Electrostatic Pushing Effect: A Prospective Strategy ... - ACS Publications

Deboleena Sarkar, Debanjana Ghosh, Paramita Das, and Nitin Chattopadhyay*. Department of Chemistry, Jadavpur University, Kolkata 700 032, India. J. Ph...
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J. Phys. Chem. B 2010, 114, 12541–12548

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Electrostatic Pushing Effect: A Prospective Strategy for Enhanced Drug Delivery Deboleena Sarkar, Debanjana Ghosh, Paramita Das, and Nitin Chattopadhyay* Department of Chemistry, JadaVpur UniVersity, Kolkata 700 032, India ReceiVed: May 28, 2010; ReVised Manuscript ReceiVed: August 17, 2010

Contrary to the expected quenching, unprecedented and remarkable enhancement is observed in the fluorescence of an anionic fluorophore, 8-anilino-1-naphthalene sulfonate, with the addition of bromide ion in cationic cetyltrimethylammonium bromide micellar medium. Electrostatic pushing effect of the halide ion on the anionic fluorophore that forces the probe to penetrate further into the micellar interior has been assigned to be responsible for the novel observation. Experiments with other probes and surfactants propose that the electrostatic pushing effect is rather a general phenomenon. While applying to the ionic drugs in real biosystems, potential application of the unorthodox effect remains in enhancing the solubilization of the drugs into the active target region leading to a radical enhancement in the drug efficacy. 1. Introduction Along with other criteria, quality of a drug is judged in terms of efficacy that refers to the potential maximum therapeutic response that a drug can provide without producing toxicity. This is often related to the packing of the drug with the target, commonly a protein or DNA. Apart from the specificity of the mutual interaction, it is often found that greater is the contact between the two greater is the efficacy of the drug. Drug delivery systems provide a beneficial vehicle for increasing the efficacy of chemotherapeutics through improved pharmacokinetics and biodistribution.1 They modify the drug release profile, absorption, distribution, and elimination of the drugs for the purpose of improving drug efficacy and safety. Current endeavors in the arena of drug research include the development of targeted delivery leading to a situation that the drug is only active in the target domain of the body (for example, in cancerous tissues).2 A wide variety of nanoscale materials such as liposomes, micelles, dendrimers, etc. have been employed as drug carriers.3 A key attribute to the drug delivery systems is their ability in regulating the drug release, minimizing the side effects, and improving the therapeutic efficacy of the conventional pharmaceuticals.4 Two approaches are adopted to regulate the release of the therapeutic payload from the carrier, namely, endogenous activation and exogenous activation.5 The endogenous activation strategy exploits specific physicochemical characteristics of the pathological microenvironment, providing biologically controlled release. Exogenous activation provides a complementary approach, employing an external stimulus to make the drug release effective.6 This allows the drug to penetrate more into the bioenvironment from the bulk aqueous phase. This leads to an enhanced contact between the drug and its target, resulting in an increased drug efficacy. Although numerous examples have shown successful drug encapsulation and cellular internalization, the release of the drugs in a controlled and targeted fashion without a toxic effect still prevails as a challenge.6 The present work focuses in this direction. Water being the bulk solvent in the biological systems, our intention has been to perform the study in aqueous biomimicking environments. For the purpose, we opted for the * To whom correspondence should be addressed. Fax: 91-33-2414-6584. E-mail: [email protected].

aqueous micellar systems. We project toward improving solubilization of the probes/drugs in the less polar target regions relative to that in water through introduction of some exogenous activating agent and hence to develop a strategy to improve the efficacy of the drugs. Extension of the unorthodox idea contained in the work is expected to lead to an electrostatic pushing of the ionic drugs inside the DNA and biomembranes so that the interaction of the latter with the drugs increases significantly leading to a radical enhancement in the drug efficacy. This should effectively reduce the dose of the drug and eventually reduce the unwanted toxic side effects arising out of it. Since micelles mimic the biomembranes and biological cell walls, we have exploited micelles as our study ground. Among the microheterogeneous environments, micelles are the simplest type of the self-assembled systems, formed from the amphiphilic surfactant molecules in aqueous solution. Micelles show a number of advantages toward the delivery of feebly water-soluble drugs.7 The hydrophobic core of the micelles is sometimes used as a space for solubilization of various therapeutic and diagnostic agents. Micelles, because of their ability to solubilize the membrane proteins, are important in simulating the complex environmental condition present in larger bioaggregates.7-11 Study of the binding interaction of numerous fluorophores with micelles stems from the fact that micellar systems are regarded as simple models of biomembranes.7-11 Interest in this regard has received attention in recent years, with an explosive growth in research targeting on various water-soluble drugs.5 As a model study for the improved cellular internalization of the ionic drugs, here we have demonstrated the penetration of an anionic probe from the micelle-water interface to the interior of the micelle simply by the introduction of a salt. This work is an endeavor to make the foundation for exploiting the strategy to enforce penetration of ionic drugs into the target region. It is a common observation that heavy ions exert a quenching effect on the fluorescence of probes in aqueous and microheterogeneous environments.11,12 In this respect, bromide ion (Br-) is known to be an efficient quencher.12-15 However, the novel observation obtained here is a remarkable enhancement in the fluorescence intensity of the anionic probe, 8-anilino-1-

10.1021/jp1049099  2010 American Chemical Society Published on Web 09/14/2010

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SCHEME 1: Schematic and Optimized Structures of ANS

naphthalene sulfonate (ANS), in cationic alkyltrimethylammonium bromide (nTAB) micellar media with the addition of bromide ion. This observation together with the associated hypsochromic shift and the enhancement in the fluorescence lifetime confirms that the probe penetrates more into the interior of the micelle in the presence of the bromide ion. The unprecedented observation, contrary to the existing purview, has been ascribed to the electrostatic pushing effect operative between Br- and ANS, both being negatively charged. The fluorophore, ANS, (Scheme 1) is known to be an excellent probe as polarity sensor for the biomimicking as well as biological environments.16-21 ANS is nearly nonfluorescent in water, but it fluoresces strongly when trapped in a region of lower polarity in the self-assembled environments.20,21 2. Experimental Section ANS, coumarin-153 (C153), cetyltrimethylammonium bromide (CTAB), tetradecyltrimethylammonium bromide (TTAB), dodecyltrimethylammonium bromide (DTAB), Triton X-100, and Tween-40 (all from Sigma-Aldrich (USA)) and analytical grade KCl, KBr, and KI (all from Merck, India) have been used as received without further purification. 1-Anthracene sulfonate (1AS) was received as a kind gift from Prof. S. C. Bhattacharya of our department. Triply distilled water is used for making the solutions. The concentration of ANS is kept at ca. 1 × 10-5 M throughout the experiment. Absorption and steady state fluorescence measurements have been performed using a Shimadzu UV 2450 spectrophotometer and a Spex fluorolog II spectrofluorimeter respectively. Fluorescence lifetimes are determined from the time-resolved intensity decays by the method of timecorrelated single-photon counting (TCSPC) using a diode source at 370 nm (IBH, UK, nanoLED-03) and TBX-04 as the detector. The instrument response function of our setup is ∼1 ns. The decays are analyzed using IBH DAS-6 decay analysis software. The quality of fits is evaluated by χ2 criterion and visual inspection of the residuals of the fitted function to the data. Mean fluorescence lifetimes (τf) for the biexponential iterative fittings are calculated from the decay times (τi) and the relative amplitudes (ai) using the following relation

〈τf〉 ) a1τ1 + a2τ2

(1)

The dynamic light scattering (DLS) measurements have been performed employing a Malvern Nano-ZS instrument equipped with a 4mW He-Ne laser (λ ) 632.8 nm) and a thermostatted sample chamber. The sample is poured into a DTS0112 low volume disposal sizing cuvette of 1.5 mL (path length 1 cm). The operating procedure is programmed by the DTS software in such a way that there are averages of 25 runs, each run being averaged for 15 s. A particular hydrodynamic diameter and size distribution is then evaluated from the data of the runs. All the experiments were performed at 25 °C temperature with airequilibrated solutions.

Figure 1. Fluorescence spectra of ANS with increasing concentration of CTAB. λexc is 370 nm. Inset shows the variation of the fluorescence maximum as a function of CTAB concentration.

3. Results and Discussion 3.1. Steady State Absorption and Emission Studies. In aqueous medium ANS absorbs in the ultraviolet region (absorption maximum being at ∼370 nm) and emits poorly in the green region (broad emission around 520 nm). In the CTAB environment the emission maximum of ANS shows a large enhancement in the fluorescence yield along with an appreciable hypsochromic shift moving the band maximum to 490 nm (Figure 1). Inset of Figure 1 shows the variation of the fluorescence maximum of ANS with CTAB concentrations. Addition of cationic CTAB results in a significant increase in the fluorescence intensity reflecting a reduction in the polarity of the microenvironment around the probe suggesting the trapping of the anionic probe into the micellar environment. Addition of bromide ion (Br-) to an aqueous solution of ANS leads to an insignificant quenching of the fluorescence of the latter (Figure 2a) in spite of the fact that Br- is known to be a good quencher.12-15 This is ascribed to the mutual electrostatic repulsion operative between the fluorophore and the quencher (both being negative). Gradual addition of KBr to the CTAB micellar solutions containing the probe (ANS) does not lead to a perceptible change in the absorption spectrum. This, however, results in a dramatic enhancement in the fluorescence intensity contrary to the possible quenching of the fluorescence, which should have been observed due to the introduction of the cationic micelles into the environment which assists in bringing the anionic quenching partners in proximity, making the quenching process more effective.11 This effect prevails over the entire range of CTAB concentrations, although to different extents (to be discussed later). This is accompanied by a further blue shift (∼7 nm) of the emission maximum relative to its position in the CTAB micelle in the absence of Br- (Figure 2b), which is already blue-shifted from the observed emission maximum of ANS in water. The large enhancement in the emission intensity of ANS in the presence of Br- is contrary to the normal expectation of quenching of the fluorescence of a probe by the said ion.12-15 The interesting feature is that the extent of fluorescence enhancement in the presence of a definite concentration of Br- varies with a variation in the concentration of CTAB. We have performed a thorough experiment where at a particular CTAB concentration the variation of the fluorescence intensity of ANS is measured as a function of concentration of the added Br-. With the addition of KBr, the fluorescence

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Figure 2. Fluorescence spectra of ANS in (a) water and (b) 0.8 mM CTAB with increasing concentration of Br-. Concentrations of Br- for both (a) and (b) are provided in the legends of (a). Inset of (b) depicts the variation of fluorescence maximum with the addition of KBr. λexc is 370 nm.

Figure 3. Plot of the maximum enhancement in the relative fluorescence intensity of ANS with the addition of Br- as a function of CTAB concentration. The solid line is not a fitted line. It simply guides the eyes to the pattern of variation.

intensity increases, eventually achieving a plateau at a sufficiently high concentration of Br- (g124 mM). Fmax/F0 was then calculated, where F0 is the fluorescence intensity in the absence of the KBr, and Fmax is the maximum value of it in the presence of sufficient concentration of KBr. The study was repeated at different CTAB concentrations to get the various values of the Fmax/F0. Taking account of the concentrations of CTAB in different solutions and the concentrations of the external bromide ion added to achieve Fmax, the variation of Br- concentration (coming from the counterion of the surfactant) because of the change in the surfactant concentration is considered to be negligibly small. Figure 3 depicts a plot of Fmax/F0 as a function of the concentration of CTAB. The plot shows a typical pattern with a CTAB maximum around 0.6 mM. This is close to the critical micellar concentration (CMC) of CTAB in the presence of the added ions.22 In the post CMC range, the relative increase in the fluorescence intensity of ANS is found to drop again with a further increase in the surfactant concentration. The unprecedented observation of the fluorescence enhancement of ANS in CTAB micelles with the addition of bromide ion is rationalized in the light of a rather new concept, coined as an electrostatic pushing effect. In aqueous medium, because of the mutual electrostatic repulsion between the two species of like charges (both being anionic), the quencher can not come closer and communicate to the fluorophore efficiently, and as a result, we observe only an insignificant quenching of the fluorescence of ANS with the addition of Br-. In the premicellar region, CTAB surfactants are known to be stacked parallely presumably forming a cylindrical structure, surrounded by the counterions.23 In the lower range of concen-

trations, both ANS and CTAB move around randomly in solution, but at any given time ANS molecules are exposed to some hydrophobic interactions provided by the alkyl chains of CTAB. In this arrangement the negatively charged sulfonate group of ANS remains closer to the positively charged trimethylammonium headgroup of CTAB and the alkyl chain of CTAB folds back to provide a hydrophobic environment for the ANS molecule.23 Thus, in the premicellar range of CTAB concentrations, clusters of CTAB lie around a given ANS molecule. Under this situation added Br- ions are not allowed to penetrate effectively inside the CTAB cluster to get access to the ANS molecule. Thus the effect of Br- ions on ANS in the CTAB premicellar condition is less significant. At higher concentrations of Br-, however, some of them can penetrate into the cluster, gain access to the ANS molecule, and succeed in pushing it into the interior of the premicellar stacked aggregates. Because the fluorophore is electrostatically pushed by the Br- ions into the premicellar aggregates, the probe experiences a lowering in the polarity. Since the fluorescence of ANS is highly sensitive to the solvent polarity, a slight change in the polarity and/or hydrophobicity of the microenvironment is reflected immensely in its emission characteristics. As the concentration of CTAB increases enhancement of the fluorescence intensity of ANS in the presence of Br- ion becomes more prominent and attains its maximum value at a concentration where micelles are formed. Upon going from premicellar to micellar phase the location of the ANS molecule changes from “inside the cavity” to “outside the micelle”, i.e., at the micelle-water interface.23 The naphthalene chromophore of the ANS molecule carries a negatively charged sulfonate group, and this part of the molecule resides outside the positively charged CTAB micelles.23 Thus, ANS resides at the micellewater interface projecting the anionic part toward water since the interior of the micelle is more hydrophobic.24 Because of the negative charge of the fluorophore, at lower concentrations, Br- ions can not come closer to the fluorophore to interact. With the addition of more and more bromide ions to the solution of ANS in CTAB micelles, its local concentration in the immediate vicinity of ANS increases (since Br- ions are electrostatically attracted toward the cationic surface of the CTAB micelles). This leads to an enhanced electrostatic repulsion to be experienced by the fluorophore molecule forcing it to penetrate into the interior of the micellar units. The CT fluorescence of ANS being highly sensitive to the solvent polarity and the micellar interior being less polar the emission spectrum bears its signature in terms of a remarkable enhancement in the intensity as well

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Sarkar et al. TABLE 1: Fluorescence Lifetimes of ANS in 0.8 mM CTAB with Varying Concentrations of Br- (λexc ) 370 nm and λem ) 490 nm)

Figure 4. Time-resolved fluorescence decays of ANS in water (λexc ) 370 nm and λem ) 520 nm) and in 0.8 mM CTAB (λexc ) 370 nm and λem ) 490 nm). Legends describe the experimental solutions. The solid black line on the left represents the instrument response function (IRF).

as a noticeable hypsochromic shift (∼7 nm) of the emission band maximum. At even higher CTAB concentrations, the number of micellar units increases and some of them remain devoid of the fluorophore. Under this situation, as Br- ions are added to the system, some of the ions are attached to the micelles which are free of ANS and hence they remain passive in terms of the interaction with the fluorophore. This leads to a decrease in the extent of enhancement of ANS fluorescence with the added Br-. Finally Fmax/F0 attains a plateau at high enough concentration of CTAB (Figure 3). It is found from Figure 3 that the maximum enhancement of fluorescence is achieved at a CTAB concentration of 0.6 mM. However, to establish further the electrostatic pushing effect we have performed the subsequent experiments (described in the forthcoming sections) at the literature CMC value of CTAB, i.e., 0.8 mM.22 This is done in order to be on the safe side to assure that micelles are really formed and the complications arising out of the formation of the premicellar aggregates do not perturb the experimental data. 3.2. Time-Resolved Fluorescence Decay Study. Ultrafast spectroscopic studies of ANS with micelles of varying surface charges and reverse micelles are available in literature.25-28 The fluorescence emission decay of ANS has been investigated by time-resolved and frequency domain fluorometric studies.25-28 The dependence of ANS fluorescence anisotropy on the emission wavelength has also been investigated.26 The result is interpreted from the heterogeneity of the medium leading to a difference in the rate of the reorientation process of water molecules around the excited state of ANS or exchange of the probe among the different locations, occurring on a time scale longer than the fluorophore lifetime. Thus, ultrafast spectroscopy has been applied in many cases to interrogate the dynamics and motion of the probe in microheterogeneous environments. ANS shows a single-exponential fluorescence decay pattern in water with a lifetime of ∼0.4 ns.29 Single-exponential fluorescence decay of ANS in aqueous solution indicates that the fluorescent species exists in a single environment.30,31 In CTAB micellar solutions, however, the decay becomes biexponential. Progressive addition of CTAB results in an increase in the fluorescence lifetime of the probe. In 0.8 mM CTAB, the average lifetime of ANS fluorescence is found to be 3.18 ns. Interestingly, with an increase in the concentration of the added Br- ion in 0.8 mM CTAB, the fluorescence lifetime of

[Br-] (mM)

a1

τ1 (ns)

a2

τ2 (ns)

τav (ns)

χ2

0 4 36 72 124

0.35 0.19 0.16 0.08 0.06

2.52 ( 0.2 2.66 ( 0.2 2.58 ( 0.2 2.62 ( 0.2 2.58 ( 0.2

0.65 0.81 0.84 0.92 0.94

3.54 4.41 4.61 4.85 5.12

3.18 ( 0.2 4.08 ( 0.2 4.29 ( 0.2 4.67 ( 0.2 4.97 ( 0.2

1.16 1.13 1.02 1.05 0.99

ANS increases further (Figure 4). The deconvoluted lifetime values are given in Table 1. Deviations from the single-exponential nature of the decay behavior in aqueous CTAB media imply that either the fluorescent species exists in multistable environments where exchange rate is slow or simultaneous relaxation from the multiple excited electronic states occurs. Such behavior is often attributed to the distribution of the probe in different regions of the microheterogeneous environments. The biexponential fluorescence decays, thus, suggest that in CTAB environments (both in the absence and in the presence of the added Br-) ANS molecules are distributed in the different regions of the micellar media. The biexponential decays of ANS in 0.8 mM CTAB environments are characterized by a shorter component with lifetime τ1 and a longer decay component τ2. The short and the long components in the fluorescence decay of ANS are ascribed to arise from the distribution of the dye in the different regions of the micelles. Consistent with the literature, the shorter lifetime component is ascribed to represent the fraction of the dye molecules present in the micelle-water interface, while the longer one to the dye molecules present in the micellar interior. It is pertinent to mention here that fluorescence spectral identification of the different species corresponding to the different lifetimes (short and long) would have been interesting. For the purpose, we have tried with the probe in different concentrations of CTAB for different concentrations of added Br- in each of the CTAB solutions. The fluorescence decay analyses are done at different emission wavelengths looking for a change in the pre-exponential factors. The experiment does not produce a pronounced differential result. This might be due to the fact that the shift in the band maximum is small within the broad emission envelope (Figure 2b). Time-resolved emission spectral (TRES) analysis might be a better option to confirm the specificity of the species. Unfortunately, we do not have the experimental facility to perform this. All the observations suggest that the probe moves not too deep into the micellar cavity upon electrostatic pushing by the added Br- ion. The forthcoming discussion on the polarity dependence study of ANS fluorescence in dioxane-water mixed solvents also corroborates only a small decrease in the ET(30) value (from ∼52 to ∼50) in 0.8 mM CTAB solution by the addition of 124 mM Br-. It is already known that in CTAB micellar solutions ANS exists mostly in the micelle-water interfacial region.23 Upon addition of the Br- ions, the lifetime of the shorter component remains almost invariant, while that of the longer component (and hence, the average lifetime) is found to increase appreciably. The value of ais (relative contribution of the individual lifetime components to the total fluorescence) gives an idea about the partitioning of ANS in the two regions of the micelle. With an increase in the Br- concentration, a1 is found to decrease considerably with the concomitant increase in the a2 value. This implies that the local concentration of ANS in the surface (represented by a1) decreases while that in the micellar

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TABLE 2: Fluorescence Intensity and Lifetime of ANS in 0.8 mM CTAB with Added Br[Br-] (mM)

Fl intensity (F, au.)

Lifetime (τ2, ns)

F/F0

τ/τ0

0 4 36 72 124

1.27 1.60 1.69 1.77 1.93

3.54 ( 0.2 4.41 ( 0.2 4.61 ( 0.2 4.85 ( 0.2 5.12 ( 0.2

1 1.26 1.33 1.39 1.52

1 1.24 1.30 1.37 1.44

interior (represented by a2) increases with the progressive addition of Br-, corroborating the Br- induced electrostatic pushing effect on ANS forcing it to penetrate deeper into the micellar interior. The lifetime of ANS is known to depend on the solvent polarity and it increases with a reduction in the polarity of the environment, ascribed to the reduction in the rate of the nonradiative transitions.32-34 Thus, an increase in the concentration of Br- helps in solubilizing ANS into the micelles due to the electrostatic pushing effect. Fluorescence decay analysis of ANS in CTAB solutions reveals that with an increase in the CTAB concentration the a2 component increases relative to a1. This goes parallel to the experiment where a2 increases with an increase in the Br- concentration at a fixed CTAB concentration (Table 1) and ensures that the probe is entrapped more into the micelles with the addition of Br-. A significant point to mention here is that the relative enhancement in the fluorescence intensity of ANS in 0.8 mM CTAB upon addition of a given amount of Br- (Figure 3) agrees well with the enhancement in the longer component of the fluorescence lifetime (which reflects the fraction of ANS molecules that are able to penetrate into the micellar interior) (Table 2). The correspondence of the relative enhancement of fluorescence intensity and fluorescence lifetime signifies that the observed enhancements are primarily due to a reduction in the polarity in the vicinity of the probe. An increase in the viscosity of the microenvironment because of the penetration of ANS into the micellar environment in the presence of the added Br- might also contribute to the enhancement of the steady state fluorescence intensity and the lifetime of ANS since increase in these two parameters have been observed with an increase in the viscosity of the solvent.35 At the moment, we can not resolve the individual contributions from the two factors toward the change in the fluorometric observation (also not very important in terms of the present objective). Nevertheless, the whole set of results confirms that the fluorophore is pushed into the micellar environment by the added Br- ion and reinstates the proposition of the electrostatic pushing effect. The essence of the present work is depicted and conveyed through Scheme 2. We have made a general study on the effect of polarity on the fluorescence of ANS to ratify the electrostatic pushing effect of the Br- ion on the probe in the micellar media. Steady state and time-resolved fluorometric experiments are performed with the probe in dioxane-water solvent mixtures. An increase in the fluorescence intensity and the fluorescence lifetime of ANS is observed with an increase in the dioxane proportion in the solvent mixtures. Figure 5 depicts the observations. Figure 5 implies that it is the polarity of the medium that plays the vital role for the enhancement in the fluorescence intensity and the fluorescence lifetime of ANS. This corroborates that the enhancement in the fluorescence intensity (and lifetime) of ANS upon addition of Br- in CTAB micellar system is because of the reduction in the polarity of the microenvironment and substantiates that Br- pushes the probe toward the interior of the micelle (region of lower polarity). Figure S1 given in

the Supporting Information depicts the plot of variation of the relative fluorescence enhancement as a function of the polarity parameter, ET(30). The figure reveals an increase in the relative fluorescence intensity as the percentage composition of dioxane is increased in the solvent mixture, i.e., as the polarity is lowered. Interpolating the relative fluorescence enhancement (relative to the fluorescence in water) of ANS in 0.8 mM CTAB on the curve gives an estimate of the polarity of the microenvironment around the probe in CTAB micelles. Addition of KBr to this solution modifies the fluorescence ratio (cf. Figure 2b). The microscopic polarity around ANS in the presence of 124 mM Br- is obtained by interpolating the relative fluorescence on the plot of Figure S1. The study reveals that addition of KBr leads to a reduction in the polarity (in terms of ET(30)) around the probe to ∼50 compared to the value ∼52 obtained in 0.8 mM CTAB solution in the absence of KBr. The results convincingly establish that the anionic fluorophore is pushed toward the micellar interior because of Br- induced electrostatic pushing effect. This polarity dependence experiment using dioxane-water solvent mixtures further suggests that there is no significant specific interaction between the anionic fluorophore and the surfactant monomer, since in dioxane-water mixed solvents CTAB is absent. 3.3. Effect of Other Halides. To check if size of the halide ion plays any role in the pushing effect, we have performed the steady state experiment with other halide ions such as chloride and iodide. The corresponding steady state fluorescence spectra are provided in Figure S2 of Supporting Information. With Clion, an enhancement in the fluorescence of ANS was observed in 0.8 mM CTAB, but to a lesser extent compared to that observed for the same concentration of Br- ion, with a smaller hypsochromic shift amounting to ∼4 nm in the emission maximum. With I- the situation is rather complex. At lower concentration of I-, the fluorescence enhancement is much more compared to those observed with Br- and Cl- with a profound hypsochromic shift of ∼20 nm. This indicates an effective penetration of the probe (ANS) into the micellar interior. The differential observations in terms of the fluorescence enhancement as well as the blue shift reveal that the probe experiences a lesser polarity upon addition of the bulkier halide ion and corroborate an improved penetration into the micellar interior. Above a concentration of 1 mM KI, however, the solution becomes turbid, and precipitation commences resulting in the disruption of the micelles.15 The study, thus, reveals that the extent of enhancement in the fluorescence of ANS follows the order I- > Br- > Cl- and indicates that a bulkier ion has a stronger and more effective pushing effect than a smaller ion. It is known that the relative quenching efficiency of the halide ions follows the same order as given above. Despite a greater quenching expected, a greater degree of fluorescence enhancement suggests that the quenching effect is superseded by the reduction in the polarity of the microenvironment around the probe and reinstates a more effective pushing effect by the bulkier halide ions. At a given CTAB concentration, the fluorescence intensity of ANS increases with an increase in the halide (KBr and KCl) concentration until a plateau is achieved in the value of F/F0 (Figure 6) where F and F0 are the fluorescence intensities of ANS in the presence and in the absence of the added halide. One may argue and assign the enhancement in the fluorescence and fluorescence lifetime of ANS in the CTAB micelles upon addition of Br- ions to the salting in/salting out effects because of the possibility of a change in the solubility of the

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Figure 5. Steady-state fluorescence spectra (a) and time-resolved fluorescence decays (b) of ANS in varying composition of dioxane-water mixtures. λexc ) 370 nm. Composition of the dioxane-water mixtures for both (a) and (b) are provided in the legends of (a). The sharp black profile in (b) represents the IRF.

SCHEME 2: Schematic Representation of the Electrostatic Pushing Effect of the Probe by the Br- Ion in the CTAB Environment

probe upon increase in the ionic strength. However, the following argument rules out this possibility, at least for the present system. Had it been like that, one should not expect a differential fluorometric enhancement of the probe for the different halide ions at a particular concentration of the KX salts. For a definite concentration of KCl, KBr, and KI the ionic strengths will be same (all being 1:1 type salts). This should lead to similar salting

Figure 6. Variation of the relative fluorescence intensity (F/F0) of ANS as a function of concentration of the halide ion in 0.8 mM CTAB solution.

in/salting out effects on the probe (ANS) upon addition of the same amount of KX. A distinct differential effect of the halides on ANS fluorescence, as evident from the above discussion and Figures 6 and S2, thus rules out the involvement of salting in/ salting out effects in the present case. 3.4. DLS Studies. ANS happens to be a very useful CT probe since its emission is highly sensitive to the polarity of its immediate environment. Thereby, from the variation of its fluorescence characteristics the probe can sense its penetration into the micellar interior with the addition of bromide, reflecting the electrostatic pushing effect. It may, of course, be argued that Br- ion induced structural change of the CTAB micellar units might disturb the distribution of the fluorophore between the different regions of the micellar phase resulting in the observed fluorescence enhancement with the addition of the halide ion. However, the small amount of Br- ion required for observing the remarkable fluorescence enhancement is insufficient to modify the micellar structure enough to perturb the distribution of ANS appreciably to manifest the observation to this extent.36-38 Literature suggests that spherical CTAB micelle

Electrostatic Pushing Effect

Figure 7. Normalized size distribution of 0.8 mM CTAB containing 1 × 10-5 M ANS with varying concentrations of the added Br-. Concentrations of Br- are provided in the legends.

changes to a rod shaped one at a Br- concentration g0.1 M which is too high compared to the concentration of Br- required here to observe the appreciable fluorescence enhancement of ANS.36 To confirm whether a change in the micellar shape is indeed playing a role or not in our observation, we have carried out the DLS studies on 0.8 mM CTAB solution with varying concentrations of Br- ion in the absence as well as in the presence of ANS. Presence of ANS does not put any signature on the DLS data. The DLS spectra are found to be monomodal in general and the measured hydrodynamic radius of 0.8 mM CTAB micellar units come out to be 34 ( 3 Å which agrees with the literature value of ∼27 Å.38 Upon gradual addition of Br- to 0.8 mM CTAB in the absence as well as in the presence of ANS it is found that the band pattern and the hydrodynamic radius remains unchanged within experimental error limit ((3 Å) (Figure 7). This confirms that there is no detectable change in the shape and/or size of the CTAB micelle upon addition of the Br- ion until a concentration of ∼80 mM (all the DLS figures are not given for the sake of clarity). The possibility of electrolyteinduced micellar structural change to be responsible for the unprecedented observation in the present case is, thus, conclusively ruled out. At higher Br- concentrations, however, the DLS band changes its pattern. This is revealed from Figure 7 for a solution of 0.8 mM CTAB containing 180 mM Br- where formation of the larger aggregates is indicated. This is consistent with the existing literature36 that reports a structural change in the CTAB micelle at a bromide concentration higher than 100 mM. 3.5. Observations with Other Surfactants and Probes. To establish the generality of the technique, similar experiments are conducted with other surfactants and probes. Similar enhancement in the fluorescence of ANS has also been observed in other analogous cationic (nTAB) micelles such as tetradecyltrimethylammonium bromide (TTAB) and dodecyltrimethylammonium bromide (DTAB). Experimenting with these nTAB micellar systems, the extent of relative enhancement is found to increase with an increase in the chain length of the surfactant which can be rationalized from the consideration of the two factors, namely, aggregation number and the compactness of the micelles.22,39,40 Experiments with nonionic micelles such as Triton X-100 and Tween 40 reveal that the effect is remarkably less. The micelles being nonionic, this is ascribed to the lack of electrostatic attraction between the micellar surface and the Br- ion, restricting the accumulation of the latter to manifest the pushing effect significantly. Experiments have also been performed with C153 and 1AS in 0.8 mM CTAB environment. The former probe is nonionic while the latter one is anionic. Both the experiments indicate a lowering of polarity in the microenvironment of the probes with the addition of KBr to the solutions. A representative plot for C153 is given in Figure

J. Phys. Chem. B, Vol. 114, No. 39, 2010 12547 S3 of Supporting Information. For a cationic probe, phenosafranin (PSF), a parallel effect is observed using a cationic quencher (Cu2+) only at premicellar concentrations of anionic sodium alkyl sulfate (SnS) micellar systems.11,41 At higher concentrations, however, a similar observation could not be noticed. We ascribe that to the lack of sensitivity of the probe (PSF) toward the polarity of the environment, the effect of penetration of PSF into SnS micellar interior in terms of the fluorescence enhancement is masked by the quenching effect of the added copper ion. The experiments encourage us to propose that the electrostatic pushing effect is rather a general phenomenon. Further experiments are invited to confirm this point. Happens to be general, the effect is expected to be potentially useful toward enhancing the solubilization of drugs in the less polar target region compared to the bulk biological fluid, namely, water. For example, a majority of drugs intercalate into the DNA strands.42-45 Because of the presence of the phosphate bases the DNA helices are negatively charged. The strategy described herein can be extended and applied to push the ionic drugs more inside the DNA so that the interaction of the latter with the drugs increases significantly leading to a radical enhancement in the drug efficacy. This should reduce the dose of the drug and eventually reduce the unwanted side effects arising out of it. 4. Conclusion The work demonstrates an unprecedented fluorescence enhancement of the anionic probe, ANS in cationic CTAB micellar medium with the addition of bromide ion. The observation has been ascribed to the electrostatic pushing effect operating between ANS and the halide ion, both being negatively charged. The effect has been corroborated from the lifetime measurements as well. That Br- induced micellar structural change is not responsible for the remarkable observation has been established from dynamic light scattering. The added anion helps to push the probe, electrostatically, toward the micellar interior. Works with other probes and micellar systems propose that the electrostatic pushing effect is a general phenomenon. Since the micellar systems mimic the biomembranes, the simple strategy has the potential to be exploited successfully in real biosystems. Increase in the concentration of the permissible salts is projected to lead to an electrostatic pushing effect to the ionic drugs toward the less polar target region, resulting in a substantial enhancement in their solubilization/delivery and hence efficacy. Extension of this work with drug molecules in biological systems can directly provide a demonstration for enhancing the drug efficacy substantially. Acknowledgment. This research is supported by DBT and CSIR, Government of India. We appreciate the cooperation received from Prof. K. P. Das of Bose Institute, Kolkata, for the DLS measurements. Thanks are due to D. Bose for experimental assistance. Supporting Information Available: Figures depicting a plot of the variation of the relative fluorescence enhancement of ANS as a function of ET(30) in dioxane-water solvent mixture, fluorescence spectra of ANS in 0.8 mM CTAB with the addition of Cl- and I- ions, and fluorescence spectra of C153 in water, 0.8 mM CTAB, and 0.8 mM CTAB + 124 mM KBr. This material is available free of charge via the Internet at http:// pubs.acs.org. References and Notes (1) Kim, C. K.; Ghosh, P.; Pagliuca, C.; Zhu, Z.-J.; Menichetti, S.; Rotello, V. M. J. Am. Chem. Soc. 2009, 131, 1360.

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