Interaction of the Nonsteroidal Anti-inflammatory Drug Indomethacin

Feb 10, 2015 - Department of Chemistry, Indian Institute of Technology Patna, Patna 800013, Bihar, India. J. Phys. Chem. B , 2015 ... Deciphering the ...
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Interaction of the Nonsteroidal Anti-inflammatory Drug Indomethacin with Micelles and Its Release Banibrata Maity, Aninda Chatterjee, Sayeed Ashique Ahmed, and Debabrata Seth* Department of Chemistry, Indian Institute of Technology Patna, Patna 800013, Bihar, India S Supporting Information *

ABSTRACT: In this study, we have reported the binding interaction and photophysics of a nonsteroidal anti-inflammatory drug (NSAID) indomethacin (IMC) in the presence of different micelles. We have used several spectroscopic techniques such as UV−vis absorption, steady state fluorescence and timeresolved fluorescence emission spectroscopy. The spectral properties of IMC were modulated in the presence of micelles compared to that in neat water. The weak emitting drug molecule (IMC) becomes highly fluorescent after binding with the micelles. The fluorescence quantum yield and fluorescence lifetime increase in the presence of micelles compared to those in neat water. The isothermal titration calorimetry (ITC) method was used to study the binding interaction of IMC with different micelles. The thermodynamic parameters and the nature of binding between IMC and different micelles have been estimated. Moreover, addition of KCl salt in the respective micelles releases IMC molecule from the micelles to the aqueous medium. This study will help elicidate the binding behavior of IMC in the presence of different micelles for possible use as potential drug delivery systems.

1. INTRODUCTION The interaction of organic fluorophores with different biomimic organized media by using different spectroscopic techniques is an important topic of interest.1−6 Surfactants have drawn attention due to manifold applications in pharmaceutics, drug delivery, emulsification, development for energy storage devices, nanometer sized electronic devices, membrane mimetic media, etc.6−10 A micelle is one of the typical membrane mimetic models.1 Research attention on the micelles has been enhanced in modern days due to their resemblances with proteins, enzymes, liposomes, bilayers, and biological membranes.1−10 Micelles are spherical or nearly spherical aggregates of the amphiphilic surfactant molecules in aqueous solution. Micelles are formed above a specific concentration of surfactants, which is termed as the critical micellar concentration (CMC). In the Stern-layer of micelles, the nonpolar hydrocarbon parts of the ionic surfactants are surrounded by hydrophilic polar head groups and counterions. The microenvironment of the organic fluorophore (micropolarity, microviscosity, etc.) inside the micellar system is considerably different compared to the aqueous medium. The nanoscopic size of the micelles has a capability to solubilize hydrophobic drug molecules and enhances its bioavailability and use as drugdelivery systems.11−13 Micellar solubilization of hydrophobic drug molecules in aqueous environments plays a vital role in both applied and fundamental sciences.11 Owing to their anisotropic water distribution inside the arranged structure, the concentration of water content decreases from the surface to the core of the micelles.11 The hydrophobic core of the micelles flock to the interior to diminish their water contact with tail © 2015 American Chemical Society

groups and is a completely water excluded region. On the other hand, the hydrophilic core of the micelles has a propensity to maximize the water contact with the head groups. The hydrophobic drug molecules get solubilized in the spatial regions of the micelles depending on the polarity of those regions.11 The nonpolar hydrophobic parts of the molecules get solubilized to the hydrophobic micellar core through hydrophobic interaction, whereas the substances with intermediate polarity reside along the surfactant molecules in certain intermediate regions. Therefore, in aqueous media to study the drug delivery and drug targeting application, it is an urgent need to solubilize the hydrophobic drug. Henceforth, micelles are utilized as drug carriers in aqueous medium and have paramount significance in pharmaceutical biotechnology.11,14 The drug molecules solubilized in micelles result in enhancement of the water solubility and bioavailability, render the molecule toxic, and have other adverse effects.11,14 The micelles forming surfactants used in this article have manifold applications in drug delivery and drug targeting systems, and commonly they have been used as drug carriers.11,14−19 Apart from the inherent resemblance with the biomimic models, micelles are optically transparent, scatter-free and spectroscopically silent. Therefore, contemporary research on the organic fluorophores in the presence of the micelles via spectroscopic techniques provides molecular level dynamics in modern days. Indomethacin (IMC) is one of the nonsteroidal antiinflammatory drugs (NSAIDs). IMC is commonly prescribed Received: January 16, 2015 Published: February 10, 2015 3776

DOI: 10.1021/acs.jpcb.5b00467 J. Phys. Chem. B 2015, 119, 3776−3785

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The Journal of Physical Chemistry B as a medicine to reduce pain, fever, swelling, stiffness, and tenderness. It also acts as prominent anti-inflammatory, analgesic and antipyretic agent. IMC has a significant contribution to reduce osteoarthritis, rheumatoid arthritis, ankylosing spondylitis, bursitis, tendinitis pain, migraine, dysmenorrhea, tumor growth, gout and collagen diseases.20,21 IMC also acts as an inhibitor in the production of prostaglandins, and it has a strong tocolytic property.22 It also acts as a strong inhibitor of the cyclooxygenase enzyme (COX-1 and COX-2). 23 This drug molecule also has paramount significance on gastrointestinal disorders, anticancer activity,24 and Alzheimer’s disease.25 Due to its low solubility and the high permeability, IMC is categorized in the Class II drug family according to the biopharmaceutical classification system (BCS).20,21 Most drug molecules are poorly soluble and lack bioavailability. In this regard, research has been focused to enhance the solubility and bioavailability of the hydrophobic IMC molecule, which can assist potential drug delivery systems. The studied surfactants are also used in drug delivery applications and safe drug carriers, reported in the literature.11,14−19 Over the past few years, the photophysics of IMC has witnessed promising prospects on enhancing its solubility and bioavailability in different environments.26−32 The kinetics of alkaline hydrolysis of IMC and related compounds were analyzed in the presence of anionic and cationic surfactants.33 The anionic micelles inhibit and the cationic micelles enhance the decomposition of the N-acylazoles moiety of IMC in alkaline media.33 In our earlier report, we had endeavored the supramolecular host−guest interaction between IMC with molecular containers (CDs) and enhanced the solubility and fluorescence intensity of IMC upon complex formation.32 The drug molecules in the presence of several biomimic media can enlarge its solubility in the aqueous phase, making it intravenously injectable in biological systems.34 In this work, we have studied the photophysics of a NSAID molecule (IMC) in different anionic and cationic micellar assemblies. The main aim of this study is to show the effect of the hydrophobic alkyl chain length of the surfactants on the binding interaction of IMC with micelles by using spectroscopic and calorimetric techniques. In this experiment, we have shown the release of a trapped IMC molecule from the micellar environment in the presence of KCl by using steady state fluorescence and time-resolved fluorescence emission spectroscopic techniques. These encouraging results may find some applications of targeted delivery of the potent drug molecule (IMC) in clinical pharmacology and make IMC injectable in physiological systems.

Scheme 1. Schematic Representation of IMC Molecule and Different Surfactantsa

a

“n” is the length the alkyl chain of the respective surfactants.

2.2. Instrumentation and methods. The steady state absorption spectra have been measured using an ultravioletvisible (UV−Vis) spectrophotometer (Model: UV-2550, Shimadzu). The steady-state fluorescence emission measurements were carried out using Fluoromax-4P spectrofluorometer (Horiba Jobin Yvon) respectively. For both absorption and emission spectral measurement, the path length of the used quartz cell is 1 cm. All the samples were excited at 295 nm to collect the fluorescence emission spectra. The fluorescence quantum yield of IMC was measured using quinine sulfate solution in 0.1(N) H2SO4 (ϕR = 0.546) as reference.35 We have calculated the fluorescence quantum yield (ϕS) value by using the following equation: ⎛A n2 ⎞ AbsR φS = φR ⎜ S × × S2 ⎟ AbsS nR ⎠ ⎝ AR

(1)

where, “ϕ” denotes the fluorescence quantum yield, “A” is the integrated area under the fluorescence curve, “Abs” is the absorbance of the respective solution at the excitation wavelengths, and “n” stands for the refractive index of the medium. The subscripts “S” and “R” stand for sample and reference, respectively. The fluorescence time-resolved emission decays were collected by using the picosecond time-correlated singlephoton counting (TCSPC) technique. We have used a timeresolved fluorescence spectrophotometer from Edinburgh Instruments (model: LifeSpec-II, U.K.). We have used a light emitting diode (LED) at an excitation wavelength of 295 nm. The instrument response function (IRF) of our setup using an LED is ∼800 ps. The fluorescence emission decays are fitted by multiexponential functions after deconvoluting IRF by using the following equation:

2. MATERIALS AND METHODS 2.1. Materials. The surfactants (Scheme 1) sodium octyl sulfate (SOS), sodium dodecyl sulfate (SDS), dodecyl trimethylammonium bromide (DTAB), myristyl trimethylammonium bromide (MTAB) and cetyltrimethylammonium bromide (CTAB), were procured from Sigma-Aldrich and used as received. Indomethacin (Scheme 1) was procured from Sigma−Aldrich and used as received without any further purification. The salt potassium chloride (KCl) was purchased from CDH, India. We have used Millipore water for preparation of all solutions. The concentration of IMC solution was maintained 35 μM in each of the experiments. To avoid any photochemical changes we have used fresh solution of IMC in all experiments.

N

I (t ) = A +

∑ Bi exp(t /τi) i=1

(2)

where Bi denotes the pre-exponential factors with the characteristic lifetimes τi and A is the background. We have used a MCP PMT as detector (Model no: 3809U, 3777

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The Journal of Physical Chemistry B Table 1. Photophysical Parameters, Partition Coefficient and Binding Constant of IMC in Different Micelles Medium neat water SOS SDS DTAB MTAB CTAB

CMC (in mM)

[Surfactant] 3 times of CMC 20 times of CMC

134 8.2 15.6 4.2 0.92

λemi max (nm) 380 364 364 360 360 365

ϕf

Partition coefficient (KM−W)

Binding Constant (K) (M−1)

250 1350 165 1180 15825

2.12 (±0.38) × 103 24.35 (±1.64) × 103 0.36 (±0.14) × 103 18.50 (±4.80) × 103 41.75(±4.75) × 103

−4

2 × 10 1.25 × 10−3 3.65 × 10−3 3.70 × 10−3 4.10 × 10−3 7.60 × 10−3

Figure 1. Plot of fluorescence intensity against [Micelle] in (a) anionic and (b) cationic surfactants.

Figure 2. Fluorescence emission spectral profiles of IMC (λexi = 295 nm) in neat water and in (a) SOS (anionic); (b) DTAB (cationic) surfactants and their change with addition of 20 mM KCl.

Table 2. Components of the Fluorescence Emission Decay of IMC in Different Micelles and due to Addition of KCl Solution (20 mM) and in Neat Water (λexi = 295 nm, λemi = 360 nm) Medium

τ1 (ns)

a1

τ2 (ns)

a2

τ3 (ns)

a3

⟨τf⟩ (ns)

χ2

neat water SOS SOS+KCl SDS SDS+KCl DTAB DTAB+KCl MTAB MTAB+KCl CTAB CTAB+KCl

1.05 1.07 0.62 0.55 0.46 0.60 0.46 0.63 0.48 0.86 0.54

0.76 0.45 0.52 0.40 0.50 0.50 0.60 0.52 0.60 0.48 0.52

6.07 4.70 4.32 4.55 3.98 3.90 2.50 4.52 4.15 5.78 3.66

0.24 0.45 0.44 0.50 0.44 0.35 0.38 0.34 0.30 0.37 0.36

23.26 17.17 26.50 24.20 22.86 15.70 25.60 22.75 26.83 24.55

0.10 0.04 0.10 0.06 0.15 0.02 0.14 0.10 0.15 0.12

4.92 2.90 5.15 3.44 5.10 1.54 5.45 3.80 6.58 4.55

1.22 1.11 1.04 1.13 0.95 1.04 1.18 1.15 0.92 1.06 1.11

Hamamatsu). The time-resolved emission decays were analyzed using F900 software. For all measurements temperature was

kept constant at 298 K. For time-resolved measurement, the temperature was controlled by using a Peltier-controlled 3778

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Figure 3. Time-resolved fluorescence emission decays (λexi = 295 nm) of IMC in (a) SOS (anionic) and (b) DTAB (cationic) surfactants, in the presence of 20 mM KCl and in neat water.

was observed, with a shoulder ∼270 nm as shown in Figure S1. In the case of cationic surfactants (DTAB, MTAB, and CTAB), the two distinct absorption peaks of IMC in aqueous medium are modified. The first absorption peak position remains unchanged, whereas the second peak modified to a shoulder at ∼270 nm (Figure S2). Incremental addition of the cationic surfactants to the aqueous solution of IMC causes enhancement of the absorbance values. The enhancement of the absorbance values of IMC in the presence of micelles is a clear manifestation that the environment around the drug molecule becomes modified due to the formation of the micelles.36,37 The absorption profile of IMC in different surfactants at same micellar concentration is shown in Figure S3. The enhancement of the absorbance of IMC in all micelles indicates the greater solubilization of the hydrophobic IMC molecule. In the presence of the micelles, the polarity of the medium becomes lower than that of the aqueous medium of IMC, which is responsible for the significant modification of the absorbance values.36,37 In the case of cationic surfactants, we have assumed that the electrostatic interaction between the positively charged micelle surface and the negatively charged carboxylate moiety of IMC molecule, in the Stern layer of the micelles, causes enhancement of the absorbance values. 3.2. Steady state fluorescence emission studies. In neat water IMC shows unstructured fluorescence emission maxima at 380 nm.32 In different micellar media the emission maxima of IMC is blue-shifted compared to that in neat water (Table 1). In all the micellar media the fluorescence intensity of IMC gradually increases with addition of surfactants. The blueshifted emission spectra of IMC in all the micellar environments may indicate that the microenvironments around the probe molecule are different compared to that in neat water and that the polarity of the micelles is less compared to neat water. The emission spectral features of IMC in anionic (SOS) and cationic (DTAB) micelles are shown in Figure S4. We have calculated the fluorescence quantum yield of IMC in different micellar media. In neat water the fluorescence quantum yield of IMC was 2 × 10−4.32 In all micellar media the fluorescence quantum yield of IMC increased compared to that in water. In both cationic and anionic micellar media, we have observed that, with gradual increase in chain length of the surfactant, the fluorescence quantum yield (ϕ) of IMC significantly increases (Table 1). The increase of ϕ in the case of CTAB surfactants is higher compared to other surfactants at the same micellar concentration, and it is ∼38 times higher compared to neat water. This

cuvette holder from Quantum Northwest (Model: TLC-50). For steady state measurement temperature was controlled by using a Jeiotech refrigerated bath circulator (Model: RW0525G). The isothermal titration calorimetry measurements were carried out by using iTC200 microcalorimeter from GE healthcare. Small angle X-ray scattering (SAXS) was performed using Rigaku X-ray diffractometer (model: TTRAX III) using a Cu Kα (λ = 1.54 Å). SAXS studies were carried out using 1.0 mm diameter glass capillary. Conductivity measurements were carried out using Systronics conductivity meter (model: 306).

3. RESULTS AND DISCUSSION 3.1. Steady state absorption studies. IMC showed two distinct absorption maxima at 320 and 267 nm respectively, in Table 3. Thermodynamic Parameters of the Binding Interaction of IMC with Different Micelles at 298 K Obtained from Isothermal Titration Calorimetry (ITC) Measurement Medium SOS (n = 8, anionic) SDS (n = 12 , anionic) DTAB (n = 12, cationic) MTAB (n = 14, cationic) CTAB (n = 16, cationic)

ΔH (kcal mol−1)

ΔS (cal mol−1 K−1)

Binding Constant (K) (M−1)

ΔG298 K (kcal mol−1)

210 (±40)

710

13 (±1.5)

−1.58

226 (±40)

765

35 (±5.2)

−1.97

292 (±60)

985

10 (±2.0)

−1.53

106 (±20)

375

2.74 (±0.8) × 103

−5.75

67 (±10)

245

22.4 (±0.6) × 103

−6.01

neat water. The two characteristic absorption maxima of IMC in neat water are reported in the literature.30−32 The pKa value of IMC is 4.5, and it is stable in neutral or slightly acidic medium. We have measured the pH of an aqueous solution of IMC, and it is found to be 5.1 and the pH of different micellar media in the presence of IMC are in the range of 6−7. Addition of anionic surfactants (SOS and SDS) in the aqueous solution of IMC enhances the absorbance of the two absorption bands of IMC, followed by significant modification of the absorption spectra of IMC compared to that in aqueous medium. With the addition of anionic surfactants one absorption peak at 320 nm 3779

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Figure 4. Isothermal titration calorimetric (ITC) profiles of (a) SOS (3 times of CMC), (b) SDS (20 times of CMC), (c) DTAB (20 times of CMC), (d) MTAB (20 times of CMC), and (e) CTAB (20 times of CMC) surfactants with 0.035 mM IMC at 298 K. Each peak in the upper panel indicates each single injection, and each lower panel shows an integrated heat profile against a molar ratio. The solid line represents the line of best fit.

remarkable modification of the ϕ value of IMC in micellar system may be used as a beneficial tool for detection analysis and sensors in pharmaceutical applications. 3.3. Determination of IMC−micelles binding constant and partition coefficient. From the variation of the fluorescence emission intensity of IMC in different micelles we have found out the binding constant with different micelles. The binding constant (K) values have been determined using the 1:1 nonlinear least-squares regression analysis method:38 F=

Fwater + FmicelleK1[Micelle] 1 + K1[Micelle]

where “S” represents the concentration of respective surfactant, CMC is the critical micellar concentration, and “N” stands for the aggregation number of the micellar system. The values of CMC and “N” are taken from literature reports.3,39,40 Bhatia and co-workers reported by using small-angle neutron scattering (SANS) experiments that the aggregation number of Pluronic F127 micelles significantly decreases in the presence of various hydrophobic drug molecules, thereby increasing the number density of the micelles. They also observed that in the presence of the drug molecules very small change takes place in the critical micelle concentration (CMC) of the F127 surfactant.41,42 The presence of hydrophobic drug molecules results increases the size of the micelles cores and coronas.41,42 We have calculated from their work that, per F127 micelle, the number of IMC molecules was ∼41 (when the concentration of F127 is 6.22 mM). So, a large number of drug molecules per micelle changes the aggregation number of the micelles after addition of IMC molecule. Alexander et al. also observed that, upon addition of the hydrophobic drug flurbiprofen to the pluoronic triblock copolymers, the aggregation number and the

(3)

where Fwater and Fmicelle are the fluorescence intensities of IMC in water and in micelles, when complete binding of dye with the micelle has occurred. K1 is the binding constant. The micellar concentration is determined by [Micelle] =

(S − CMC) N

(4) 3780

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The Journal of Physical Chemistry B CMC value significantly change.43−45 This is in sharp contrast to our experiment. According to their experiments,43−45 the concentration of pluoronic micelles was kept constant and the concentration of drug molecule was varied such that the population of the drug molecule becomes high compared to respective micelles, whereas in our case the situation is just the opposite. In our case, the concentration of micelles is several times higher than drug (IMC). The water solubility of IMC in neat water is very low and is reported as 14 μg/mL (39 μM).46 Therefore, we have maintained the concentration of IMC solution in neat water as 35 μM. The motivation of our study is to endeavor the photophysical properties of IMC in micelles. Therefore, we have taken a fixed concentration of IMC molecule, varying the concentration of the micelles. By using conductometric titration and small-angle X-ray scattering (SAXS) experiments, we have observed that there is no significant change of the CMC value as well as the aggregation number (N) of the respective micelles in the presence of the 35 μM of IMC molecule (shown in Figures S5 and S6). Therefore, from these experiments, it may be confirmed that the presence of a small concentration of IMC molecule cannot change the CMC value and, hence, the aggregation number (N) of the respective micelles. In our system, the ratios of micelles:IMC are as follows: [SDS-Micelles]:[IMC] = 45:1; [SOS-Micelles]: [IMC] = 177:1; [DTAB-Micelles]:[IMC] = 142:1; [MTABMicelles]:[IMC] = 43:1; [CTAB-Micelles]:[IMC] = 6:1 Therefore; in our case we are assuming no change of the aggregation number of a mixed system containing IMC. Figure 1 shows the plot of variation of fluorescence intensity against [Micelle]. From this plot we have estimated the binding constant of IMC with different micelles (Figure 1, Table 1). From Table 1 we have observed that with increase of the hydrophobic alkyl chain length of the surfactants (in the case of both anionic and cationic surfactants), the binding constant value is significantly enhanced. Among all the surfactants, the IMC molecule is strongly bound with the CTAB micellar phase. A similar result was found in our earlier report in the case of cationic micelles.47,48 The pH of the aqueous solution of IMC is 5.1, signifying that the molecule remains in anionic form. In the case of cationic micelles, the degree of penetration of the drug molecule is very low compared to the anionic micelles. In addition, the water content is very much lower in the case of the cationic micellar periphery compared to anionic micelles.49 With increase of the hydrophobic alkyl chain length of the surfactants, the compactness of the micellar headgroup enhances, causing low penetration of water molecule.50 The greater compactness of the micelles in CTAB causes greater electrostatic interaction and strong binding with anionic IMC molecule. In the case of cationic micelles, with the gradual increase in chain length of surfactants, the micelles become more and more compact and at the same time the hydration of micelle decreases. This causes the greater positive charge felt by the anionic IMC molecule (since the pKa value of IMC is 4.5). Moreover, as the hydrophobic character of the micelle increases, the hydrophobic part of the IMC molecule which is immersed in the nonpolar oil like core and experiences greater hydrophobic interaction. Therefore, the dye molecule experiences both the cumulative effect of hydrophobic interaction together with the electrostatic interaction and enhancing the interaction between the dye and the cationic

micelles. Similar results have also been observed in our earlier reports.47,48 In the case of anionic micelles, direct interaction between the dye and the negatively charged stern layer is quite improbable to happen by means of electrostatic interaction. Again, it is also noteworthy that with the increase in micellar size the binding interaction is increasing, like our previous result.48 Hence, it is quite probable that the interaction which is stabilizing the micelle−dye complex is mainly a hydrophobic interaction. As the dye is negatively charged, so it is impossible that the negatively charged COO− moiety will be pointing toward the hydrophobic core. So, we can expect that the hydrophobic part of the dye molecule will remain in the core of the micelle with its charged COO− group pointing outward to be stabilized by the positively charged counterions by means of electrostatic interaction. So, as the hydrophobic character of the micelle is increased, the dye−micelle complex will be more and more stable to be visualized by its binding affinity. The drug molecule (IMC) is poorly soluble in aqueous medium, and its solubility is enhanced in the presence of micelles, causing modification of the absorption and fluorescence emission spectra and their binding interaction. The partition coefficient of IMC with micellar phase from the aqueous medium is very high, resulting in IMC molecule completely residing in the micellar phase. We have calculated the micelle−water partition coefficient (KM−W) of IMC as follows: KM − W =

[IMC ]micelle [IMC ]water

(4a)

where [IMC]micelle and [IMC]water are the concentration of IMC in micelle and water, respectively. From fluorescence intensity data we have quantitatively estimated the penetration of IMC molecule into the micellar system and the partition coefficient value by using the following equation: F=

Fwater + FmicelleKM − W γMicelle[Micelle] 1 + γmicelle[Micelle]

(5)

γMicelle is the molar volume of the respective micelles, and [Micelle] is the micellar concentration. From the plot of fluorescence intensity (F) with [Micelle] we have determined the partition coefficient (KM−W) values of IMC in different micelles. The values are tabulated in Table 1 and are quite high. This supports favorable interaction between IMC and the respective micelles. In all the studied micelles, we have found that >99.3% drug molecule is strongly bound with the micellar phase leaving a small portion of IMC in free form in aqueous medium. The partition coefficient (KM−W) value significantly enhances with increase of the hydrophobic alkyl chain length of the surfactants. This demonstrates strong drug−micelle interaction, and it is higher in the case of CTAB micelles. 3.4. Effect of addition of 20 mM KCl salt. In this section, we have shown the release IMC molecule from strongly bound micellar environments. The addition of salt in the respective micelles reduces their CMC values.51 Addition of salt to the micellar solution dehydrates the surfactant head groups, causing destruction of the hydration layer of the surfactants.52−54 As a result, the electrostatic repulsion decreases and counterion binding increases in the presence of salt. Therefore, the surfactant molecules are more closely packed by themselves and increase their aggregation number.52−54 Lianos and Zana55 reported that increase of the concentration of NaCl salt 3781

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micelles, the fluorescence emission decays of IMC are fitted by three exponential functions, comprising a small relative population of long components (τ3 > 20 ns). We have also observed that, with a gradual increase of the hydrophobic alkyl chain length of the surfactants, the average lifetime values of IMC are gradually enhanced. The appearance of a long component in the presence of the surfactants may show that the drug−micelle interaction takes place. Therefore, from this observation we can conclude that the emission behavior of an IMC molecule in the presence of micelles is different compared to water, and IMC becomes highly fluorescent. The excited state fluorescence emission behavior of IMC bound to micelles is modulated due to addition of 20 mM KCl solution as additive. Addition of salt solution in the studied system causes remarkable modification of the lifetime components and their relative population. The average lifetime value quenches in the presence of KCl solution. In all the studied micellar systems, we have noticed from Table 2 that addition of 20 mM KCl solution changes the lifetime components and their relative population, causing quenching of the average lifetime values of IMC in the respective micelles. The fast component value decreases (τ1), and its relative population (a1) increases in the presence of the additive, signifying that the drug molecule (IMC) bound with the respective micelles has propensity to come out to the aqueous medium. The decrease of the slow component (τ2) and the long component (τ3) and their relative population also clearly suggests the migration of IMC from the micellar interface to the aqueous medium. The decrease of the average lifetime value is more in anionic SOS micelles and cationic DTAB micelles (Table 2). The variation of the emission decay of IMC is shown in Figure 3. These interesting results give evidence that the release of drug molecule (IMC) from the loosely bound micellar interface is more compared to the more tightly bound micelles. To further confirm the effect of KCl salt on the micelles bound IMC molecule, we have studied the fluorescence emission and time-resolved emission decay of IMC in neat water in the presence of 20 mM KCl. We have found that after addition of 20 mM KCl, there is no significant change of the intensity as well as emission decay of IMC in neat water (Figure S9). These results clearly confirmed that due to the addition of 20 mM KCl solution, IMC molecules migrate from the micellar interface to the bulk water. 3.6. Isothermal Titration Calorimetric Measurements. The isothermal titration calorimetric (ITC) method was used to obtain the drug−micelles binding interaction. The thermodynamic parameters and the nature of binding between IMC and the cationic and anionic micelles have been determined from ITC measurements. The data obtained from the ITC measurements are best fitted by using a one site binding model for the titration of respective surfactants by IMC (1:1 nature). The binding constant values of IMC in different micelles media are tabulated in Table 3, obtained from ITC measurements. The peak in the upper panel of Figure 4 shows that the entire reaction is a highly endothermic process (ΔH > 0). The upper panel of Figure 4 represents a single injection of drug molecule (IMC) into the respective surfactant solutions. The lower panel of Figure 4 shows the heat changes as a function of the molar ratio of the IMC/surfactant. The binding between the drug molecule and the respective surfactants is a spontaneous and entropy driven process (ΔS > 0, ΔG < 0). The highly endothermic process is governed by the increase of entropy in the respective systems, and as a result

increases the aggregation number and caused conformational/ structural change of the micelles. In our case, with gradual increment of KCl salt solution up to 20 mM concentration, the fluorescence intensity of IMC in respective micelles decreases. This interesting result may be due to the release of IMC molecule from the strongly bound micellar interface to aqueous medium. We have corrected the fluorescence intensities of IMC for the slight dilution effect of adding KCl solution in the respective micelles using the following equation: Icorr = Iexp ×

Vtotal VF

(6)

where Icorr and Iexp denote the corrected and the experimental emission intensities, respectively. Vtotal and VF represent the volume of total solution after addition of 20 mM KCl and micelles bound IMC solution, respectively. From Table 1 we have observed that the binding affinity of IMC is very strong in CTAB micelles and very weak in DTAB micelles. The decrease of emission intensity is more in the case of DTAB micelles and is less in the case of CTAB micelles. Figure 2 displays the change of fluorescence emission profiles of IMC in anionic (SOS) and cationic (DTAB) micelles upon addition of 20 mM KCl solution. Therefore, the percentage (%) of IMC molecule released from micelles is calculated as follows: (%) of IMC molecule releases ⎛F − Fsalt ⎞ ⎛ Vtotal ⎞ = ⎜ micelle ⎟×⎜ ⎟ × 100 ⎝ Fmicelle ⎠ ⎝ Vmicelle ⎠

where Vtotal and Vmicelle denote the volume of total solution after addition of 20 mM KCl and micelles bound IMC solution, respectively. Fsalt is the fluorescence intensity of IMC after addition of 20 mM KCl salt to the micellar solution. Fsalt is the fluorescence intensity of IMC in the respective micelles. Here, we have found that the release of IMC molecule from the loosely bound micelles is maximum compared to strongly bound micelles. In SOS and SDS micelles the release of IMC molecule are 48% and 26%, respectively. In DTAB, MTAB and CTAB micelles, the release of IMC molecule are 58%, 26% and 16%, respectively (shown in Figure S7). From the binding constant value of the drug−micelles interaction study, we may interpret that the loosely bound IMC molecule is more easily released from the trapped micellar environments to the aqueous medium. The release rate of IMC molecule decreases with increasing the hydrophobic alkyl chain length of the surfactants. The drug−micelles binding interaction decreases in an opposite manner. 3.5. Time resolved fluorescence emission study. The fluorescence lifetime of the probe molecule is a sensitive indicator for analyzing the local environment around the fluorophore, and it gives an idea about the excited state behavior of the fluorophore. To elucidate the microenvironment around the IMC molecule, we have studied the timeresolved fluorescence emission behavior of IMC in different micellar media. The fluorescence emission decay profiles of the IMC molecule in different media are collected by exciting the molecule at 295 nm and collecting emission at respective emission wavelengths (Figure S8). In neat water, the fluorescence emission decay of IMC was fitted by a biexponential function, consisting of fast (τ1) and slow (τ2) components. The fluorescence lifetime components of IMC in water and micelles are tabulated in Table 2. In the presence of 3782

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The Journal of Physical Chemistry B

from the micellar interface due to addition of IMC. The binding capability of IMC and the changes of free energy gradually enhance with increase of the hydrophobic tail group of the surfactants. A similar type of results was obtained from the fluorescence measurements. We have also shown the release of an IMC molecule from the micellar environments in the presence of 20 mM KCl solution as additive by using fluorescence emission spectroscopy. The release of drug molecule (IMC) is high in the case of weakly bound micelles. The photophysics and the calorimetric study on the drug molecule entrapped in the biomimic micellar system and its release would be of great interest for application of the IMC molecule in the fields of potential targeted drug design and its delivery, clinical pharmacology, and physiological systems.

reaction becomes spontaneous (ΔG < 0). In all the cases, we have found |ΔH| < |TΔS|. This suggests the reaction is entropically favorable and not enthalpically. The high positive value of the entropic contribution in all the micelles suggests that binding of IMC with the micellar surfaces involves disruption of a hydrogen bonded water molecule from the micellar surface causing increase of entropy and making the thermodynamic system energetically favorable (ΔG < 0).56−59 All the thermodynamics parameters of IMC in different surfactants are tabulated in Table 3. From the ITC measurements, we have found that with gradual increase of the alkyl chain length of the cationic and anionic surfactants, the binding constant (K) and the change in free energy (ΔG298K) gradually enhance. A similar type of result was also obtained from the fluorescence measurements. 3.7. Difference between ground state and excited state binding. The ground state binding constant values of IMC in different micelles obtained from ITC measurements are significantly lower compared to the excited state binding obtained from fluorescence measurements. This may account for the difference between the ground state and the excited state binding interactions. The drug molecule (IMC) noncovalently interacts and binds with the micellar environments. The rate of binding interaction (kin) of the IMC molecule (guest) into the micellar environments (host) must be significantly higher as compared to the rate of exclusion (kex) of the IMC from the micellar surface.60−62 The binding constant value is represented as K = kin/kex = τes/τin. The parameters τin and τex denote the time scale for inclusion and exclusion of guest molecule in the hosts.60 Since the excited state lifetime value is high, the guest molecule (IMC) is excited and entrapped in the micelles during the excited state lifetime. The excited state lifetime value enhances with increase of the hydrophobic alkyl chain length of the surfactants. The exclusion of IMC from micelles to water is extremely slow. Hence, the binding constant determined from fluorescence measurement is higher than from ITC measurement. For this reason, we have found a significant difference between the ground state and the excited state binding.

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ASSOCIATED CONTENT

S Supporting Information *

The absorption and fluorescence emission spectral features of IMC in different micellar media; the release profile of IMC molecule from different micelles with addition of 20 mM KCl; time-resolved fluorescence emission decays in different media and the fluorescence intensity and the time-resolved fluorescence emission decays of IMC in the presence of 20 mM KCl. Conductometric titration plots and small-angle X-ray scattering (SAXS) plots are shown for different micelles in the presence and absence of IMC. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: 91-612-2277383. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS All the authors are thankful to the Indian Institute of Technology Patna, India, for the research facilities. B.M. and S.A.A. are thankful to IIT Patna for research fellowships. A.C. is thankful to CSIR, New Delhi, for research fellowships.

4. CONCLUSIONS In this study, the interaction of a drug molecule (IMC) with anionic and cationic micellar environments has been reported, by using UV−vis absorption, steady-state and time-resolved fluorescence emission spectroscopy, and isothermal titration calorimetric methods. The ground state and excited state properties of IMC are modulated in the presence of micellar environments compared to those in water. The weak emitting IMC becomes highly fluorescent in micellar media. The fluorescence emission decay of IMC in an aqueous medium shows two lifetime components. In the micellar media, with gradual increase of the alkyl chain length of the surfactants, the average lifetime value becomes significantly enhanced. In the micellar environments, the fluorescence emission decay of IMC shows three components. The appearance of a long component in the emission decay (τ3 > 20 ns) of IMC showed that the drug−micelle interaction takes place. The isothermal titration calorimetry (ITC) method was used to study the binding interaction of IMC with different micellar environments. The binding between IMC and the respective surfactants is a spontaneous, highly endothermic, and entropy driven process. The positive value of change of entropy in the respective micellar media suggests that the water molecules are expelled

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REFERENCES

(1) Fendler, J. H. Membrane Mimetic Chemistry; Wiley-Interscience: New York, 1982. (2) Ramamurthy, V., Ed. Photochemistry in Organized and Constrained Media; VCH: New York, 1991. (3) Kalyanasundram, K. Photochemistry in Microheterogenous Systems; Academic Press: New York, 1987. (4) Kalyanasundram, K. Photophysics of Molecules in MicelleForming Surfactant Solutions. Chem. Soc. Rev. 1978, 7, 453−472. (5) Moulik, S. P. Micelles: Self-organised Surfactant Assemblies. Curr. Sci. 1996, 71, 368−376. (6) Tanford, C. The Hydrophobic Effect and the Organization of Living Matter. Science 1978, 200, 1012−1018. (7) Onyuksel, H. A.; Ramkrishnan, S.; Chai, H. B.; Pezzuto, J. M. A Mixed Micellar Formulation Suitable for the Parenteral Administration of Taxol. Pharm. Res. 1994, 11, 206−212. (8) Nasongkla, N.; Bey, E.; Ren, J.; Ai, H.; Khemtong, C.; Guthi, J. S.; Chin, S. F.; Sherry, A. D.; Boothman, D. A.; Gao, J. Multifunctional Polymeric Micelles as Cancer-Targeted, MRI-Ultrasensitive Drug Delivery Systems. Nano Lett. 2006, 6, 2427−2430. (9) Friberg, S. E., Lindman, B., Eds. Organized Solutions, Surfactants in Science and Technology; Dekker: New York, 1992. 3783

DOI: 10.1021/acs.jpcb.5b00467 J. Phys. Chem. B 2015, 119, 3776−3785

Article

The Journal of Physical Chemistry B (10) Kataoka, K.; Harada, A.; Nagasaki, Y. Block Copolymer Micelles for Drug Delivery: Design, Characterization and Biological Significance. Adv. Drug Delivery Rev. 2001, 47, 113−131. (11) Rangel-Yagui, C. O.; Pessoa, A., Jr.; Tavares, L. C. Micellar Solubilization of Drugs. J. Pharm. Pharm. Sci. 2005, 8, 147−163. (12) Kwon, G. S.; Okano, T. Polymeric Micelles as New Drug Carriers. Adv. Drug Delivery Rev. 1996, 21, 107−116. (13) Gu, F.; Zhang, L.; Teply, B. A.; Mann, N.; Wang, A.; RadovicMoreno, A. F.; Langer, R.; Farokhzad, O. C. Precise Engineering of Targeted Nanoparticles by using Self-assembled Biointegrated Block Copolymers. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 2586−2591. (14) Torchilin, V. P. Micellar Nanocarriers: Pharmaceutical Perspectives. Pharm. Res. 2007, 24, 1−16. (15) Caetano, W.; Gelamo, E. L.; Tabak, M.; Itri, R. Chlorpromazine and Sodium Dodecyl Sulphate Mixed Micelles Investigated By Small Angle X-Ray Scattering. J. Colloid Interface Sci. 2002, 248, 149−157. (16) Gao, Z.; Lukyanov, A. N.; Chakilam, A. R.; Torchilin, V. P. PEG-PE/Phosphatidylcholine Mixed Immunomicelles Specifically Deliver Encapsulated Taxol to Tumor Cells of Different Origin and Promote their Efficient Killing. J. Drug Targeting 2003, 11, 87−92. (17) Hirlekar, R.; Jain, S.; Patel, M.; Garse, H.; Kadam, V. Hexosomes: A Novel Drug Delivery System. Curr. Drug Delivery 2010, 7, 28−35. (18) He, Q.; Gao, Y.; Zhang, L.; Zhang, Z.; Gao, F.; Ji, X.; Li, Y.; Shi, J. A pH-Responsive Mesoporous Silica Nanoparticles-Based MultiDrug Delivery System for Overcoming Multi-Drug Resistance. Biomaterials 2011, 32, 7711−7720. (19) He, Q.; Shi, J.; Chen, F.; Zhu, M.; Zhang, L. An Anticancer Drug Delivery System Based on Surfactant-Templated Mesoporous Silica Nanoparticles. Biomaterials 2010, 31, 3335−3346. (20) Shen, T. Y.; Winter, C. A. Chemical and Biological Studies on Indomethacin, Sulindac and their Analogs. Adv. Drug Res. 1977, 12, 90−245. (21) Hardman, J. G., Limbird, L. E., Eds. In Goodman and Gilman’s the Pharmacological Basis of Therapeutics, 10th ed.; McGraw-Hill: New York, 2001. (22) Macones, G. A.; Marder, S. J.; Clothier, B.; Stamilio, D. M. The Controversy Surrounding Indomethacin for Tocolysis. Am. J. Obstet. Gynecol. 2001, 184, 264−272. (23) Blobaum, A. L.; Uddin, M. J.; Felts, A. S.; Crews, B. C.; Rouzer, C. A.; Marnett, L. J. The 2′-Trifluoromethyl Analogue of Indomethacin is a Potent and Selective COX-2 Inhibitor. ACS Med. Chem. Lett. 2013, 4, 486−490. (24) Lundholm, K.; Gelin, J.; Hyltander, A.; Lonnroth, C.; Sandstrom, R.; Svaninger, G.; Korner, U.; Gulich, M.; Karrefors, I.; Norli, B.; Hafstrom, L. O.; Kewenter, J.; Olbe, L.; Lundell, L. Antiinflammatory Treatment May Prolong Survival in Undernourished Patients with Metastatic Solid Tumors. Cancer Res. 1994, 54, 5602− 5606. (25) Hoozemans, J. J. M.; Veerhuis, R.; Janssen, I.; Elk, E. V.; Rozemuller, A. J. M.; Eikelenboom, P. The role of Cyclo-oxygenase 1 and 2 Activity in Prostaglandin E2 Secretion by Cultured Human Adult Microglia: Implications for Alzheimer’s Disease. Brain Res. 2002, 951, 218−226. (26) Moore, D. E.; Chappuis, P. P. A Comparative Study of the Photochemistry of the Non-steroidal Anti-inflammatory Drugs, Naproxen, Benoxaprofen and Indomethacin. Photochem. Photobiol. 1988, 47, 173−180. (27) Weedon, A. C.; Wong, D. F. The Photochemistry of Indomethacin. J. Photochem. Photobiol., A 1991, 61, 27−33. (28) Fronza, G.; Mele, A.; Redenti, E.; Ventura, P. 1H NMR and Molecular Modeling Study on the Inclusion Complex β-CyclodextrinIndomethacin. J. Org. Chem. 1996, 61, 909−914. (29) Bogdan, M.; Pirnau, A.; Floare, C.; Bugeac, C. Binding Interaction of Indomethacin with Human Serum Albumin. J. Pharm. Biomed. Anal. 2008, 47, 981−984. (30) Ghatak, A.; Mandal, P. C.; Sarkar, M. Indomethacin: A NSAID Sensitive to Micro Heterogeneity in Alcohol-water Mixtures. Chem. Phys. Lett. 2008, 460, 521−524.

(31) Chakraborty, S.; Sehanobish, E.; Sarkar, M. A Traditional Painkiller as a Probe for Microheterogeneity in 1-Propanol-water Mixtures. Chem. Phys. Lett. 2010, 501, 118−122. (32) Maity, B.; Chatterjee, A.; Ahmed, S. A.; Seth, D. Supramolecular Interactions of Nonsteroidal Anti-inflammatory Drug in Nanochannels of Molecular Containers: A Spectroscopic, Thermogravimetric and Microscopic Investigation. ChemPhysChem 2014, 15, 3502−3514. (33) Cipiciani, A.; Ebert, C.; Germani, R.; Linda, P.; Lovrecich, M.; Rubessa, F.; Savelli, G. Micellar Effects on the Basic Hydrolysis of Indomethacin and Related Compounds. J. Pharm. Sci. 1985, 74, 1184− 1187. (34) Shieh, Y. A.; Yang, S. J.; Wei, M. F.; Shieh, M. J. Aptamer-based Tumor-targeted Drug Delivery for Photodynamic Therapy. ACS Nano 2010, 4, 1433−1442. (35) Crosby, G. A.; Demas, J. N. Measurement of Photoluminescence Quantum Yields. J. Phys. Chem. 1971, 75, 991−1024. (36) Das, P.; Chakrabarty, A.; Mallick, A.; Chattopadhyay, N. Photophysics of a Cationic Biological Photosensitizer in Anionic Micellar Environments: Combined Effect of Polarity and Rigidity. J. Phys. Chem. B 2007, 111, 11169−11176. (37) Chakrabarty, A.; Das, P.; Mallick, A.; Chattopadhyay, N. Effect of Surfactant Chain Length on the Binding Interaction of a Biological Photosensitizerwith Cationic Micelles. J. Phys. Chem. B 2008, 112, 3684−3692. (38) Zoltewicz, J. A.; Munoz, S. Binding of Heterocyclic Cations to SDS Micelles. Compensatory Effects of the Charge on Binding. J. Phys. Chem. 1986, 90, 5820−5825. (39) Ranganathan, R.; Tran, L.; Bales, B. L. Surfactant-and Saltinduced Growth of Normal Sodium Alkyl Sulfate Micelles well above their Critical Micelle Concentrations. J. Phys. Chem. B 2000, 104, 2260−2264. (40) Weidemaier, K.; Tavernier, H. L.; Fayer, M. D. Photoinduced Electron Transfer on the Surfaces of Micelles. J. Phys. Chem. B 1997, 101, 9352−9361. (41) Sharma, P. K.; Bhatia, S. R. Effect of Anti-Inflammatories on Pluronic® F127: Micellar Assembly, Gelation and Partitioning. Int. J. Pharm. 2004, 278, 361−377. (42) Sharma, P. K.; Reilly, M. J.; Jones, D. N.; Robinson, P. M.; Bhatia, S. R. The Effect of Pharmaceuticals on the Nanoscale Structure of PEO−PPO−PEO Micelles. Colloids Surf., B 2008, 61, 53−60. (43) Alexander, S.; Cosgrove, T.; Prescott, S. W.; Castle, T. C. Flurbiprofen Encapsulation Using Pluronic Triblock Copolymers. Langmuir 2011, 27, 8054−8060. (44) Alexander, S.; de Vos, W. M.; Castle, T. C.; Cosgrove, T.; Prescott, S. W. Growth and Shrinkage of Pluronic Micelles by Uptake and Release of Flurbiprofen: Variation of pH. Langmuir 2012, 28, 6539−6545. (45) Alexander, S.; Cosgrove, T.; Castle, T. C.; Grillo, I.; Prescott, S. W. Effect of Temperature, Cosolvent, and Added Drug on PluronicFlurbiprofen Micellization. J. Phys. Chem. B 2012, 116, 11545−11551. (46) Rojas-Oviedo, I.; Retchkiman-Corona, B.; Quirino-Barreda, C. T.; Cárdenas, J.; Schabes-Retchkiman, P. S. Solubility Enhancement of a Poorly Water Soluble Drug by Forming Solid Dispersions using Mechanochemical Activation. Indian J. Pharm. Sci. 2012, 74, 505−511. (47) Chatterjee, A.; Maity, B.; Seth, D. Photophysics of 7(diethylamino)coumarin-3-Carboxylic Acid in Cationic Micelles: Effect of Chain Length and Head Group of the Surfactants and Urea. RSC Adv. 2014, 4, 34026−34036. (48) Maity, B.; Chatterjee, A.; Seth, D. Photophysics of Lumichrome in Anionic and Cationic Micellar Media. RSC Adv. 2015, 5, 3814− 3824. (49) Krishnamoorthy, G.; Dogra, S. K. Twisted Intramolecular Charge Transfer Emission of 2-(4′-N,N-dimethylaminophenyl) Benzimidazole in Micelles. J. Colloid Interface Sci. 1999, 213, 53−61. (50) Paul, B. K.; Ray, D.; Guchhait, N. Binding Interaction and Rotational-Relaxation Dynamics of a Cancer Cell Photosensitizer with Various Micellar Assemblies. J. Phys. Chem. B 2012, 116, 9704−9717. 3784

DOI: 10.1021/acs.jpcb.5b00467 J. Phys. Chem. B 2015, 119, 3776−3785

Article

The Journal of Physical Chemistry B (51) Corrin, M. L.; Harkins, W. D. The Effect of Salts on the Critical Concentration for the Formation of Micelles in Colloidal Electrolytes. J. Am. Chem. Soc. 1947, 69, 683−688. (52) Tanford, C. The Hydrophobic Effect: Formation of Micelles and Biological Membranes, 2nd ed.; Wiley: New York, 1980. (53) Mazer, N. A.; Benedek, G. B. An Investigation of the Micellar Phase of Sodium Dodecyl Sulfate in Aqueous Sodium Chloride Solutions Using Quasielastic Light Scattering Spectroscopy. J. Phys. Chem. 1976, 80, 1075−1085. (54) Vlachy, N.; Drechsler, M.; Verbavatz, J. M.; Touraud, D.; Kunz, W. Role of the Surfactant Headgroup on the Counterion Specificity in the Micelle-to-Vesicle Transition through Salt Addition. J. Colloid Interface Sci. 2008, 319, 542−548. (55) Lianos, P.; Zana, R. Use of Pyrene Excimer Formation to Study the Effect of Sodium Chloride on the Structure of Sodium Dodecyl Sulfate Micelles. J. Phys. Chem. 1980, 84, 3339−3341. (56) Patra, A.; Hazra, S.; Kumar, G. S.; Mitra, R. K. Entropy Contribution toward Micelle-Driven Deintercalation of Drug−DNA Complex. J. Phys. Chem. B 2014, 118, 901−908. (57) Pan, A.; Maiti, S. S.; Naskar, B.; Bhattacharya, S. C.; Moulik, S. P. Self-Aggregation of MEGA-9 (N-Nonanoyl-N-methyl-d-glucamine) in Aqueous Medium: Physicochemistry of Interfacial and Solution Behaviors with Special Reference to Formation Energetics and Micelle Microenvironment. J. Phys. Chem. B 2013, 117, 7578−7592. (58) Abraham, T.; Lewis, R. N. A. H.; Hodges, R. S.; McElhaney, R. N. Isothermal Titration Calorimetry Studies of the Binding of the Antimicrobial Peptide Gramicidin S to Phospholipid Bilayer Membranes. Biochemistry 2005, 44, 11279−11285. (59) Jelesarov, I.; Bosshard, H. R. Isothermal Titration Calorimetry and Differential Scanning Calorimetry as Complementary Tools to Investigate the Energetics of Biomolecular Recognition. J. Mol. Recognit. 1999, 12, 3−18. (60) Wagner, B. D. The Use of Coumarins as EnvironmentallySensitive Fluorescent Probes of Heterogeneous Inclusion Systems. Molecules 2009, 14, 210−237. (61) Wagner, B. D. Hydrogen Bonding of Excited States in Supramolecular Host-Guest Inclusion Complexes. Phys. Chem. Chem. Phys. 2012, 14, 8825−8835. (62) Chatterjee, A.; Maity, B.; Seth, D. Supramolecular Interaction Between a Hydrophilic Coumarin Dye and Macrocyclic Hosts: Spectroscopic and Calorimetric Study. J. Phys. Chem. B 2014, 118, 9768−9781.

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