An Investigation into the Effect of the Structure of Bile Salt Aggregates

Article pubs.acs.org/JPCB

An Investigation into the Effect of the Structure of Bile Salt Aggregates on the Binding Interactions and ESIHT Dynamics of Curcumin: A Photophysical Approach To Probe Bile Salt Aggregates as a Potential Drug Carrier Sarthak Mandal, Surajit Ghosh, Debasis Banik, Chiranjib Banerjee, Jagannath Kuchlyan, and Nilmoni Sarkar* Department of Chemistry, Indian Institute of Technology, Kharagpur 721302, West Bengal, India S Supporting Information *

ABSTRACT: This work demonstrates the utilization of bile salt aggregates as a potential biological host system for studying the binding interactions and dynamics of the poorlywater-soluble drug curcumin by means of photophysical techniques. We found that the level of degradation of curcumin is greatly suppressed upon encapsulation into the nanocavities of three different bile salt aggregates. However, NaTC aggregates are more effective to suppress the level of degradation of curcumin than NaCh and NaDC aggregates. We also report the modulation of the photophysical and dynamical properties of curcumin into the nanocavities of bile salt aggregates using steady-state and time-resolved fluorescence spectroscopy. The reduced level of interaction of curcumin with water upon incorporation into the different binding sites of bile salt aggregates results in an enhanced fluorescence intensity along with the blue shift in the emission maxima of curcumin. However, the observation of higher fluorescence quantum yield as well as longer fluorescence lifetime in NaTC aggregates compared to that in NaCh and NaDC aggregates clearly indicates a more effective decrease in the excited-state intramolecular hydrogen atom transfer (ESIHT) mediated nonradiative deactivation of curcumin by the interaction with the anionic headgroup of NaTC. The binding and location of curcumin into the bile salt aggregates has been further confirmed from the steady-state fluorescence anisotropy measurements. In addition, we have shown the effect of addition of salt on the photophysical properties of curcumin in the confined environments of bile salt aggregates. Our results indicate that on addition of salt the time scale of ESIHT process of curcumin in bile salt aggregates is markedly increased.

1. INTRODUCTION Curcumin, a natural polyphenolic compound isolated from rhizomes of the Indian spice turmeric (Curcuma longa), has long been known for its medicinal properties which include anticancer, anti-inflammatory, antioxidant, antimicrobial, antiamyloid, anti-Alzheimer, anticystic fibrosis, and wound healing properties.1−9 As a result, curcumin is undergoing many clinical trials in humans to test the safety and effectiveness for the various cancer treatments.10−12 However, its poor aqueous solubility and lack of bioavailability are the two major difficulties which need to be overcome to increase the effectiveness of curcumin as a treatment agent.13 Moreover, curcumin is reported to be unstable, decomposing to trans-6(4′-hydroxy-3′-methoxyphenyl)-2,4-dioxo-5-hexanal, vanillin, ferulic acid, and feruloylmethane in the aqueous solution of neutral and alkaline pH.14−16 Wang et al.14 reported that deprotonation is one of the main reasons for the degradation of curcumin. Over the past few years, many efforts have been made to increase the solubility and bioavailability of curcumin through encapsulation into various biological assemblies such as micelles, mixed micelles, reverse micelles, vesicles, proteins, and cyclodextrins.17−31 In a very recent study, we showed that © 2013 American Chemical Society

nonionic surfactant forming vesicles (niosomes) are a better model system than micelles to enhance the solubility and stability of curcumin.32 In continuation of our keen interest in the study of the interaction of drug with biomimicking assemblies, in this work we have investigated the interaction of curcumin with three different bile salt aggregates of varying structural properties by means of photophysical techniques. Bile salts are one of the most widely studied naturally occurring amphiphilic molecules with a convex hydrophobic surface and a concave hydrophilic surface in their nonplanar steroidal skeleton.33,34 These bioactive molecules are synthesized in the liver from cholesterol and play an important role in the solubilization of lipids in living organisms, which allows them to be used as a potential drug delivery system. Bile salts spontaneously form aggregates in the aqueous solution because of their amphiphilic nature. The complex aggregation behavior of bile salts in aqueous solution has been extensively studied by many experimental and theoretical simulation methods.35−37 Received: August 5, 2013 Revised: October 7, 2013 Published: October 8, 2013 13795

dx.doi.org/10.1021/jp407824t | J. Phys. Chem. B 2013, 117, 13795−13807

The Journal of Physical Chemistry B

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Though several models are proposed to explain the aggregation process of bile salts, the primary and secondary aggregate model proposed by Small et al.35 is the most widely accepted one. According to this model, the primary aggregates with hydrophobic binding sites are formed at lower concentration of bile salt by the hydrophobic interaction between the convex surfaces of monomers. With increasing concentration of bile salt, the primary aggregates agglomerate to form secondary aggregates of larger size by hydrogen-bonding interactions. Small-angle X-ray scattering (SAXS) and small-angle neutron scattering (SANS) studies have indicated that the secondary aggregates resemble an elongated rod where the central hydrophilic core is filled with water and ions.38 For NaDC aggregates, the reported length and radius of the rod are ∼40 and ∼8 Å, respectively.38 It has also been shown that trihydroxy bile salts (NaCh/NaTC) form smaller micelles than dihydroxy bile salts (NaDC).39 The spin-label studies Kawamura et al.40 also indicated the difference in the micellar structures between trihydroxy bile salts and dihydroxy bile salts despite their similar shapes. Very recently, Miranda and co-workers41 showed the varying distribution of the two types of aggregates (primary/ secondary) at different concentrations using fluorescent dansyl derivative of sodium cholate (NaCh). Owing to the presence of these two different types of binding sites, bile salt aggregates are suitable for carrying both hydrophobic and hydrophilic drug molecules.42,43 Although a number of photophysical studies are available in the literature on the host−guest interactions using bile salt aggregates as a potential biological host system, the studies on the interactions of drugs with these systems are limited.44−50 In a very recent study, Miranda and co-workers43 demonstrated the utilization of cholic acid aggregates as drug carriers using an effective nonsteroidal antiinflammatory drug, naproxen, and its methyl ester derivative as photophysical probes. They have shown a very high affinity of the hydrophobic drug to the cholic acid aggregates. Moreover, by singlet and triplet state quenching studies of the bound probe molecules, they have indicated that with an increase in the hydrophobicity of the probe the binding affinity increases. Curcumin being a hydrophobic drug which is almost insoluble in water, we expect its strong binding with bile salt aggregates. In this work, we have therefore exploited the potential of three different bile salt aggregates as an effective drug delivery media with enhanced solubility, stability, and bioavailability of curcumin. We demonstrate that a slight variation in the structural properties of bile salts, in particular the anionic group at the hydrophilic surface, largely affects the binding dynamics and stability of curcumin. It has been found that the anionic group of the bile salts significantly interacts with the acidic group of curcumin to modulate its photophysics. The keto−enol tautomer with strong intramolecular hydrogen bond (Scheme 1) is the most stable conformer of curcumin in solution as confirmed by many theoretical and experimental studies.51 The photophysical properties of curcumin have been extensively studied in various homogeneous and heterogeneous systems by using picosecond and subpicosecond time-resolved fluorescence spectroscopy.52−57 These studies revealed that the excited-state intramolecular hydrogen atom (proton) transfer (ESIHT/ESIPT) and solvation dynamics are the two major photophysical processes that take place during the deactivation of curcumin in the first singlet excited state. Since ESIHT is one of the major nonradiative deactivation pathways of curcumin, the perturbation of the intramolecular hydrogen bond by the

Scheme 1. Chemical Structure of the Keto−Enol Form of Curcumin with Strong Intramolecular Hydrogen Bond Involved in ESIPT Process

surrounding media significantly modulates its photophysics and photodynamics. Using transient absorption spectroscopy, Ghosh et al.57 have shown that both hydrogen bond donating and accepting solvents perturb the intramolecular hydrogen bond associated with the curcumin to affect its ESIHT process. According to their study, in polar solvents the perturbation of the intramolecular hydrogen bond by dipole−dipole interactions results in a reduced nonradiative deactivation of curcumin. On the contrary, in protic solvents, although the ESIHT-mediated nonradiative deactivation is reduced but a new deactivation pathway is opened through the intermolecular proton transfer of curcumin with solvent. Erez et al.58 also observed the solvent-mediated proton transfer reaction of curcumin in polar protic solvents ethanol and 1-propanol. The same group also studied the effect of mild base (sodium acetate) on the ESIHT of curcumin in order to understand the effect of proton-accepting ability of solvent.59 In a very recent study, Saini and Das60 have demonstrated the modulation of the nonradiative decay rates of curcumin in the binary solvent mixture of toluene and methanol. The ESIHT of curcumin in the confined environments of micelles, vesicles, proteins, and cyclodextrins is reported to be largely affected by the surface charge, structure, interaction, and dynamics of the microenvironment.56,61 Here, we have shown the modulation of the ESIHT dynamics of curcumin upon confinement into the bile salt aggregates. While our group was engaged in preparing the manuscript of this article, Patra and co-workers62 reported the interaction of curcumin with NaDC and NaCh bile salts but not with the NaTC bile salt. In this work we have used three different bile salts (NaDC/NaCh/NaTC) to show how the photophysical and dynamics processes of curcumin in their aggregates are tuned by the structural properties of the bile salts. It has been found that a minute change in the structural properties of bile salt significantly modulates the photophysics of curcumin. The major finding of this work is that the fluorescence efficiency of curcumin is significantly enhanced in NaTC aggregates than in NaCh and NaDC aggregates. Moreover, the level of degradation of curcumin is suppressed more effectively in NaTC aggregates than in NaCh and NaDC aggregates. It has been revealed that, while the presence of an anionic carboxyl group in the NaDC and NaCh bile salts results in the reduced fluorescence efficiency of curcumin, the anionic headgroup of NaTC bile salt results in enhanced fluorescence efficiency. This finding can be explained on the basis of the enhanced nonradiative deactivation through the intermolecular proton transfer from acidic group of curcumin to the anionic carboxyl group of NaDC and NaCh bile salts. 13796



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2. EXPERIMENTAL SECTION 2.1. Materials. Curcumin (purity ∼80%) was purchased from Sigma Aldrich and used as received without further purification. In the earlier studies, Adhikary et al.21,56 showed that the presence of other curcuminoids (∼20%) negligibly affect the photophysics of curcumin. The bile salts, sodium deoxycholate (NaDC; purity ∼98%), sodium cholate (NaCh; purity ∼98%), and sodium taurocholate (NaTC; purity ∼97%), were also purchased from Sigma Aldrich and used as received. The chemical structures of the bile salts are shown in Scheme 2.

so that the ultimate concentration of curcumin in the solution becomes 10 μM. Methanol content in the studied solutions always remained below 0.5% (v/v). The UV−vis absorption and fluorescence studies were performed with increasing concentrations of different bile salts into this solution. However, for degradation-kinetics study, the curcumin in aqueous 0.2 M NaCl solutions with 30 mM bile salts was prepared in the cuvette after removal of methanol completely. Although curcumin is almost insoluble in water, it is solubilized upon encapsulation into the different microenvironments of the bile salt aggregates. 2.3. Instrumentation. Steady-state UV−vis absorption and fluorescence emission spectra were recorded in Shimadzu (model UV 2450) UV−vis spectrophotometer and Hitachi (Model No.F-7000) spectrofluorimeter, respectively. The timeresolved emission decays were collected using time-correlated single-photon-counting (TCSPC) setup. The detailed experimental setup of this instrument has been described in our previous publication.46 In brief, picosecond diode laser at 408 nm (IBH, UK, Nanoled) was used as light source and the signal was detected in magic angle (54.7°) polarization using Hamamatsu MCP PMT (3809U). The typical instrument response function that is the full width at half-maximum is ∼90 ps in our system. The decays were analyzed using IBH DAS-6 decay analysis software. The average fluorescence lifetimes for the decay curves were calculated from the following equation

Scheme 2. Chemical Structures of Sodium Cholate (NaCh), Sodium Deoxycholate (NaDC), and Sodium Taurocholate (NaTC)

2.2. Preparation of Solutions. First of all, a concentrated (2 mM) stock solution of curcumin was prepared in methanol. A small aliquot (∼10 μL) of that stock was taken in a quartz cuvette and diluted with 2 mL of aqueous 0.2 M NaCl solution

τav = a1τ1 + a 2τ2

(1)

Figure 1. Changes in the UV−vis absorption spectra of curcumin in the aqueous 0.2 M NaCl solution with increasing concentration of different bile salts (a) NaDC [absorption spectra (i)−(vii) correspond to concentration of NaDC = 0, 0.8, 2.2, 3.8, 4.3, 6.5, 10.1, 15 mM], (b) NaCh [spectra (i)− (x) correspond to concentration of NaCh = 0, 1.4, 4.1, 5.9, 8.1, 10.1, 12.9, 16.6, 20.3, 25 mM], and (c) NaTC [spectra (i)−(ix) correspond to concentration of NaTC = 0, 2.0, 3.5, 6, 8.5, 10.5, 13, 16.1, 19.4 mM]. 13797



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Figure 2. Changes in the absorption spectra of curcumin (∼10 μM) in (a) NaDC (30 mM), (b) NaCh (30 mM), and (c) NaTC (30 mM) aggregates with increasing time. The insets show the extent of degradation of curcumin in terms of the decrease in absorbance at 424 nm with increasing time interval.

where τ1 and τ2 are the time constants of the first and second decay components of curcumin and a1 and a2 are the corresponding relative amplitudes of these components. The errors in the experimental measurements have been calculated by repeating the experiments with new sets of solutions that are on the basis of independent experiments with independent solution preparation. The steady-state fluorescence anisotropy values of curcumin in the aqueous 0.2 M NaCl solution with increasing concentration of bile salts were measured using Perkin-Elmer LS-55 spectrofluorimeter. The steady-state anisotropy can be determined by using the following equation: r=

G=

(IVV − GIVH) (IVV + 2GIVH)

IHV IHH

ΦS A (Abs)R nS2 = S ΦR AR (Abs)S nR 2

(4)

where Φ represents quantum yield, Abs represents absorbance, A represents area under the fluorescence spectra, and n is refractive index of the medium. The subscripts S and R denote the corresponding parameters for the sample and reference, respectively.

3. RESULTS AND DISCUSSION 3.1. Steady-State UV−Vis Absorption Studies. Figure 1 shows the changes in the UV−vis absorption spectra of curcumin in the aqueous 0.2 M NaCl solution with increasing concentration of bile salts, NaDC, NaCh, and NaTC. In aqueous solution, curcumin exhibits a broad absorption spectrum which consists of a band maximum at 430 nm with a shoulder at 355 nm in accordance with the earlier literature reports.18,22 The absorption band at 430 nm is assigned to the lowest (π,π*) transitions involved in the conjugated curcumin whereas the shoulder peak at 355 nm corresponds to the π−π* transitions in the feruloyl unit.18,22,61 This shoulder band appears only in the aqueous solution as a result of the strong interaction of curcumin with water. It can be seen from Figure 1 that with increasing concentration of bile salts in the aqueous 0.2 M NaCl solution of curcumin (∼10 μM) the absorption intensity increases, suggesting an increase in the solubilization of the drug inside the different hydrophobic binding sites of bile salt aggregates. Moreover, with increasing concentration of bile salts the shoulder peak disappears gradually with the appearance of an intense peak at 424 nm. This clearly indicates that the gradual incorporation of the probe molecules into the

(2)

(3)

where IVV and IVH are the emission intensities of the sample when excitation polarizer is oriented vertically and the emission polarizer is vertically and horizontally oriented, respectively. G is correction factor for the sensitivity of the detector to the polarization direction of the emission. The fluorescence quantum yields of curcumin in the aqueous 0.2 M NaCl solution with increasing concentration of bile salt were determined using coumarin 153 with absolute quantum yield 0.56 in acetonitrile at 25 °C as secondary standard. The following equation was used for calculation 13798



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Figure 3. Changes in the steady-state fluorescence spectra of curcumin with increasing concentration of (a) NaDC [fluorescence spectra (i)−(vii) correspond to concentration of NaDC = 0, 0.8, 2.2, 3.8, 4.3, 6.5, 10.1, 15 mM], (b) NaCh [fluorescence spectra (i)−(x) correspond to concentration of NaCh = 0, 1.4, 4.1, 5.9, 8.1, 10.1, 12.9, 16.6, 20.3, 25 mM], and (c) NaTC [fluorescence spectra (i)−(ix) correspond to concentration of NaTC = 0, 2.0, 3.5, 6, 8.5, 10.5, 13, 16.1, 19.4 mM]. (d) Overlay of the normalized emission spectra of curcumin in three different bile salts (30 mM).

NaCh and NaTC aggregates. Note that similar type of broad absorption spectrum of curcumin was also observed by Adhikary et al.21 in SDS micelles due to the greater extent of water penetration in the hydration layer where the probe molecules are basically confined. However, in CTAB and TX100 micelles the absorption spectra of curcumin showed the presence of vibronic structure with the shoulder bands suggesting a lesser extent of interaction of curcumin with water in these micelles than in SDS micelles. 3.2. Degradation Kinetics of Curcumin. Curcumin undergoes rapid degradation in the aqueous solution of neutral and alkaline pH.14,15 The degradation of curcumin with time can be effectively monitored by the decrease in intensity of the absorption spectra. Harada et al.20 have shown that in aqueous phosphate buffer solution (pH 7.4) the absorption intensity of curcumin decays to approximately 40% of the initial value within 30 min. Therefore, recording the time-dependent UV− vis absorption spectra of curcumin in the solution of different bile salt aggregates allows us a better understanding of the effectiveness of the systems in protecting the drug molecule from the interaction of water. This study will also allow us to further judge the suitability of the bile salt aggregates as a potential drug delivery media for curcumin. Figure 2 shows the time-dependent changes in the absorption spectra of curcumin in three different bile salt aggregates. As we can see, the encapsulation of curcumin into the hydrophobic sites of bile salt aggregates significantly reduces the level of degradation of curcumin. The insets of Figure 2 have been given for better understanding the level of degradation of curcumin with time. Interestingly, it has been found that among the three different

nanocavities of different binding sites (primary/secondary) of the bile salt aggregates resulting in a lower level of interaction of curcumin with water. The shoulder peak at 355 nm disappears completely above the cmc of the individual bile salts. A closer look at the UV−vis absorption spectra of curcumin in three different bile salts as depicted in Figure 1 indicates that a small shoulder band at 445 nm is more prominent in NaCh and NaTC aggregates, whereas in NaDC aggregates curcumin exhibits quite a broad absorption spectrum with less vibronic structure (Figure S1, Supporting Information). It has been earlier reported that intermolecular hydrogen-bonding interaction in the ground state causes broad absorption spectrum of curcumin.21 The broad absorption spectrum of curcumin in NaDC aggregates therefore indicates the presence of significant intermolecular hydrogen-bonding interaction between the bound probe molecules and the anionic carboxyl group of the NaDC bile salt. Although both NaDC and NaCh bile salts contain anionic carboxyl group, the intermolecular hydrogenbonding interaction is more prominent in the more rigid and confined microenvironment of NaDC aggregates than in NaCh aggregates.44,45 The interaction of anionic carboxyl group with curcumin can be further supported by the observation of Huppert and co-workers.59 Very recently, they demonstrated that addition of sodium acetate into the methanolic/ethanolic solution of curcumin leads to the rapid intermolecular proton transfer from the acidic group of curcumin to the acetate. The appearance of broad absorption spectrum of curcumin in NaDC aggregates is further confirmed by degradation kinetic studies (followed later section) of curcumin that show higher level of degradation when encapsulated into NaDC than in 13799



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Scheme 2). This results in a significant difference in the shape and intensity of the emission spectra of curcumin. It has been shown that the emission intensity of curcumin in methanol remarkably decreases upon gradual addition of sodium acetate as a result of the interaction of curcumin with acetate ion.59 They have concluded that acetate acts as mild base to abstract the acidic proton of curcumin and thus prohibit the ESIHT process with the opening of a new nonradiative deactivation pathway through solvent-mediated intermolecular hydrogen atom transfer mechanism. The red-shifted shoulder band in the emission spectra of curcumin in the presence of NaDC and NaCh aggregates (see Figure 3d) probably arises from the interaction of the acidic proton of curcumin with the carboxylate moiety of the bile salts (NaDC/NaCh) in the secondary binding sites. We have also determined the change in the fluorescence quantum yield of curcumin with increasing concentration of three different bile salts. As the concentration of bile salt increases, the fluorescence quantum yield of curcumin increases and finally reaches a limiting value at higher concentrations which is clearly observed from Figure 4. In each

bile salts, NaTC aggregates provide higher stability of curcumin than that provided by NaCh and NaDC aggregates. Since deprotonation is one of the main reasons for the degradation of curcumin and the presence of anionic carboxylate group causes rapid intermolecular proton transfer from curcumin to acetate,59 we may expect a lower extent of stabilization of curcumin in NaDC and NaCh aggregates than in NaTC. This observation is quite expected from the UV−vis absorption study where we observed broad absorption spectrum of NaDC aggregates-bound curcumin due to the higher level of interaction with the anionic headgroup of bile salt. From the insets of Figure 2, it is evident that in NaTC aggregates curcumin degrades less than 5% after 50 h whereas on this same time scale it degrades ∼10% in NaCh aggregates and ∼20% in NaDC aggregates. The suppression of degradation upon encapsulation into the confined microenvironments of micelles, mixed micelles, vesicles, proteins, and cyclodextrins was previously reported by several groups.18−20,24,32 However, such a high extent of stability of curcumin as that observed in NaTC aggregates has been reported in very few organized systems.25 In an earlier study, it has been shown that the halflife of curcumin is extended from less than 30 min to greater than 16 h in the presence of diamide-linked γ-cyclodextrin dimers.20 Very recently, we also showed that in the tween surfactants forming nonionic micelles curcumin undergoes 15− 20% degradation after 12 h.32 Therefore, comparing the earlier results, we can say that bile salt aggregates in particular NaTC aggregates are highly promising systems for curcumin delivery providing the effective stability of curcumin. 3.3. Steady-State Fluorescence Studies. The steadystate emission spectra of curcumin in the aqueous 0.2 M NaCl solutions with increasing concentration of different bile salts were recorded at the excitation wavelength of 408 nm and are depicted in Figure 3. From this figure we can see that the emission intensity of curcumin increases as the concentration of bile salt increases. However, among three different bile salts, the increase in intensity is found to be higher upon addition of NaTC than NaDC and NaCh addition. The increase in fluorescence intensity is also accompanied by the gradual blue shift in the emission maxima. The blue shift in the emission maxima clearly indicates the partitioning of the probe molecules into the different aggregates (primary/secondary) of bile salts from the bulk aqueous phase. Since NaDC is more hydrophobic compared to NaCh and NaTC, the micropolarity sensed by the probe molecules in NaDC aggregates is less compared to that in NaCh and NaTC aggregates and this has been supported by the observation of higher blue shift in the emission maximum of curcumin in NaDC aggregates than in NaCh and NaTC aggregates. The emission maximum of curcumin at 496 nm in NaDC aggregates is ∼5 nm blue-shifted from that in NaTC aggregates (∼501 nm). Apart from this, the spectral feature of curcumin in NaDC and NaCh aggregates is also found to be significantly different from that in NaTC aggregates. However, the spectral feature of curcumin is found to be more or less identical in both NaDC and NaCh aggregates.62 Therefore, the difference in the spectral appearance of curcumin in NaDC and NaCh aggregates from that in NaTC aggregates as evident from the normalized emission spectra of curcumin given in Figure 3d is due to the difference in the chemical structure of the hydrophilic headgroup region of bile salt. NaDC and NaCh bile salts contain carboxylate moiety in the hydrophilic surface, whereas NaTC contains sulfonate moiety in its hydrophilic surface (see

Figure 4. Variation of quantum yield of curcumin in aqueous 0.2 M NaCl solution with increasing concentration of three different bile salts NaDC (▲), NaCh (■), and NaTC (●) at 298 K.

case, we observed a point of inflection indicating the critical micelles concentrations of the corresponding bile salts. Since the formation of primary aggregates is mainly governed by the hydrophobic interactions, NaDC form aggregates at lower monomer concentration than NaCh and NaTC. This is evident from Figure 4 where it shows the appearance of an inflection point at lower concentration of bile salt monomer for NaDC and then followed in order by NaCh and NaTC. Curcumin in NaTC aggregates exhibits considerably higher fluorescence quantum yield than in NaDC and NaCh aggregates. As can be seen from Table 1, the quantum yield value of curcumin in NaTC aggregates is approximately twice the value observed in NaCh and NaDC aggregates at any given temperature. The higher fluorescence quantum yield of curcumin in the presence of NaTC aggregates can be explained on the basis of the reduced nonradiative decay rate which arises from the perturbation of the intramolecular hydrogen bond assisted ESIHT process by the interaction of curcumin with the negatively charged sulfonate moiety present in the headgroup of the NaTC bile salt. On the contrary, the interaction of curcumin with the carboxylate moiety of NaDC and NaCh bile salts results in the reduced quantum yield. Again, the fluorescence quantum yield of curcumin in NaCh aggregate is lower than in NaDC aggregate which is mainly due to the 13800



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Table 1. Fluorescence Lifetimes, Quantum Yields (Φf), and Radiative (kr) and Nonradiative (knr) Decay Rate Constants of Curcumin in NaDC, NaCh, and NaTC Aggregates at Different Temperatures Ranging from 283 to 333 K (λex = 408 nm, and λem = 496 nm) curcumin in

T (K)

τ1/ps (a1)

τ2/ps (a2)

⟨τ⟩ava/ps

χ2

ΦX

knr (109s−1)

kr (108 s−1)

NaDC (30 mM)

283 293 303 313 323 333 283 293 303 313 283 293 303 313 323 333

71 (0.94) 64 (0.95) 54 (0.96) 44 (0.97) 38 (0.98) 31 (0.98) 50 (0.98) 40 (0.99) 30 (0.99) 22 (0.99) 102(0.93) 87 (0.93) 71 (0.93) 62 (0.95) 53 (0.97) 50 (0.97)

351(0.06) 307 (0.05) 271 (0.04) 242 (0.03) 213 (0.02) 198 (0.02) 340 (0.02) 313 (0.01) 277 (0.01) 265 (0.01) 390 (0.07) 318 (0.07) 273 (0.07) 253 (0.05) 234 (0.03) 212(0.03)

88 76 63 50 42 34 56 43 32 24 122 103 85 72 58 55

1.23 1.13 1.17 1.10 0.92 0.98 1.16 1.22 1.01 1.25 1.17 0.96 1.03 0.93 1.01 0.98

0.010 0.009 0.008 0.007 0.006 0.005 0.009 0.008 0.007 0.006 0.021 0.018 0.016 0.014 0.012 0.010

11.25 13.04 15.75 19.86 23.67 29.26 17.69 23.07 31.03 41.45 8.02 9.53 11.58 13.69 17.03 17.90

1.14 1.18 1.27 1.40 1.43 1.47 1.61 1.86 2.19 2.19 1.72 1.75 1.88 1.94 2.07 1.82

NaCh (30 mM)

NaTC (30 mM)

a

Experimental errors ±5%.

the different aggregates (primary/secondary) of bile salts. Finally, at higher bile salt concentration it ensures saturation, indicating that the equilibrium of host−guest inclusion complexation is reached. In each case, a point of inflection is observed at the cmc value of the corresponding bile salt. Since, NaDC is more hydrophobic compared to NaCh and NaTC, the inflection point appears at lower concentration of NaDC and then followed in order by NaCh and NaTC. The steady-state anisotropy of curcumin in the presence of bile salt aggregates is increased to 0.33−0.35. This observation clearly indicates that the probe molecules strongly interact with bile salt aggregates upon incorporation into the different binding sites. Considering the errors in the measurements, it has been found that there is almost no difference in the anisotropy values of curcumin in the presence of the high concentration (30 mM) of three different bile salts. This indicates that the drug molecule is incorporated into the rigid microenvironments of all the three bile salt aggregates studied. Similar extent of increase in the steady-state fluorescence anisotropy value was also observed for the binding of curcumin with bovine serum albumin.63 To understand the effect of temperature, we also performed steady-state fluorescence anisotropy measurements of curcumin in the presence of bile salt aggregates (30 mM) with increasing temperature in the range of 283−333 K. However, we observed no significant change in the anisotropy values of the aggregatebound curcumin with the change in temperature in this range. This finding is well correlated with the earlier reports in the literature.64 The binding interaction and dynamics can be better understood from the time-resolved fluorescence anisotropy studies. However, unfortunately because of very short fluorescence lifetime we were unable to perform time-resolved rotational relaxation studies of curcumin in the present systems. 3.5. Temperature-Dependent Time-Resolved Fluorescence Studies. The fluorescence lifetime measurements provide important information regarding the modulation of the nonradiative decay rates of curcumin upon interactions with bile salts in the confined environment. The time-resolved fluorescence decays of curcumin in the aqueous solution of three different bile salts have been recorded at the excitation wavelength of 408 nm and monitored at the corresponding

increased hydrophilicity and water accessibility in the binding sites of NaCh aggregates. Therefore, we can infer that, although curcumin is encapsulated into the hydrophobic nanocompartment of NaDC and NaCh aggregates, a significant portion of curcumin still interacts with the hydrophilic surfaces of bile salts which results in an effective decrease in the fluorescence quantum yield. Further evidence for the encapsulation of curcumin into the aggregates can be obtained from the steadystate fluorescence anisotropy measurements. 3.4. Steady-State Fluorescence Anisotropy Studies. Steady-state fluorescence anisotropy measurements provide important information regarding the binding and location of the probe molecules in such aggregated systems. Moreover, this study helps us to draw a comparative study on the rigidity of the surrounding microenvironments provided by different bile salt aggregates. The variation of steady-state fluorescence anisotropy of curcumin in the aqueous 0.2 M NaCl solutions with increasing concentration of three different bile salts is shown in Figure 5. From this figure we can see that as the concentration of bile salt increases the anisotropy value gradually increases due to the encapsulation of curcumin into

Figure 5. Changes in the steady-state fluorescence anisotropy of curcumin in the aqueous 0.2 M NaCl solution with increasing concentration of different bile salts NaDC (■), NaCh (▲), and NaTC (◆) at 298 K. 13801



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Figure 6. Effect of temperature (283−333 K) on the steady-state fluorescence spectra and time-resolved fluorescence decays of curcumin in NaDC (a,c) and NaTC (b,d) aggregates.

Figure 7. (a) Effect of temperature on the quantum yield of curcumin in different bile salt aggregates and (b) Arrhenius plots of ln(knr) versus 1000/ T for curcumin in NaDC and NaTC aggregates.

emission maxima. The preceding discussions revealed that a slight difference in the chemical structure in the headgroup region of the host system (bile salt aggregates) results in a large difference in the absorption and fluorescence properties of curcumin. The active role of the bile salts in bringing such alteration can be better understood from the time-resolved fluorescence studies as the fluorescence lifetime is very sensitive to the excited-state interactions especially when there is an option of partitioning of the probe molecules in the confined environment from the bulk aqueous phase. Figure 6 represents the steady-state and time-resolved fluorescence emission decays of curcumin in the aqueous 0.2 M NaCl solutions of NaDC and NaTC aggregates at temperatures 283−333 K. The steady-state and time-resolved emission decays of curcumin in NaCh aggregates at different temperatures have been given in Figure S2 of the Supporting Information. Very recently, Erez et al.58

studied the nonradiative decay process of curcumin in ethanol and 1-propanol at temperatures ranging from 175 to 250 K. They have shown that at very low temperature, i.e., below 237 K, the fluorescence emission maxima and the spectral feature of curcumin are highly affected with the change in temperature. In this work, we have reported the temperature-dependent fluorescence properties of curcumin in the range of 283−333 K. At these temperatures, the fluorescence emission maxima of curcumin are found to be unaltered. At all temperatures the time-resolved emission decays were found to fit well with a biexponential function and the fitted decay parameters are given in Table 1. The overall results showed that, as the temperature increases, the fluorescence intensity sharply decreases with the effective decrease in the fluorescence lifetime of curcumin. The change in the fluorescence quantum yield of curcumin in NaDC and NaTC aggregates with 13802



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when the average fluorescence lifetime (⟨τ⟩f) and fluorescence quantum yield (Φf) values are known

increasing temperature has been shown in Figure 7a. It has been extensively reported that the aggregation behavior of bile salts in the aqueous solution is affected by the change in concentration, pH, temperature, and ionic strength of the medium.65−67 As reported earlier, the cmc value of bile salt decreases very slightly with increasing temperature in the range of 283−333 K.65,66 By monitoring the temperature-dependent fluorescence emission of curcumin in the presence of different concentrations of NaCh, Patra and co-workers62 observed a slight but continuous decrease in the cmc of NaCh bile salt with increasing temperature in range of 288−318 K. Several studies have also indicated that the average aggregation number of bile salt gradually decreases with an increase in temperature.67 Therefore, increasing temperature may lead to increased level of water accessibility to the aggregate-bound curcumin resulting in an effective decrease in their fluorescence efficiency. This is also supported by the observation of continuous decrease in the absorbance of the aggregates-bound curcumin with increasing temperature. However, the interesting observation of the present study is that at a particular temperature the fluorescence lifetime of curcumin in NaTC aggregates is significantly higher than in NaDC and NaCh aggregates. As one can see from Table 1, the time constants of curcumin in NaTC aggregates are 71 ps (94%) and 273 ps (6%) with an average lifetime of 85 ps at 303 K, whereas in NaDC aggregates the lifetime components at the same temperature are 54 ps (97%) and 271 ps (3%) with an average lifetime of 63 ps. This difference in the fluorescence lifetime of curcumin in different bile salt aggregates arises from the differential interactions of the different anionic head groups of studied bile salts. The anionic carboxyl group of NaDC and NaCh bile salts may result in an enhanced nonradiative deactivation of curcumin through intermolecular proton transfer and thereby the fluorescence lifetime decreases.59 On the other hand, the interaction of curcumin with the anionic headgroup of NaTC bile salt prohibited the ESIHT process of curcumin to enhance its fluorescence lifetime. Moreover, since the trihydroxy bile salt, NaCh, is more hydrophilic compared to the dihydroxy bile salt NaDC, the lifetime components of curcumin are further decreased in NaCh aggregates from that in NaDC aggregates. In NaCh aggregates the fluorescence lifetime of curcumin becomes too short especially at high temperature (≥323 K) that we were unable to measure using our TCSPC setup with limited time resolution. In a very recent study, Adhikary et al.21 have shown that the time scales of the ESIHT of curcumin in conventional surfactants forming micelles varies in the range of 50−80 ps. In view of the above points, we can infer that the fast decay component of curcumin in the aqueous solution of different bile salt aggregates is attributed to the ESIHT process and the time scale of this process is longer in NaTC aggregates than in NaDC and NaCh aggregates. The longer time scale of ESIHT process of curcumin in NaTC aggregates compared to that in NaDC and NaCh aggregates is attributed to the perturbation of the intramolecular hydrogen bond of curcumin by the interaction of curcumin with the anionic headgroup of NaTC. Therefore, a slight variation in the chemical structures of the bile salts largely affects the time scale of ESIHT process of curcumin due to the strong interaction of the two concerned parties. Now, to understand the modulation of nonradiative decay rate of curcumin in bile salt aggregates (NaDC/NaCh/NaTC) with increasing temperature, we have determined the radiative and nonradiative decay rates using the following equations

kr = k nr =

Φf τ f

(5)

1 − kr τ f

(6)

where kr and knr represent the radiative and nonradiative rate constants, respectively. As we can see from the results given in Table 1, the nonradiative decay rate constants increase with increasing temperature from 283 to 333 K. Moreover, the nonradiative decay rate of curcumin in NaDC and NaCh aggregates is higher than in NaTC aggregates. The lower degree of nonradiative deactivation process of curcumin in NaTC aggregates is certainly due to the prohibition of ESIHTmediated main nonradiative pathway by the interaction of curcumin with NaTC bile salt. We have also determined the activation energy for the nonradiative decay process of curcumin in NaDC and NaTC aggregates from the slope of the Arrhenius plot of ln(knr) versus 1/T as depicted in Figure 7b. The activation energy values thus obtained for the nonradiative decay process of curcumin in NaDC and NaTC aggregates are ∼15 and ∼13 kJ/mol, respectively. 3.6. Effect of Addition of Salt. The electrolytes present in biological systems significantly affect the self-assemblies of biomolecules. Therefore, so much attention has been paid to understand the effect of electrolytes on the behavior of drug molecules confined in the biomimicking model membrane systems such as micelles, vesicles, and bile salt aggregates. Moreover, the ESIPT reactions play an important role in chemistry and biology. Therefore, studying the effect of salt addition on the photophysical behavior of biologically active PT chromophores in the model membrane systems is of more importance from the biological point of view. The protontransfer reactions in the biologically resembled supramolecular assemblies are reported to be largely affected by the addition of salt.24,68 Bhattacharyya and co-workers68 have recently demonstrated the salt-induced retardation of PT reaction of HPTS in cholesterol/TX-100 niosomes. Very recently, we showed that the addition of salt in the aqueous solution of a surface-active ionic liquid (SAIL) forming micelles results in an increase in the fluorescence intensity and lifetime of curcumin due to the reduced interaction of water.24 The presence of salt also increases the stability of curcumin in micelles. In this work, we have shown the salt-induced modulation in the dynamics of ESIHT process of curcumin resulting from the changes in the properties of bile salt aggregates, including size, shape, and polarity of the confined microenvironment. The aggregation properties of bile salts are strongly dependent on the ionic strength of the medium.44,45,69 As reported earlier, the minimum concentration of bile salt required to start the aggregation process is markedly decreased in the presence of salt.44,45 Moreover, as the concentration of salt increases, the aggregation number of the bile salts gradually increases with an increased size of the aggregates. Using pyrene as a polarity probe Fuentelba et al.69 have shown that the binding site polarity of the aggregates decreases with the addition of salt as confirmed from the decrease in the II/IIII value of pyrene. Since the fluorescence properties of curcumin are highly sensitive to the polarity and hydrogen-bonding ability of the surrounding microenvironment, we observed remarkable changes in both the steady-state and time-resolved fluorescence decays of 13803



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Figure 8. Effect of increasing salt (NaCl) concentration (0−1.6 M) on the (a) UV−vis absorption, (b) steady-state fluorescence (λex = 408 nm), (c) fluorescence quantum yield, and (d) time-resolved fluorescence decays of curcumin in 30 mM NaTC aggregates (λex = 408 nm, and λem = 496 nm).

Table 2. Effect of Addition of Salt (NaCl) on the Fluorescence Lifetimes, Quantum Yields (Φf), and Radiative (kr) and Nonradiative Rate (knr) Constants of Curcumin in the Aqueous Solution of NaTC (30 mM) Aggregates at 298 K (λex = 408 nm, and λem = 496 nm)

a

system

NaCl (M)

curcumin in NaTC (30 mM) aggregates

0 0.6 0.8 1.0 1.2 1.6

τ1/ps (a1)

τ2/ps (a2)

70 77 82 85 93 98

280 298 298 311 333 335

(0.97) (0.92) (0.91) (0.91) (0.91) (0.90)

(0.03) (0.08) (0.09) (0.09) (0.09) (0.10)

⟨τ⟩ava/ps

χ2

ΦX

knr (109 s−1)

kr (108 s−1)

76 95 101 105 115 122

1.13 1.17 1.10 0.98 1.01 1.03

0.014 0.020 0.021 0.022 0.023 0.026

12.97 10.32 9.69 9.31 8.50 7.98

1.84 2.10 2.08 2.10 2.00 2.13

Experimental errors ±5%.

curcumin upon gradual addition of salt. Such changes in the fluorescence properties of curcumin basically originate from the salt-induced modulation of the nonradiative decay process, i.e., the ESIHT process of curcumin. Figure 8, a and b, represent the changes in the UV−vis absorption spectra and steady-state fluorescence spectra of curcumin in NaTC aggregates with the presence of different concentrations of NaCl. As shown in Figure 8b, the steady-state fluorescence intensity of curcumin in NaTC aggregates increases with an increase in concentration of NaCl. As a result the fluorescence quantum yield increases to saturation at higher concentration of NaCl which is shown in Figure 8c. The fluorescence lifetime of curcumin also increases significantly as the concentration of salt increases. Figure 8d represents the time-resolved fluorescence decays of curcumin in NaTC aggregates with an increasing concentration of NaCl and the fluorescence lifetime values obtained from a biexponential fitting of the decays have been given in Table 2. The saltinduced changes in the UV−vis absorption and fluorescence properties of curcumin in NaCh aggregates have been shown in Figure S3 of the Supporting Information. In the aqueous

solution of curcumin-loaded NaDC aggregates, on addition of more than 0.6 M of NaCl, the solution becomes gel and therefore we were unable to perform the salt-induced changes in the photophysical properties of curcumin in this system over a wide range of salt concentrations. However, up to 0.6 M concentration of NaCl, the changes in the fluorescence properties of curcumin in NaDC aggregates follow the same trend as it follows in NaCh and NaTC aggregates. The results given in Table 2 indicate the salt-induced retardation of the ESIHT process of curcumin. Addition of salt reduces the accessibility of water to the probe molecules confined in the nanocavities of bile slat aggregates which also helps to reduce the nonradiative decay rate of curcumin. This results in an increase in the fluorescence intensity of curcumin along with the increase in fluorescence lifetime values. Moreover, the addition of salt also enhances the interaction of curcumin with the negatively charged headgroup of NaTC to perturb the intramolecular hydrogen bond of curcumin and effectively reduce the ESIHT-mediated nonradiative deactivation process. 13804



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(6) Yang, F.; Lim, G. P.; Begum, A. N.; Ubeda, O. J.; Simmons, M. R.; Ambegaokar, S. S.; Chen, P. P.; Kayed, R.; Glabe, C. G.; Frautschy, S. A.; et al. Curcumin Inhibits Formation of Amyloid β Oligomers and Fibrils, Binds Plaques, and Reduces Amyloid in Vivo. J. Biol. Chem. 2005, 280, 5892−5901. (7) Egan, M. E.; Pearson, M.; Weiner, S. A.; Rajendran, V.; Rubin, D.; Gloeckner-Pagel, J.; Canny, S.; Du, K.; Lukacs, G. L.; Caplan, M. J. Curcumin, a Major Constituent of Turmeric, Corrects Cystic Fibrosis Defects. Science 2004, 304, 600−602. (8) Gopinath, D.; Ahmed, M. R.; Gomathi, K.; Chitra, K.; Sehgal, P. K.; Jayakumar, R. Dermal Wound Healing Processes with Curcumin Incorporated Collagen Films. Biomaterials 2004, 25, 1911−1917. (9) Sharma, R. A.; Gescher, A. J.; Steward, W. P. Curcumin: the Story so Far. Eur. J. Cancer 2005, 41, 1955−1968. (10) Hsu, C.-H.; Cheng, A.-L. Clinical Studies with Curcumin. Adv. Exp. Med. Biol. 2007, 595, 471−480. (11) Hatcher, H.; Planalp, R.; Cho, J.; Torti, F. M.; Torti, S. V. Curcumin: From Ancient Medicine to Current Clinical Trials. Cell. Mol. Life Sci. 2008, 65, 1631−1652. (12) Dhillon, N.; Aggarwal, B. B.; Newman, R. A.; Wolff, R. A.; Kunnumakkara, A. B.; Abbruzzese, J. L.; Ng, C. S.; Badmaev, V.; Kurzrock, R. Phase II Trial of Curcumin in Patients with Advanced Pancreatic Cancer. Clin. Cancer Res. 2008, 14, 4491−4499. (13) Anand, P.; Kunnumakkara, B.; Newman, R. A.; Aggarwal, B. B. Bioavailability of Curcumin: Problems and Promises. Mol. Pharm. 2007, 4, 807−818. (14) Wang, Y. J.; Pan, M. H.; Cheng, A. L.; Lin, L. I.; Ho, Y. S.; Hsieh, C. Y.; Lin, J. K. Stability of Curcumin in Buffer Solutions and Characterization of its Degradation Products. J. Pharm. Biomed. Anal. 1997, 15, 1867−1876. (15) Tønnesen, H. H.; Karlsen, J. Z. Kinetics of Curcumin Degradation in Aqueous Solution. Lebensm.-Unters. Forsch. 1985, 180, 402. (16) Jagannathan, R.; Abraham, P. M.; Poddar, P. TemperatureDependent Spectroscopic Evidences of Curcumin in Aqueous Medium: A Mechanistic Study of Its Solubility and Stability. J. Phys. Chem. B 2012, 116, 14533−14540. (17) Podaralla, S.; Averineni, R.; Mohammed, A.; Perumal, O. Synthesis of Novel Biodegradable Methoxy Poly(ethylene glycol)− Zein Micelles for Effective Delivery of Curcumin. Mol. Pharm. 2012, 9, 2778−2786. (18) Leung, M. H. M.; Kee, T. W. Effective Stabilization of Curcumin by Association to Plasma Proteins: Human Serum Albumin and Fibrinogen. Langmuir 2009, 25, 5773−5777. (19) Leung, M. H. M.; Colangelo, H.; Kee, T. W. Encapsulation of Curcumin in Cationic Micelles Suppresses Alkaline Hydrolysis. Langmuir 2008, 24, 5672−5675. (20) Harada, T.; Pham, D.-T.; Leung, M. H. M.; Ngo, H. T.; Lincoln, S. F.; Easton, C. J.; Kee, T. W. Cooperative Binding and Stabilization of the Medicinal Pigment Curcumin by Diamide Linked γ-Cyclodextrin Dimers: A Spectroscopic Characterization. J. Phys. Chem. B 2011, 115, 1268−1274. (21) Adhikary, R.; Carlson, P. J.; Kee, T. W.; Petrich, J. W. ExcitedState Intramolecular Hydrogen Atom Transfer of Curcumin in Surfactant Micelles. J. Phys. Chem. B 2010, 114, 2997−3004. (22) Ke, D.; Wang, X.; Yang, Q.; Niu, Y.; Chai, S.; Chen, Z.; An, X.; Shen, W. Spectrometric Study on the Interaction of Dodecyltrimethylammonium Bromide with Curcumin. Langmuir 2011, 27, 14112− 14117. (23) Niu, Y.; Wang, X.; Chai, S.; Chen, Z.; An, X.; Shen, W. Effects of Curcumin Concentration and Temperature on the Spectroscopic Properties of Liposomal Curcumin. J. Agric. Food Chem. 2012, 60, 1865−1870. (24) Ghatak, C.; Rao, V. G.; Mandal, S.; Ghosh, S.; Sarkar, N. An Understanding of the Modulation of Photophysical Properties of Curcumin inside a Micelle Formed by an Ionic Liquid: A New Possibility of Tunable Drug Delivery System. J. Phys. Chem. B 2012, 116, 3369−3379. Banerjee, C.; Ghatak, C.; Mandal, S.; Ghosh, S.; Kuchlyan, J.; Sarkar, N. Curcumin in Reverse Micelle: An Example to

4. CONCLUSION In summary, we have shown the impressive ability of bile salt aggregates particularly to suppress the degradation of curcumin with time. In particular, NaTC aggregates provide greater extent of stability of curcumin (degradation less than 5% of curcumin after 50 h) than NaCh and NaDC aggregates. The effectiveness of these systems in stabilizing curcumin clearly arises from the strong binding of the drug to the different binding sites of the bile salt aggregates as evidenced from the remarkable changes in its photophysical properties upon encapsulation. The ESIHT process of curcumin has been found to be significantly modulated upon encapsulation into the aggregates. It was observed that, upon encapsulation of curcumin into the different aggregates of bile salts, the fluorescence intensity of curcumin is increased along with the significant blue shift in the emission maxima because of the reduced level of interaction with water. Moreover, the time scale of ESIHT process of curcumin in NaTC aggregates is longer than that in comparatively more hydrophobic NaDC aggregates, which can be attributed to the perturbation of the intramolecular hydrogen bond of curcumin by the interaction with the negatively charged headgroup of NaTC bile salt. The ESIHT process of curcumin is further retarded upon addition of salt.

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ASSOCIATED CONTENT

S Supporting Information *

Information on the normalized absorption spectra of curcumin in NaDC and NaCh bile salts, temperature-dependent fluorescence properties of curcumin in NaCh aggregates, saltinduced changes in the UV−vis absorption and fluorescence properties of curcumin in NaCh aggregates, and complete author list for refs 6 and 31. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: 91-3222-255303. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS N.S. is thankful to Council of Scientific and Industrial Research (CSIR), Government of India, for generous research grants. S.M. and S.G. are thankful to CSIR for research fellowships. C.B. and J.K. are thankful to UGC for research fellowships.

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An Investigation into the Effect of the Structure of Bile Salt Aggregates

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