophysical parameters of this molecular system in this membrane mimetic media to be very helpful in developing a better understanding of the nature of binding and also in assessing the biodistribution of this dye system inside the bioenvironments including the living cells. Apart from the biological aspect, attention has also been focused on another important and interesting feature of norharmane, namely, its fluorescence from different prototropic species21-23 that shows remarkable sensitivity to some parameters (e.g., polarity, pH, etc.) of the microenvironment. This has stimulated scientists to exploit this group of compounds as sensitive fluorescent molecular probes for exploring binding sites in different biological targets (e.g., micelles, cyclodextrins, proteins, etc.13,24-26). Although a large number of biochemical and molecular biological investigations have been carried out using norharmane,27-29 a large portion of these studies is associated with their phototoxic effect toward various organisms.28,30-32 Their phototoxicity again depends on the diffusion of these molecules into different regions of the biological targets or cells.28 Solubility and partitioning studies by Burrows and co-workers and our group have independently shown that the neutral form of the probe has a strong affinity for hydrophobic domains. 24-26,30,32-34 (21) Reyman, D.; Vinas, M. H.; Poyato, J. M. L.; Pardo, A. J. Phys. Chem. A 1997, 101, 768. (22) Varela, A. P.; Burrows, H. D.; Douglas, P.; Miguel, M. G. J. Photochem. Photobiol., A 2001, 146, 29. (23) Vert, F. T.; Sanchez, I. Z.; Torrent, A. O. J. Photochem. 1983, 23, 355. (24) Mallick, A.; Chattopadhyay, N. Biophys. Chem. 2004, 109, 261. (25) Mallick, A.; Haldar, B.; Chattopadhyay, N. J. Photochem. Photobiol., B 2005, 78, 215. (26) Mallick, A.; Chattopadhyay, N. Photochem. Photobiol. 2005, 81, 419. (27) Nagao, M.; Yahagi, T.; Sugimura, T. Biochem. Biophys. Res. Commun. 1978, 78, 373. (28) Larson, R. A.; Marley, K. A.; Tuveson, R. W.; Berenbaum, M. R. Photochem. Photobiol. 1988, 48, 665. (29) Chakrabarty, A.; Mallick, A.; Haldar, B.; Das, P.; Chattopadhyay, N. Biomacromolecules 2007, 8, 920. (30) Dias, A.; Varela, A. P.; da Miguel, M.; Becker, R. S.; Mac¸ anita, A. L.; Burrows, H. D. J. Phys. Chem. 1996, 100, 17970. (31) Varela, A. P.; Miguel, M. G.; Mac¸ anita, A. L.; Becker, R. S.; Burrows, H. D. J. Phys. Chem. 1995, 99, 16093. (32) Varela, A. P.; Dias, A.; Miguel, M. G.; Becker, R. S.; Mac¸ anita, A. L. J. Phys. Chem. 1995, 99, 2239. (33) Miguel, M. G.; Burrows, H. D.; Escaroupa, P.; Varela, A. P. Colloids Surf., A 2001, 176, 85. (34) Chakraborty, H.; Sarkar, M. Langmuir 2004, 20, 3551.
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In this article, we have studied the photophysics of this prototropic probe in anionic surfactants of varying chain length. The basic intention of the present spectroscopic investigation is twofold: first, to explore the potential usefulness of the spectroscopic properties of the fluorophore in understanding the structural switching between the different prototropic forms; second, to investigate whether the prototropic transformation of this molecule is modified with increasing hydrophobic chain length in biomimicking micellar systems. Despite the realization of the fact that these factors are crucial in controlling the biological activity of the dye, the structural modification and location of norharmane in different biological environments are yet to be well understood. Fluorescence quenching and time-resolved fluorescence decay analysis studies, reported herein, may shed some light on this aspect of microheterogeneous micellar environments. It is pertinent to mention here that in aqueous solution NHM exists in four different forms (neutral, cation, anion, and zwitterion) depending on the pH of the medium,23 so one may expect contributions from the individual species following the relevant equilibria. It is, however, known that within the pH range of 1-11 only cation and neutral forms exist in the ground state whereas for the same pH range only cationic species emit in the excited state.21,23 Accordingly, under the present experimental condition only two species (neutral and cation) are expected in the ground state whereas the cationic species exists only in the excited state. Therefore, the present study is concerned with only these species. 2. Experimental Section Norharmane (NHM) was procured from Aldrich and purified by recrystallization from ethanol. All of the surfactants, namely, sodium decyl sulfate (S10S), sodium dodecyl sulfate (S12S), and sodium tetradecyl sulfate (S14S), were procured from Lancaster Chemical Co., England, and used as received. Triply distilled water was used throughout the experiment. Analytical-grade hydrated copper sulfate (SRL, India) was used as received for the quenching studies. The micellar solutions were freshly prepared to avoid aging. The pH values of the aqueous experimental solutions were measured to be ∼7.0. A Shimadzu UV 1600 spectrophotometer and Spex Fluorolog-2 spectrofluorimeter were used for the absorption and steady-state fluorescence measurements, respectively. Fluorescence lifetimes were determined from time-resolved intensity decay by the method of time-correlated single-photon counting (TCSPC) using a nanoLED at 370 nm (IBH U.K., nanoLED-03) as the light source. The typical response of this excitation source was 1.2 ns. The decay curves were analyzed using IBH DAS-6 decay analysis software. The goodness of fit was evaluated by χ2 criterion and visual inspection of the residuals of the fitted function to the data. The lifetimes were measured in air-equilibrated solution at ambient temperature.
3. Results and Discussion 3.1. Absorption Study: Implication for the Structural Switchover. The absorption spectrum of norharmane in aqueous solution shows two bands with maxima at 348 and 372 nm corresponding to the neutral and the cationic species, respectively.23 The relative intensity of the two absorption spectral bands is sensitive to the nature of the solvent as well as the external additives. Gradual addition of surfactants SnS (S14S, S12S, and S10S) to the aqueous solution of NHM changes the absorption spectrum significantly. The band at 372 nm corresponding to the cationic species is enhanced with a concomitant decrease in the band at 348 nm corresponding to the neutral species, resulting in an isosbestic point at 352 nm indicating that the ground-state prototropic equilibrium is favored towards the cationic species in all of the anionic micellar environments.
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Figure 1. Variation of the absorption spectrum of NHM as a function of the concentration of S14S. Concentrations of S14S in (i) f (vi) are 0.0, 0.78, 0.97, 1.16, 1.35, and 1.53 mM, respectively. The inset shows the variation of the absorbance of the cationic band at 372 nm as a function of S14S concentration ([NHM] ) 1 × 10-4 M).
Figure 1 depicts a representative set of absorption spectra of NHM in the presence of a varying concentration of S14S. The presence of an isosbestic point establishes a direct interconversion between the neutral and cationic species of the probe in the aqueous surfactant solutions, maintaining an equilibrium. A plot of the absorbance value for the individual species against the surfactant concentration reveals break points around the cmc. Because the micellar phase is silent in the spectroscopic sense, the effect of surfactant molecules on the prototropic equilibrium is reflected through the changes in the spectral properties of the probe molecule itself. Ratios of the optical density and the individual molar extinction coefficient values of the cationic and the neutral species provide measures of the ratios of concentrations of the respective species, leading to an estimation of the equilibrium constant (Keq).34 The prototropic equilibrium can be represented as
neutral (N) h cation (C) and the equilibrium constant (Keq) can be represented as
Keq )
[C] [N]
where [C] and [N] represent the concentrations of the cationic and the neutral species, respectively. From the ratio of the optical density values of the cation to the neutral species, Keq has been evaluated at different surfactant concentrations. The free-energy change corresponding to the above equilibrium can then be determined from the following relation:
∆G ) -RT ln Keq Figure 2 shows the variation of the equilibrium constant (Keq) and the corresponding free-energy change (∆G) with increasing surfactant concentration for the three surfactant systems studied. From Figure 2, it is evident that as the surfactant concentration increases the equilibrium moves toward the cationic species for all the surfactant systems studied. This is ascribed to the electrostatic attraction between the cation of NHM and the anionic micelles as described above. It is further noted that the ∆G value at the saturation levels of the probe-micelle interaction becomes
Figure 2. Variation of the equilibrium constant (0) and free-energy change (9) as a function of (a) S14S, (b) S12S, and (c) S10S concentrations.
more negative as the surfactant chain length increases. Because with an increase in the surfactant chain length ∆G becomes more negative (starting from without surfactant to the highest surfactant concentration, i.e., when the interaction is complete), one can conclude that the switchover from the neutral to the cationic form is more favored with increasing hydrophobic chain length. This observation is contrary to our normal expectation that an increase in hydrophobicity should favor the formation of the neutral species and consequently demands the consideration of other factors. Differences in the degree of water penetration in the micellar environment, local pH at the micellar surface, and micellar aggregation number appear to be promising factors. It is known that water can enter micelles up to a certain depth depending on the compactness of the micellar units.35-37 Thus, (35) Muller, N. In Reaction Kinetics in Micelles; Cordes, E. A., Ed.; Plenum Press: New York, 1973. (36) Mallick. A.; Haldar, B.; Maiti, S.; Chattopadhyay, N. J. Colloid Interface Sci. 2004, 278, 215. (37) Berr, S.; Jones, R. R. M.; Johnson, J. S. J. Phys. Chem. 1992, 96, 5611.
Biological Photosensitizer Prototropic Transformation
micelles with compact head groups suffer smaller water penetration compared to micelles with less compact head groups. It is pertinent to mention in this regard that neutron-scattering experiments on micelles having different surfactant chain lengths reveal that the headgroup structures of the micelles differ significantly.37 As an example, in tetradecyl trimethyl ammonium bromide (TTAB) water penetrates into the headgroup region to a depth of ∼2.5 carbons, whereas in dodecyl trimethyl ammonium bromide (DTAB) water penetrates to ∼4 carbons.37 This observation reveals that the compactness of the headgroup increases with an increase in the surfactant chain length. Similarly, when we move from sodium decyl sulfate (S10S) to sodium tetradecyl sulfate (S14S) the increased chain length enhances the compactness of the headgroups gradually, which in turn decreases the extent of water penetration (micellar hydration).38 This is expected to favor the equilibrium toward the neutral form of NHM rather than the cationic species, contrary to the experimental findings. The model of water penetration/micellar hydration, which fails to explain the experimental observations, is thus ruled out to play any important role. Another plausible factor responsible for switching between the two prototropic forms of NHM is ascribed to the local pH at the micellar surface. To verify this, we have performed the same experiment in aqueous buffered surfactant solutions at pH 7.0. Use of the buffer is supposed to resist changes in the pH of the solution, and the local pH near the micellar surface does not change. Interestingly, the dependency of the structural switching of NHM on the variation of the chain length of SnS surfactants under the buffered condition was remarkably less than it was in the aqueous environment. This observation attests that a change in the local pH at the micellar surface (with the variation in surfactant chain length) plays a significant role, along with other factors, in this switchover between the prototropic species of NHM. Chakraborty et al.4 explained this issue using nonsteroidal anti-inflammatory drugs (NSAIDs) as chromophores in micelles of opposite charge. They ascribed their observation of mutual transformation between the different prototropic species to a change in the apparent pKa values of the drugs. They considered the ionic interaction of the surfactant with the different prototropic species of the probe and the hydrophobic interaction between the nonpolar region of the micelles and the probe to be possible reasons for the apparent shift in the resulting pKa values. Rottman and Avnir have also assigned a similar explanation while explaining their experimental observation with a single dopant in micelles induced by additional sol-gel entrapment.3 The recent works of Rottman et al. and Chakraborty et al. have encouraged us to explain our observation in terms of a change in the local pH of the micellar surface.3,4 We cannot, however, rule out the role of the aggregation number and the size of the three anionic micelles in our experimental observation. It is known that the micellar aggregation number increases with an increase in the chain length in a series of surfactants (Table 1).5,39 An increase in the aggregation number results in an increase in the negative surface charge of the micellar units that stabilizes the cations, moving the prototropic equilibrium to the right. We believe that both the local pH and micellar aggregation number contribute to our experimental findings. 3.2. Steady-State Fluorescence Study. For the fluorometric studies, the probe was excited at the respective isosbestic points obtained in the absorption spectra of the probe in different micellar systems (355 nm in S14S, 353 nm in S12S, and 355 nm in S10S). (38) Chakrabarty, D.; Chakraborty, A.; Seth, D.; Hazra, P.; Sarkar, N. J. Chem. Phys. 2005, 122, 184516. (39) Ranganathan, R.; Peric, M.; Bales, B. L. J. Phys. Chem. B 1998, 108, 8436.
Langmuir, Vol. 23, No. 9, 2007 4845 Table 1. Literature Values of cmc and Aggregation Numbers of the Investigated Surfactant Systems
a
surfactants
cmc (mM)a
aggregation numbera
S10S S12S S14S
30 8.1 1.8
64 92 120
From ref 39.
The room-temperature fluorescence spectrum of the aqueous solution of NHM (at pH 7) shows a single and unstructured band peaking at 450 nm ascribed to the cationic species.23 With an increase in the concentration of SnS in an aqueous solution of the fluorophore, the fluorescence band shows an initial decrease followed by an increase along with a small hypsochromic shift of about 10 nm (Figure 3). The addition of SnS does not lead to the development of a second emission band corresponding to the neutral species of NHM as was observed in the case of CTAB and TX-100.24 In conformity with the absorption study, the observation of only the cationic emission band reflects the fact that the cationic species is stabilized in the SnS environment, and the effect can be assigned to an electrostatic interaction between the anionic surface charge of the SnS micellar units and the cationic species of the fluorophore. Similar types of results were reported by Pal et al. while investigating the photophysical properties of rhodamine derivatives in SDS micelle and also by Ghosh et al. during their study of the interaction of 1-methylaminopyrene with anionic surfactants having different chain lengths.40-42 It is important to mention here that the cationic band intensity decreases before micelle formation and it depends on the surfactant chain length. The formation of premicellar aggregates41 and a slight lowering of the environmental polarity due to the addition of SnS until the cmc is attained24 may be responsible for the initial decrease in the fluorescence of the cationic species of NHM. It was reported that the formation of premicellar aggregates becomes more effective in the case of surfactants with longer chain lengths.41 The increase in the cationic emission of NHM in the micellar environment suggests that the species does not penetrate into the micellar core but rather sits closer to the micellar surface where it is electrostatically stabilized. 3.3. Accessibility of NHM toward the Quencher in Micellar Environments. Quenching of the fluorescence of micelle-bound NHM by an external heavy metal ion has been studied using ionic quencher Cu2+ with an intention to see the degree of accessibility of the fluorophore in different surfactant media toward the quencher and hence to see the impact of the variation of surfactant chain length on the degree of exposure of the probe.43-46 The idea behind the experiment is the following. The ionic quencher is not supposed to be available in the micellar core because of the very low polarity in the said region. It is expected to be available in the aqueous phase as well as in the micelle-water interface.13,36 Moreover, because the quencher is (40) Pal, P.; Zeng, H.; Durocher, G.; Girard, D.; Giasson, R.; Blanchard, L.; Gaboury, L.; Villeneuve, L. J. Photochem. Photobiol., A 1996, 98, 65. (41) Ghosh, S. K.; Pal, A.; Kundu, S.; Mandal, M.; Nath, S.; Pal, T. S. Langmuir 2004, 20, 5209. (42) Malik, W. U.; Pal Verma, S. J. Phys. Chem. 1966, 70, 26. (43) Mallick. A.; Mandal, M. C.; Haldar, B.; Chakrabarty, A.; Das, P.; Chattopadhyay, N. J. Am. Chem. Soc. 2006, 128, 3126. Mallick. A.; Mandal, M. C.; Haldar, B.; Chakrabarty, A.; Das, P.; Chattopadhyay, N. J. Am. Chem. Soc. 2006, 128, 10629. (44) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 2nd ed.; Plenum: New York, 1999. (45) Mallick, A.; Haldar. B.; Maiti, S.; Bera, S. C.; Chattopadhyay, N. J. Phys. Chem. B 2005, 109, 14675. (46) Martin, L.; Olives, A.; Castillo, B.; Martin, M. A. Luminescence 2005, 20, 152.
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Figure 4. Variation of the relative fluorescence intensity (F0/F) of NHM (10 × 10-5 M) as a function of Cu 2+ concentration in S14S (6.0 mM), S12S (25 mM), and S10S (80 mM) micelles.
Figure 5. Variation of kq of NHM against Cu2+ quenching in micelles as a function of the number of carbons in the surfactant. Table 2. Stern-Volmer and Fluorescence Quenching Rate Constants for the Fluorescence Quenching of NHM by Cu2+ in Aqueous and Aqueous Micellar Media
Figure 3. Fluorescence spectra of NHM in SnS micelles as a function of surfactant concentration. Curves i-vii correspond to 0.0, 0.30, 0.78, 3.06, 3.41, 3.75, and 4.09 mM for S14S; 0.0, 3.0, 10, 12, 15, 18, and 19 mM for S12S; and 0.0, 24, 36, 48, 60, 72, and 80 mM for S10S. Insets show the variations of the fluorescence intensity of NHM in the respective media as a function of surfactant concentration ([NHM] ) 1 × 10-4 M).
positive in nature, its availability in the interfacial zone is supposed to depend on the surface charge of the micellar units and it is expected to be remarkably greater in the peripheral region of the anionic micellar systems with negative surface charge.13 Hence, had the fluorophore been located in the micellar core, there should have been no appreciable fluorescence quenching as a result of the lack of availability of the quencher (Cu2+). However, if the probe is located in the micelle-water interface, then the Cu2+induced quenching of its fluorescence is supposed to be remarkably greater than that in a pure aqueous medium. The quenching experiment of NHM has been performed at the saturation level of NHM-micelle interactions (which corresponds to the high concentration of surfactants in the plateau region of the insets in Figure 3). In a recent report, we have shown that the quenching efficiency is very much dependent on the surfactant concentration.43 The specific concentrations of the surfactants used here for the quenching studies are indicated in Table 2.
environment
KSV/M-1
kq × 10-9/M-1 s-1
S14S (6.0 mM) S12S (25 mM) S10S (80 mM) water
2210 ( 50 990 ( 50 560 ( 10 20 ( 2
86.0 38.4 24.3 1.00
These concentrations were chosen because the addition of surfactants beyond these concentrations failed to bring about any further changes in the spectral pattern and quenching efficiency. The quenching of the fluorescence of NHM with the addition of quencher (Cu2+) was followed using the SternVolmer relation
F0 ) 1 + KSV[Q] ) 1 + kqτ0[Q]... F where F0 and F are the fluorescence intensities in the absence and presence of quencher, respectively, [Q] is the molar concentration of the quencher, KSV is the Stern-Volmer constant, kq is the bimolecular quenching constant, and τ0 is the lifetime of the probe molecule in the absence of the quencher. The slopes of the Stern-Volmer plots (KSV) are related to the degree of exposure (accessibility) of the probe molecule to the aqueous phase and also to the surface charge and surface charge density of the individual micelles. The higher the slope, the greater the degree of exposure assuming that there is not a large difference in the fluorescence lifetime. The bimolecular quenching constant (kq) is a more accurate measure of the degree of exposure because it takes into account the differences in the values of the fluorescence lifetime. The quenching parameter (kq) obtained are plotted in Figure 5 and tabulated in Table 2. From Figure 5 and Table 2, it is clear that an increase in the hydrophobic chain length increases the accessibility of the probe molecule to the quencher.
Biological Photosensitizer Prototropic Transformation
Langmuir, Vol. 23, No. 9, 2007 4847
Table 3. Lifetimes of NHM in Aqueous and Micellar Environments environment
τf/ns
χ2
S14S (6.0 mM) S12S (25 mM) S10S (80 mM) water
25.7 25.8 23.0 20.0
1.30 1.25 1.28 1.20
The quenching rates of NHM in all of the anionic micellar environments are significantly higher than in the pure aqueous phase, presumably owing to the high local concentration of the quencher ion near the surface because of the electrostatic interaction.43 The quenching experiments clearly suggest that the fluorescence moiety of NHM is very much accessible to the quencher. Figure 5 reveals that the quenching process becomes more favored as the surfactant chain length increases. The efficiency of the diffusion-controlled quenching process is dependent on the rate of diffusion of the interacting species. Any factor that enhances the rate of diffusion of the interacting species should activate the quenching process. In the present case, the electrostatic factor might play a role of this sort. A similar increase in the KSV values in anionic and reverse micellar environments has recently been reported in the literature.13,36,47 The gradual increase in the Stern-Volmer and/or quenching constant with an increase in the surfactant chain length can be rationalized easily by considering, as before, a gradual increase in the aggregation number.39 3.4. Time-Resolved Fluorescence Study. The fluorescence lifetimes of NHM have been measured at the saturation level of NHM-micelle interaction. (This corresponds to a high concentration of surfactants in the plateau region of the insets in Figure 3.) The specific concentrations used for the micelles are indicated in Table 3. These concentrations were chosen because the addition of greater amounts of surfactants failed to bring about any further change in the spectral pattern or lifetime. Excited-state lifetime measurements serve as a sensitive parameter for exploring the local environment around a fluorophore and also provide information on probe-micelle interactions.48-51 For the same reason that the emission spectra of the fluorophores are sensitive to the local environment, their fluorescence lifetimes reflect intermolecular interactions. In water and also in all of the micelles, NHM exhibits a single-exponential decay. Typical decay profiles of NHM in aqueous environments and different micellar environments are shown in Figure 6. The fluorescence lifetimes of NHM in different SnS micelles are tabulated in Table 3 along with the lifetime of the probe in pure water. Table 3 reflects that an increase in the chain length of the surfactant constituting the micellar microenvironments affects the fluorescence lifetime only marginally. The single-exponential decay characteristics in the micellar environments suggest that the probe molecule is located in a single type of site.52-55 From the significant differences in the lifetime values of the NHM in micelles from that of the fluorphore in water, it is reasonable to rule out the assignment of the aqueous phase as this site. As is (47) Panda, M.; Behera, P. K.; Misra, B. K.; Behera, G. B. J. Photochem. Photobiol., A 1995, 90, 69. (48) Mallick, A.; Haldar, B.; Chattopadhyay, N. J. Phys. Chem. B 2005, 109, 14683. (49) Chattopadhyay, A.; Mukherjee, S.; Raghuraman, H. J. Phys. Chem. B 2002, 106, 13002. (50) Prendergast, F. G. Curr. Opin. Struct. Biol. 1991, 1, 1054. (51) Das, P.; Mallick, A.; Haldar, B.; Chakrabarty, A.; Chattopadhyay, N. J. Chem. Phys. 2006, 125, 044516. (52) Matzinger, S.; Hussey, D. M.; Fayer, M. D. J. Phys. Chem. B 1998, 102, 7216. (53) Cang, H.; Brace, D.; Fayer, M. D. J. Phys. Chem. B 2001, 105, 1007. (54) Shannigrahi, M.; Bagchi, S. Chem. Phys. Lett. 2004, 396, 367. (55) Dutt, G. B. J. Phys. Chem. B 2003, 107, 10546.
Figure 6. Time-resolved fluorescence intensity decay of NHM in water and in SnS micelles (λexc ) 370 nm). The inset shows the respective environments. The sharp profile on the left is the lamp profile.
known, micelles are characterized by three distinct regions: a nonpolar core formed by the hydrocarbon tails of the surfactant, a compact stern layer having the head groups, and a relatively wider Gouy-Chapman layer containing the counterions.56 Depending on the nature of the probe and the micelle, a probe molecule can bind either to the headgroup region or to the nonpolar core of the micelles. The two microenvironments (the Stern layer and the core region) of the micelles have quite different properties. The core region is usually characterized by a highly viscous hydrocarbon-like environment with a very low degree of water penetration. The Stern layer mainly consists of polar head groups, bound counterions, and largely structured water molecules. Because of the differences in the properties of the head group and core regions of the micelles, different types of molecules are expected to be located selectively in the two regions. Although there exists different microscopic environments in a micellar solution, a single-exponential fluorescence decay indicates that either the fluorescent species exists in a single environment or that there is a rapid exchange between multiple environments on the time scale of measurement. Very often, the interfacial region and the core region of aqueous micelles resemble the wateralkanol system and the 1,4-dioxane-like environment, respectively.52,54 We have determined the lifetime of NHM both in pure methanol and pure dioxane media. The decay curves are best fitted by a single exponential in both the cases, and the lifetimes are determined to be 20 and 2.3 ns in methanol and dioxane, respectively. The decay times of NHM in the studied micellar environments are much different from the measured lifetime of the probe in the dioxane medium. This suggests that the probe is not located in the interior of the micellar units and proposes the Stern layer as the possible location of the fluorophore. The steady-state fluorescence and fluorescence quenching study also supports this conjecture. An increase in the fluorescence lifetime of the micelle-bound NHM can be ascribed to the additional stabilization of the cationic species of the probe as a result of the binding interaction with the anionic micellar surface charge.
4. Conclusions The present investigation reports the effect of hydrophobic chain length on the prototropic switching of a biological (56) Kalyanasundram, K. Photochemistry in Microheterogenous Systems; Academic Press: New York, 1987.
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photosensitizer, NHM, in anionic micellar environments with varying surfactant chain lengths. As the surfactant chain length increases, the prototopic equilibrium is favored towards the cationic species. A change in the local pH in the micellar surface region and an increase in the aggregation number of the micelles formed from surfactants of longer chain length leading to more compact environment are ascribed to be responsible. Both the steady-state and the time-resolved fluorescence studies indicate the Stern layer of the micelles as the probable site for the location of the fluorophore. The present study reveals that the composition
Chakrabarty et al.
of different prototropic species of a suitable drug can be modulated through control over the chain length of the surfactant. An understanding of this composition and the probable location of the drug molecules in phospholipids or membranes will help in optimizing the PDT efficiency. Acknowledgment. Financial support from DST and CSIR, Government of India, are gratefully acknowledged. P.D. is thankful to CSIR for the fellowship. LA0700063
