Enhanced Binding of Phenosafranin to Triblock Copolymer F127

Mar 3, 2016 - The ITC raw data were analyzed using the NanoAnalyze v2.4.1 software using the independent site-binding model. .... Location of the Prob...
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Enhanced Binding of Phenosafranin to Triblock Copolymer F127 Induced by Sodium Dodecyl Sulfate: A Mixed Micellar System as an Efficient Drug Delivery Vehicle Ramakanta Mondal, Narayani Ghosh, and Saptarshi Mukherjee* Department of Chemistry, Indian Institute of Science Education and Research Bhopal, Bhopal Bypass Road, Bhauri, Bhopal 462 066, Madhya Pradesh, India S Supporting Information *

ABSTRACT: In this study, we explored the interaction of a cationic phenazinium dye, phenosafranin (PSF, here used as a model drug), with pluronic block copolymer F127, both in the presence and in the absence of the anionic surfactant sodium dodecyl sulfate (SDS), which forms mixed micelles with F127. We applied both steady-state and time-resolved spectroscopic techniques, along with isothermal titration calorimetry (ITC), to demonstrate the binding of the probe PSF to both the pluronic and F127/SDS mixed micelles. Dynamic light scattering (DLS) study revealed that, upon interaction with SDS, the hydrodynamic diameter (dH) of F127 micelles decreased due to the formation of the mixed micelles. The PSF penetrated to the more hydrophobic interior of the mixed micellar system as compared to F127 micelles alone. Micropolarity and fluorescence-quenching experiments revealed that PSF is more deeply seated in the case of F127/SDS mixed micelles. Through a partition coefficient, lifetime measurements, and timeresolved anisotropy experiments, we also established that the partitioning of the probe within the F127 micelles in the presence of SDS is almost double than that in its absence. ITC data corroborates the fact that the binding of PSF is the strongest and most thermodynamically favorable when mixed micelles are formed, which enables our system to serve as an excellent drug delivery vehicle when compared to F127 alone.

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INTRODUCTION Recently, nonionic pluronic triblock copolymers have received considerable attention because of their rapidly expanding contributions to the fields of biological and medical science as modern drug delivery carriers1−7 and also for their industrial applications.8,9 Amphiphilic triblock copolymers, having the general formula PEOx-PPOy-PEOx, are composed of a hydrophobic poly(propylene oxide) (PPO) block and two units of a hydrophilic poly(ethylene oxide) (PEO) block, in which the PPO block is in the center.10−12 Because of their amphiphilic character, these block copolymers show surfactant properties, which includes the ability to interact with hydrophobic surfaces and biological membranes. In aqueous solutions above the critical micelle concentration (CMC), these copolymers self-assemble and form micelles. Due to the presence of their unique core−shell structure, polymeric micelles such as F127 have the ability to solubilize hydrophobic drugs without using any organic solvent or covalent bond formation. Because these polymeric micelles are also nontoxic in nature, they can be safely used for the controlled release of drugs.5,6 A general method for designing a drug delivery system is to incorporate the drug within the nanocarrier, resulting in enhanced solubility. Copolymers are efficient in intracellular delivery because of the presence of the oxyethylene groups in © 2016 American Chemical Society

the corona: the hydrophilic PEO corona prevents aggregation and protein adsorption, and the hydrophobic PPO incorporates lipophilic drugs.6 Therefore, pluronic micelles can solubilize many poorly soluble drugs and protect them from inactivation in biological media.7 Triblock copolymeric micelles are also known to be an excellent delivery system for hydrophobic anticancer drugs6,7 and nucleic acids3 and are also used in the treatment of multidrug-resistant tumors.6 These pluronic block copolymers have the unique ability to translocate into the cells after becoming incorporated inside membranes. By doing so, they affect many cellular functions, such as mitochondrial respiration, ATP synthesis, gene expression, etc.4 As a consequence, these block copolymers cause drastic sensitization of multidrug-resistant tumors to various anticancer agents,6 trigger and execute an increased rate of drug delivery across the blood, brain, and intestinal barriers, and augment the transcriptional activation of gene expression.4,7 Although triblock copolymers are nondegradable in nature, the molecules with low molecular weight (less than 15 kDa) are relatively easily eliminated from the body.5,6 However, the stability of Received: January 23, 2016 Revised: February 26, 2016 Published: March 3, 2016 2968

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interaction(s). The results obtained from our experiments suggest that the encapsulation efficiency of the probe with the F127/SDS mixed micelles is much higher and more stable than that of the polymeric micelles.

these micelles is low due to the high critical micelle concentration (CMC for F127 has been reported to be ∼0.56 mM11,12). These micelles are often dissociated by dilution or interaction with different components in the blood.13 Through the mixture of pluronic micelles with other polymers/surfactants, the stability of the resulting micelles can be enhanced, and thus the bioavailability of the encapsulated drugs can also be improved. Recently, to reduce the CMC and consequently enhance the stability of the micelles, pluronic L121 copolymeric micelles have been cross-linked through their hydrophilic shells.14 This prevents the micelles from becoming dissociated at low concentrations in the bloodstream upon dilution.14 There are several reports on mixed micellar systems, but these works focus mostly on the effect of various additives on the micellization and structure of block copolymers, thermodynamics of the formation of mixed micelles, determination of the CMC of mixed micelles, and solubilization of poorly soluble hydrophobic drugs into mixed micelles.12−19 However, the detailed characterization of the interaction of a drug with mixed micelles is scarcely reported. In this present work, we have focused mainly on the drug-binding and encapsulation capability of the F127/SDS mixed micellar system compared to F127 micelles alone through the use of phenosafranine (PSF, Figure 1a, 3,7-diamino-5-phenyl phenazinium chloride) as a

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EXPERIMENTAL SECTION Materials. PSF, pluronic F127, SDS, Rhodamine 6G, and potassium iodide (KI) were purchased from Sigma-Aldrich Chemical Co. and used without further purification. Spectroscopic grade 1,4-dioxane was purchased from Spectrochem and used as received. All solutions were prepared in triply distilled deionized Milli-Q water. The concentration of PSF used for spectroscopic measurements was kept at ∼3 μM. Instruments. Steady-State Spectroscopic Measurements. The absorption and fluorescence spectral measurements were carried out on a Cary 100 UV-vis spectrophotometer and a Fluorolog 3-111 fluorometer, respectively. The temperature was maintained at 298 K. Time-Resolved Fluorescence Measurements. The fluorescence lifetime of PSF was recorded using the time correlated single photon counting (TCSPC) technique.26 The samples were excited at λex = 470 nm using a picosecond diode laser (IBH-NanoLED N-470L, full width at half-maximum (fwhm), ∼140 ps), and the signals were collected at the magic angle polarization using a Hamamatsu MCP Photomultiplier (model R-3809U-50).27,28 The lifetime decays were deconvoluted using DAS-6 decay analysis software. Isothermal Titration Calorimetry (ITC) Measurements. The ITC experiments were carried out on a Nano ITC (TA Instruments) at 298 K. A total of 25 aliquots of degassed PSF solution (4 mM, 2 μL for each injection) were injected from a rotating syringe (300 rpm) into the ITC sample chamber containing a 1 mM F127 solution. The interval between two injections was kept at 120 s. The control experiments to determine the heat of the dilution of PSF were performed by injecting the same concentration of PSF into water. The ITC raw data were analyzed using the NanoAnalyze v2.4.1 software using the independent site-binding model. The heat of dilution was subtracted from the raw data of the interaction of PSF with the surfactant before analysis. Similarly, the thermodynamics of PSF-mixed micelles and the F127/SDS interaction were evaluated by the titration of 4 mM PSF with the mixed micelles (2 mM F127 + 2 mM SDS) and 4 mM SDS with 1 mM F127 under the same experimental conditions mentioned above. It is pertinent to mention that, according to the convention of the associated software with our instrument (that is, Nano ITC, TA Instrument), the endothermic and exothermic processes are represented by “downward” and “upward” heat bursts, respectively.29−31 However, it is important to note that the upward/downward trend of the raw ITC data is not universal. The sign of ΔH obtained from the fitting of the experimental data provides the genuine thermodynamic signature of the concerned process, and we have interpreted our data accordingly. Dynamic Light Scattering (DLS) Measurements. DLS measurements were performed on a Beckman Coulter Delsa Nano C instrument employing 658 nm dual 30 mW laser diodes and equipped with a thermostatic sample chamber maintained at 298 K. Subsequently, the scattering intensity data were processed using the instrumental software to estimate the hydrodynamic diameter (dH) and the size distribution of the scatterer in each sample.

Figure 1. (a) Structural representation of phenosafranine (PSF) and (b) size distribution of 2 mM F127 (olive green) and F127/SDS mixed micelles (formed by the addition of 2 mM SDS to 2 mM F127) (red) obtained from DLS measurements.

model drug. PSF, a biologically potent cationic phenazinium dye, has been used extensively as a semiconductor20 and sensitizer in various biological systems where the processes of energy and electron transfer are operational21−23 and exhibits various photophysical and photobiological applications.24,25 The additive sodium dodecyl sulfate (SDS), which becomes adsorbed into the F127 micelles, alters the photophysical properties of PSF, which are otherwise very different in the presence of F127 micelles alone. Our spectroscopic analyses reveal that, for the F127/SDS mixed micellar system,15−19 the binding of the probe to the F127 micelles is augmented remarkably, even at low concentrations of SDS. The variation in the size of the micelle upon addition of SDS has been studied using the DLS technique. In order to quantify the different binding interactions involved, we have determined the associated thermodynamic parameters involved in such interactions through isothermal titration calorimetry (ITC). Our experiments show some unexplored results regarding the thermodynamics of the interaction of PSF with both the F127/ SDS mixed micelle and F127 micelle, which is crucial for understanding the nature of the force underlying such 2969

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Figure 2. (a) Absorption profiles of PSF in the presence of increasing concentrations of F127 in an aqueous medium (curves (i) → (vii) correspond to 0, 0.25, 0.75, 1, 2, 3, and 5 mM F127, respectively). (b) Absorption profiles of PSF with increasing concentrations of SDS in the presence of 2 mM F127 (curves (i) → (vi) correspond to 0, 0.2, 0.4, 0.6, 1, and 2 mM SDS, respectively). (c) Excitation spectral profiles of PSF (λmonitored = λmax em ) in the presence of varying concentrations of F127 (curves (i) → (vii) correspond to 0, 0.5, 1, 2, 3, 4, and 5 mM F127, respectively). (d) Excitation spectral profiles of PSF (λmonitored = λmax em ) with increasing concentrations of SDS in the presence of 2 mM F127 (curves (i) → (vi) correspond to 0, 0.4, 0.6, 1, 1.6, and 2 mM SDS, respectively).

Figure 3. (a) Emission profiles of PSF (λex = 520 nm) in the presence of (i) → (x): 0, 0.25, 0.75, 1, 1.5, 2, 2.5, 3, 4, and 5 mM F127, respectively, in aqueous medium. (b) Emission profiles of PSF (λex = 520 nm) in 2 mM F127 with increasing concentrations of SDS: curves (i) → (ix) represent 0, 0.2, 0.4, 0.6, 0.8, 1, 1.2, 1.6, and 2 mM SDS, respectively. (c) Plot of 1/(I − I0) vs [F127]−1 (mM−1).

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decreased to ∼8 nm, which revealed that addition of SDS results in the formation of a copolymer−SDS complex (i.e., a mixed micelle (F127/SDS)).15 Because we are using 2 mM SDS and its CMC is ∼8 mM at 298 K, micelles of SDS are not formed. Thus, we can rationally conclude that F127/SDS mixed micelles are formed at very low concentrations of SDS. The formation of mixed micelles is due to the solubilization of hydrophobic hydrocarbon chains of the monomeric SDS

RESULTS AND DISCUSSION

Dynamic Light Scattering Studies. We determined the variation in the micellar size of F127 upon addition of SDS through a DLS experiment. From Figure 1b, the hydrodynamic diameter of pure F127 micelles at 2 mM concentration (i.e., post-micellar concentration) is ∼22 nm, which is in good agreement with reports in the literature.16 Upon addition of 2 mM SDS to the same F127 solution (2 mM), the micellar size 2970

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Figure 4. Variation of steady-state fluorescence anisotropy of PSF (λex = 520 nm and λmonitored = λmax em ) with increasing concentrations of (a) F127 in aqueous medium and (b) SDS in the presence of 2 mM F127.

emission maxima compared to that in F127 micelles alone max (from λmax em ∼ 585 nm in water to λem ∼ 576 nm in the presence max of 2 mM F127 and λem ∼ 565 nm upon the addition of 2 mM SDS to 2 mM F127). These results clearly reveal that the probe is embedded in the more hydrophobic interior of the mixed micelles rather than the F127 micelles in the absence of SDS. The degree of penetration of the probe into the polymeric micelle is estimated from the knowledge of the partitioning of the probe between the micellar environment and the aqueous medium. The partition coefficient of a solute between the micellar environment and the aqueous solution is defined by the following relation:25,33,34

surfactant molecules in the core of the F127 micelle, i.e., the PPO units of the block copolymer.17,32 Steady-State Absorption and Emission Studies. The absorption profiles of PSF with increasing concentrations of F127 are shown in Figure 2a. In water, PSF shows an absorption peak centered at ∼520 nm.25 Upon the addition of F127 to a ∼3 μM solution of PSF, the absorption profile undergoes a significant modification as displayed in Figure 2a. As seen in Figure 2, 5 mM F127 results in an enhancement of the absorption intensity along with a red shift of ∼7 nm. Such a modification in the absorption profile indicates that, upon addition of F127, PSF experiences an environment which is different from that of bulk water.25 To explore the effect of SDS on the F127 polymeric micelle, we monitored the variation in absorption of PSF in the presence of varying concentrations of SDS. The absorption maximum of PSF was found to be further red-shifted (from ∼525 nm in 2 mM F127 to ∼534 nm upon the addition of 2 mM SDS), accompanied by an increment in absorbance (Figure 2b). Thus, the bathochromic shift of ∼9 nm is a clear signature of the fact that, upon the formation of mixed F127/ SDS micelles, PSF experiences an environment which is substantially different from that of the F127 micelles alone. We also monitored the excitation spectra of PSF with increasing concentrations of the F127 solution and upon addition of varying concentrations of SDS in a 2 mM F127 solution. Excitation spectra of PSF show a ∼7 nm red shift in the presence of 2 mM F127 (Figure 2c) and a further ∼8 nm red shift upon the addition of 2 mM SDS in 2 mM F127 (Figure 2d). These results successfully corroborate the aforementioned absorption spectral changes as exemplified by Figures 2a and 2b. The emission characteristics of PSF in the presence of varying concentrations of F127 are illustrated in Figure 3a. In water, PSF shows a single unstructured broad-emission band at ∼585 nm25 and, upon incorporation within the F127 micelles, the emission spectra of PSF become remarkably modified. As seen in Figure 3a, the emission profile of PSF undergoes a significant enhancement of emission intensity with a 10 nm blue shift (λmax em ∼ 575 nm) following the addition of 5 mM F127. This blue shift in the emission maxima of PSF in the presence of 5 mM F127 can be attributed to the hydrophobic environment the fluorophore experiences upon its incorporation inside the hydrophobic corona of the pluronic micelles. The marked modulations of the emission profiles of PSF in the presence of the F127/SDS mixed micelles are displayed in Figure 3b. Figure 3b illustrates a remarkable increase in emission intensity followed by a further blue shift of the

KP =

(CM /C T)/[polymer] (C W /C T)/[water]

(1)

where CT is the total concentration of PSF and CM and CW represent the concentrations of PSF in polymer and water, respectively. The partition coefficient (KP) of PSF in the micelle is evaluated by analyzing the fluorescence data according to the following equation:25,33,34 1 1 1 [water] = + × ΔI ΔImax ΔImaxKP [polymer]

(2)

where ΔI = (I − I0), ΔImax = (I∞ − I0), and I0, I, and I∞ are the fluorescence intensities of the fluorophore in the absence of polymer, at an intermediate polymer concentration, and at the saturation level of interaction, respectively. Using eq 2, the partition coefficient for the PSF-F127 interaction is estimated from the 1/ΔI vs [polymer]−1 plot and is found to be 1.59 × 104 (Figure 3c). Thus, a large KP value indicates efficient partitioning of PSF within the F127 micelles. We determined the percentage of probe bound to F127 micelles using the following equation:25 C0 =

CT 1 + KP[polymer]/[water]

(3)

where C0 and CT are the concentrations of free and total probes, respectively. Thus, it can be estimated from eq 3 that ∼58% of PSF remains in the bound state in the presence of 5 mM F127 micelles. These data support our time-resolved results, which are discussed under Fluorescence Lifetime Decay Measurements. It must be stated here that PSF also binds to SDS when micelles are formed. Because we are mainly concerned with low concentrations of SDS (∼2 mM, which is much lower than the CMC of SDS in water), we have ruled out any partitioning of PSF in SDS at such low concentrations (please refer to Figure 2971

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Figure 5. (a) Modulation in emission profiles of PSF in a varying composition of water/dioxane mixtures. Curves (i) → (x) correspond to % dioxane (v/v) = 0, 10, 20, 30, 40, 50, 60, 70, 80, and 90, respectively. (b) Calibration curve for the micropolarity determination by plotting the relative variation of PSF emission maxima as a function of ET(30) of the water/dioxane reference solvent mixture.

SDS mixed micelles on the calibration curve, we found the micropolarity values of PSF in the environments of F127 alone and F127/SDS mixed micelles to be ∼58.5 and ∼51.5 kcal mol−1, respectively (Figure 5b). The value of micropolarity in the immediate vicinity of PSF signifies that the probe resides in an environment whose polarity is substantially less than that of the bulk aqueous phase (for the bulk aqueous phase, ET(30) = 63.1 kcal mol−1).35,36 The reduction of polarity around PSF in mixed micelles compared to that in F127 micelles indicates that the probe is buried deeper in the interior of the micelles, which supports our previous results. Fluorescence Quenching. The binding locale of PSF within the F127 and F127/SDS mixed micelles has been further substantiated by fluorescence-quenching experiments using KI as the quencher.37 The quenching results have been analyzed using the Stern−Volmer equation:26,27

S1 of the Supporting Information). It is evident from Figure S1 that PSF does not significantly bind to the surfactant at concentrations less than ∼8 mM SDS. Equation 3 provides an estimate of the amount of PSF bound to the polymeric micelle F127 and is valid when the polymer is dissolved in water. To estimate the percentage of probe bound to the F127/SDS mixed micellar system, we need to know the concentration of polymer (here, F127/SDS mixed micelle) pursuant to eqs 2 and 3. This information is not available; therefore, such an estimation is not possible here. Steady-State Fluorescence Anisotropy. Figure 4a depicts the variation of steady-state fluorescence anisotropy (r) of PSF in varying concentrations of F127 in an aqueous medium. From Figure 4a, it is clear that the fluorescence anisotropy of PSF increases sharply with F127 concentration and then reaches saturation. In the presence of F127, the motional degrees of freedom of PSF are restricted due to the transfer of the probe from bulk water to the confined micellar environment, which is evident from the rising trend of anisotropy values with increasing concentrations of F127.25,26 Figure 4b shows that the steady-state fluorescence anisotropy of PSF in F127/SDS mixed micelles further increases sharply in the presence of 2 mM F127 and varying concentrations of SDS. This result suggests that PSF experiences a relatively more constrained environment within the mixed F127/SDS micelles as compared to that in F127 micelles alone. A saturation of the anisotropy is observed at around 2 mM SDS. The fluorescence anisotropy results thus confirm that the binding of the probe is substantially stronger in the F127/SDS mixed micellar environment which is discussed in detail under Isothermal Titration Calorimetry (ITC). Location of the Probe. Micropolarity Study. To determine the binding location of the probe, we investigated the micropolarity in and around the fluorophore in two different micellar systems.12,33 The micropolarity value is generally estimated by comparing the spectral modulations of the probe in the corresponding microheterogeneous environment with those obtained in pure solvents or solvent mixtures of known polarity.12,33,35 Figure 5a shows the fluorescence spectra of PSF as a function of a varying composition of a water/dioxane mixture. Due to a decrease in polarity of the medium (achieved by an increase in the percentage of dioxane), the emission intensity of PSF increases along with a remarkable blue shift of emission maxima. A calibration curve was generated by plotting the variation in the emission maxima of PSF in a water/dioxane solvent mixture against the solvent polarity equivalent parameter (ET(30)).35 By interpolating the emission maxima of PSF in F127 polymeric micelles and F127/

F0 = 1 + KSV[Q] F

(4)

where F0 and F are the fluorescence intensities of PSF in the absence and presence of the quencher KI, respectively, [Q] is the concentration of KI, and KSV is the Stern−Volmer quenching constant. The KI-quenching studies monitor the accessibility of the probe: the more buried the probe, the lower the extent of quenching. The Stern−Volmer plots for quenching PSF in different micellar environments are shown in Figure S2 in the Supporting Information. The iodide ioninduced fluorescence Stern−Volmer quenching constants of the cationic probe PSF in bulk water, 5 mM F127, and F127/ SDS mixed micelles (formed by 2 mM F127 and 2 mM SDS) have been estimated at 26.5, 19.5, and 5.5 M−1, respectively. It can be inferred from the KSV values that the probe is most deep-seated in the case of F127/SDS mixed micelles. Fluorescence Lifetime Decay Measurements. The variation in time-resolved fluorescence decay profiles of PSF upon addition of F127 is depicted in Figure 6a, and the fitted data are summarized in Table S1 in the Supporting Information. Table S1 shows that PSF exhibits a monoexponential decay with a time constant of 0.85 ns in bulk water,25 which is a clear signature of the fact that, in bulk water, PSF experiences an environment which is rather homogeneous. However, the decay transients of PSF in the presence of F127 micelles fit to biexponential decay functions indicate the heterogeneity thereby introduced.25,38,39 As previously discussed, PSF partitions between aqueous and micellar phases; hence, a biexponential decay may be expected. Table S1 also shows that the relatively shorter lifetime component (τ1) of 2972

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Figure 6. Fluorescence decay profiles of PSF in the presence of (a) increasing concentrations of F127 (curves (i) → (vii) represent 0, 0.25, 0.5, 1, 2, 3, and 5 mM F127 solution, respectively) and (b) increasing concentrations of SDS in the presence of 2 mM F127 (curves (i) → (vi) represent 0, 0.2, 0.4, 0.6, 1, and 2 mM SDS, respectively.

Figure 7. Time-resolved fluorescence anisotropy decay profiles of PSF in various environments as marked in the figure.

low initial anisotropy value, whereas in micellar environments, it shows a biexponential pattern with a slow reorientation time and a fast reorientation time (Table S2); the decay profiles are vastly different from those obtained for bulk water. Also, as seen in Figure 7, the nature of the anisotropy decay of PSF in F127/SDS mixed micelles is much slower compared to that of the F127 micelles alone. The short and long components in the anisotropy decay can be explained by the rotational diffusion of the free probe in solution and the micelle-bound probe, respectively. Our time-resolved anisotropy data are in excellent agreement with our lifetime parameters. As evidenced in Tables S1 and S2, the amplitudes, which signify the micelle-bound probe (α2 for lifetime studies and α2r for time-resolved anisotropy studies), are almost similar. Hence, from both of these independent experiments, we conclude that ∼55 ± 2% of the probe molecules remain bound to the F127 micelles, and the contribution increases to ∼93 ± 2% when F127/SDS mixed micelles are formed. Also, these values agree well with the data obtained from our partition coefficient experiments detailed above. The values of the rotational-relaxation times thus suggest that PSF experiences the most constrained environment when F127/SDS mixed micelles are formed because of a greater degree of penetration within the hydrophobic scaffolds of the mixed micelles. Isothermal Titration Calorimetry (ITC). The thermodynamic parameters for PSF-F127, PSF-F127/SDS mixed micelle, and SDS-F127 micelle (in the absence of PSF) binding interactions have been determined by ITC. Such a technique offers a direct method to evaluate the thermodynamic parameters by considering the entire system compared to fluorometric approaches that probe the immediate microenvironment in and around the fluorophore.30 We kept the surfactant in the calorimeter cell and subsequently added PSF from a syringe so that the concentration of surfactant remained above CMC throughout the titration and contributions from the monomeric surfactant binding to PSF could be canceled out. Figures 8a, 8b, and 8c depict the ITC enthalpograms during the titration of PSF with F127, PSF with mixed F127/ SDS, and mixed micelles formed between SDS and F127 in the absence of PSF, respectively. The heat changes during the titrations were fit to an independent site-binding model to estimate the thermodynamic parameters.30,40−43 The binding of PSF to both F127 micelles and F127/SDS mixed micelles is endothermic in nature (ΔH > 0) and characterized by a large positive entropic change (ΔS > 0) (Table 1). The free energy changes of binding for all three systems were negative (ΔG < 0), indicating that the processes are thermodynamically favorable. When PSF binds to the F127 micellar system, ΔH and ΔS are positive, which highlights the role of the hydrophobic force involved during the binding interac-

PSF in F127 is very close to the lifetime of PSF in bulk water. This can be attributed to the unbound probe, whereas the longer lifetime component (τ2) is attributed to the F127-bound probe. The regular decrement in the amplitude (α1) of the shorter lifetime component τ1 and simultaneous increment in the amplitude (α2) of the longer lifetime component τ2 of PSF upon addition of F127 indicates a gradual encapsulation of the probe within the micelles in a bulk aqueous medium. Thus, it is evident from Table S1 that ∼54% of the probe molecules are bound to F127 (the contribution of the longer lifetime component α2 increased from 0 to 54% in the presence of 5 mM F127). This corroborates our previous results, determined from the partition coefficient, that 58% of the probe is bound in 5 mM F127 copolymer. It is interesting to note that, upon addition of SDS to F127 micelles (Figure 6b, Table S1), the contribution of the longer lifetime α2 increases from ∼40% (in 2 mM F127) to ∼90% in the presence of 2 mM SDS (Table S1). This indicates that, with the addition of SDS to F127 micelles, almost all of the probe molecules penetrate the mixed micelle from bulk aqueous medium. We also estimated the fluorescence quantum yields (Φf) of PSF (using Rhodamine 6G as the standard) in different environments, which are shown in Table S1.12 It is clear from Table S1 that the quantum yield of PSF increased from 0.038 in water22,36 to 0.065 in the presence of 5 mM F127 to 0.151 upon the addition of 2 mM SDS. In mixed micelles, the probe thus becomes more emissive, and this is supported by the increased fluorescence intensities and lifetimes. This increment of quantum yield may be attributed to the suppression of the nonradioactive decay pathways of PSF due to the increase of motional restriction around the probe. This result indicates that, in mixed micelles, the binding of PSF is stronger compared to that of F127 micelles, and the microenvironment becomes more rigid. Rotational-Relaxation Dynamics. The time-resolved fluorescence anisotropy decay is expressed as26,28 r (t ) =

∑ r0iαir e(−t /τ ) ir

i

(5)

where αir describes the pre-exponential factor for the ith rotational correlation time τir and r0 = ∑ir0i describes the initial anisotropy.26 Figure 7 displays the time-resolved fluorescence anisotropy decay profiles of PSF in various environments, and the associated rotational dynamic parameters are summarized in Table S2 in the Supporting Information. PSF exhibits monoexponential time-resolved anisotropy decay, having a reorientation time of ∼145 ps in water with a 2973

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Figure 8. ITC profiles for the titration of (a) PSF-F127, (b) PSF with F127/SDS mixed micelles, and (c) SDS with F127 at 298 K. The upper panel of each graph corresponds to the raw data of the integrated heat after correction for heat of dilution. The lower panel shows the integrated heat data against the molar ratio. The solid lines are the fitted curves. According to the power convention of the accompanying software of our instrument, the exothermic and endothermic processes are shown by upward and downward heat burst curves, respectively. For more details, please see Instruments.

Table 1. Thermodynamic Parameters of PSF-F127, PSF-Mixed Micelles, and the SDS-F127 Interaction Obtained from ITC Measurements system

ΔH (kJ mol−1)

ΔS (J mol−1 K−1)

ΔG (kJ mol−1)

Ka (M−1)

PSF-F127 PSF-mixed micelles SDS-F127

1.03 ± 0.08 91.9 ± 5.0 188.5 ± 9.1

84.1 ± 4.1 407.5 ± 22.2 714.2 ± 35.1

−24.03 ± 1.14 −29.56 ± 1.60 −24.33 ± 1.36

(1.63 ± 0.06) × 104 (1.55 ± 0.07) × 105 (1.91 ± 0.09) × 104

tions.29,44,45 The primary cause of hydrophobic interaction is the rearrangement of the water structure.46,47 Because of the interaction of PSF with F127 micelles, the structured water molecules surrounding the micelles collapse and attain a more random conformation; therefore, the entropy of the system increases (ΔS > 0). Thus, the overall interaction of PSF with F127 micelles is an entropy-driven, thermodynamically favorable process. In the PSF-mixed micelle interaction, similar types of thermodynamic parameters are obtained (ΔH > 0, ΔS > 0, and ΔG < 0). It can be seen in Table 1 that the magnitudes of both ΔH and ΔS are much higher for the PSF-mixed micelle interaction compared to those estimated for the PSF-F127 micelles. When PSF interacts with mixed micelles formed by SDS and F127, three possible interactions may take place: interaction of PSF with SDS, interaction of PSF with F127, and interaction of PSF with the mixed micelles formed between SDS and F127. As stated earlier, the interaction of SDS with PSF at 2 mM SDS is almost negligible (please refer to Figure

S1). The magnitudes of all thermodynamic parameters and the binding constant K values (Table 1) for the PSF-mixed micelle interaction are found to be greater than those observed for the PSF-F127 interaction, which proves the thermodynamic feasibility and stronger binding of PSF when mixed micelles are formed. To estimate the thermodynamics of the formation of mixed micelles in the absence of PSF, we also studied the interaction of SDS with F127 micelles (Figure 8c, Table 1). Table 1 shows that the interaction of SDS with F127 is characterized by a binding constant value of K = 1.91 × 104 M−1, a large positive enthalpy change, and a high positive entropy change (ΔH > 0 and ΔS > 0). This indicates that hydrophobic interaction is the major driving force in the formation of mixed micelles. The release of structured water from F127 micelles contributes to a large positive entropy change46,47 upon binding of SDS to the interior of the F127 micelles17 and forms the well-packed, mixed micellar system. The net negative free energy change (ΔG < 0) signifies the spontaneity of the interaction. 2974

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CONCLUSIONS In this study, we investigated the interaction of a cationic probe, PSF, as a model drug, with pluronic F127 micelles and mixed micelles, which are formed by the addition of low concentrations of the anionic surfactant SDS to F127 micelles. Steady-state results show that PSF experiences a less polar and more constrained environment in the presence of F127/SDS mixed micelles compared to that of F127 micelles alone. Partition coefficient data prove that ∼58% of PSF becomes embedded in the micellar matrix of F127. Time-resolved study confirms that the fraction of the probe bound to F127 micelles increases significantly upon adsorption of very low concentrations of SDS below its CMC. From the iodide ion-induced Stern−Volmer quenching constant values, it can be concluded that the mixed micellar system can provide greater protection to the probe from external agents (such as an external quencher) compared to that of the normal micelles. The binding constant of PSF with F127/SDS mixed micelles which is estimated from ITC experiment is an order of magnitude higher than that of with F127 alone. This result suggests that the interaction of the probe with the F127/SDS mixed micelles is stronger than the interaction with the F127 micelles. We also proved that the hydrophobic force is the driving parameter of the interaction of PSF with both the F127 micelles and also the F127/SDS mixed micelles. Thus, our F127/SDS mixed micellar system can serve as a vehicle for drug delivery (with PSF being the model drug) with an efficiency higher than what would have been achieved with F127 micelles alone, which is the alternative.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.6b00759. Variations in emission intensities of PSF in the presence of SDS, Stern−Volmer plots of iodine-ion quenching, lifetime decay parameters, and time-resolved rotational anisotropy parameters (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS R.M. acknowledges IISER Bhopal for a research fellowship. N.G. acknowledges a research fellowship from the Government of India through CSIR-NET. S.M. sincerely thanks DST, Government of India, for financial support.

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REFERENCES

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