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Contrasting Effects of Salt and Temperature on Niosome-Bound Norharmane: Direct Evidence for Positive Heat Capacity Change in the Niosome:β-Cyclodextrin Interaction Bijan K. Paul, Narayani Ghosh, Ramakanta Mondal, and Saptarshi Mukherjee* Department of Chemistry, Indian Institute of Science Education and Research Bhopal, Bhopal Bypass Road, Bhauri, Bhopal 462066, Madhya Pradesh India S Supporting Information *

ABSTRACT: The modulation of the prototropic equilibrium of a cancer cell photosensitizer, norharmane (NHM), within a niosome microheterogeneous environment has been investigated. The contrasting effects of temperature and extrinsically added salt on the photophysics of niosome-bound drug have been meticulously explored from steady-state and time-resolved spectroscopic techniques. The cation ⇌ neutral prototropic equilibrium of NHM is found to be preferentially favored toward the neutral species with increasing salt concentration, and the results are rationalized on the basis of water penetration to the hydration layer of niosome. The effects are typically reversed with temperature. The differential rotational relaxation behavior of NHM under various conditions has also been addressed from fluorescence anisotropy decay. Further, the study delineates the application of β-cyclodextrin (βCD) as a potential host system, leading to drug sequestration from the niosome-encapsulated state. To this end, a detailed investigation of the thermodynamics of the niosome:βCD interaction has been undertaken by isothermal titration calorimetry (ITC) to unravel the notable dependence of the thermodynamic parameters on temperature. Consequently, a critical analysis of the variation of the enthalpy change (ΔH) of the process with temperature leads to the unique observation of a positive heat capacity change (ΔCp) marking the hallmark of hydrophobic hydration.

1. INTRODUCTION Over the past few years, β-carbolines have envisaged enormous impetus within the scientific community on a variety of research avenues. This mainly arises from the broad-spectrum of biological and medicinal applications of this class of natural products, including their functionality in reversible inhibition of monoamine-oxidase, which in turn plays a vital role in governing the psychoactivity of the indigenous psychedelic drug ayahuasca. Interaction of these drugs with a large number of neurotransmitters and neuromodulators of the central nervous system1−5 has been argued to have crucial consequences, such as regulation of the sleep-wake cycle, governing convulsiveness and anxiety, memory enhancing effects, and so forth. Besides, the cytotoxicity of β-carbolines and its enhancement following photoexcitation by long-wavelength UV radiation have been intensively investigated.1,6−9 However, the ability of β-carbolines to produce singlet oxygen (which is detrimental to malignant tumorous/cells) has pushed the frontiers of the medical applications of these naturally occurring alkaloids with the prospect of application as photosensitizer in photodynamic therapy.10,11 Such a vast range of applications has naturally triggered dynamic research endeavors toward the study of the interaction of β-carbolines with relevant biological/ biomimicking targets and drug delivery vehicles. Apart from being recognized as a simplistic model system for cell membranes, vesicles have gathered considerable research © XXXX American Chemical Society

attention, owing to their promising prospect as targeted drug carriers.12−14 Vesicles can be made up of phospholipids, nonionic or ionic surfactants, mixed surfactants, etc.15−17 Niosomes are vesicles composed of nonionic surfactants having reasonable similarity with liposomes in terms of structure and properties. Liposomes prepared from phospholipids contain a hydrophilic core and a hydrophobic bilayer region. Consequently, liposomes have been tested for solubilizing, loading, and targeted delivery of hydrophobic as well as hydrophilic drug molecules. However, phospholipid molecules are not easily degradable by hydrolysis in aqueous medium,12,18 which severely restricts prolific application of liposomes in drug delivery, as the optimization of the overall ADME (administration−distribution−metabolism−elimination) profile of drugs becomes challenging. The relatively high cost of phospholipids further impedes their applications to this effect. Thus, in search of a complementary candidate, niosomes have captured significant attention. The present work reports a detailed investigation of the interaction of an anticancer biological photosensitizer, norharmane (NHM), with niosome prepared from nonionic Triton X 100 (TX100) surfactant and cholesterol. Our steady-state absorption and fluorescence Received: March 1, 2016 Revised: April 5, 2016

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DOI: 10.1021/acs.jpcb.6b02168 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry B spectroscopic results unveil the modulation of prototropic transformation of NHM following interaction with niosome. Subsequently, the effects of salt (NaCl) and temperature on the photophysical characteristics of the niosome-encapsulated drug have been explored. The dynamical aspects of the interaction have been addressed by time-resolved fluorescence decay measurements. In addition, in order to achieve an in-depth understanding of the molecular forces governing the interaction, a detailed thermodynamic landscape of the phenomenon has been elucidated by isothermal titration calorimetry (ITC). In the overall context of drug delivery research, controlled release of the loaded drug from the carrier cargo is of profound importance. To this end, our endeavor has been extended toward studying the effect of β-cyclodextrin (βCD)19−21 on the niosome carrying the bound drug. In this context, the ability of βCD to form a host:guest inclusion complex has been explored to function as a tool to release the niosome-bound drug molecules by forming an inclusion complex with the constituents of the niosome membrane. The study of the interaction of niosome with CDs accompanies further relevance in the context of looking into the effect of CD on cell membranes (employing niosome structures as a simplistic model) inasmuch as a disruptive influence of CDs would considerably restrict their applications.22,23 Despite the considerable volume of research in these directions, a-priori predictions about such inherently complex interactions can be counterintuitive. This necessitates meticulous design of experiments for apt rationalization of such phenomena.22,23 To this effect, our efforts are framed to focus on the characterization of different possible equilibria involved within the complex microheterogeneous environments and thus to inquire into the actual interaction mechanism.

Figure 1. (a) Size distribution profile (intensity versus hydrodynamic diameter) of niosome as obtained from DLS measurement. The inset shows a simplified schematic of the prototropic equilibrium of NHM. (b) Fluorescence confocal microscopic image of niosome (a magnified view is shown in the inset).

measurement. The DLS profile shows a fairly monomodal distribution having an average hydrodynamic diameter, dh (±5%) ∼ 962 nm, which is in good agreement with literature reports.26,27 A simplified cartoon displaying the prototropic equilibrium of NHM is also presented in the inset of Figure 1a. Furthermore, the shape and morphology of the niosomes were characterized by confocal fluorescence microscopy (Figure 1b). 3.2. Absorption Spectroscopic Study. The absorption profile of NHM in aqueous buffer shows two bands centered at λabs ∼ 348 nm and ∼370 nm, characteristic of the neutral and cationic species of NHM, respectively.28−30 The absorption profile of NHM undergoes only marginal change with added niosome in terms of a slight increase in absorbance (figure not shown). Figure 2a represents the modulations of the absorption

2. EXPERIMENTAL SECTION Norharmane (NHM), Rhodamine 6G (Rh6G), Cholesterol, Triton X 100 (TX100), βCD, and sodium chloride (NaCl) were used as procured from Sigma-Aldrich Chemical Co., USA. Phosphate buffer was obtained from Sigma-Aldrich Chemical Co., USA, and 10 mM buffer solution of pH 7.40 was prepared in triply distilled deionized Milli pore water. The absorption and fluorescence spectra/steady-state fluorescence anisotropy were recorded on a Cary 100 UV−vis spectrophotometer and Fluorolog 3-111 fluorometer, respectively. Fluorescence lifetimes were recorded by the time-correlated single photon counting (TCSPC) technique.24 Dynamic light scattering (DLS) was performed on a Beckman Coulter Delsa Nano C instrument. The ITC experiments were performed on a Nano ITC, TA Instruments.25 A Carl-Zeiss LSM 510 META confocal laser scanning microscope was used for imaging studies. Fluorescence correlation spectroscopic (FCS) measurements were performed on a Holmarc FCS setup equipped with an inverted optical microscope (Olympus IX-71). The niosome was prepared by the literature reported method.26,27 The details of the experimental protocols and methodologies have been elaborated in the Supporting Information (SI).

Figure 2. Absorption spectra of niosome-bound NHM (∼2.0 μM) at 25 °C (a) in the presence of increasing NaCl concentration: curves 1 to 6 indicate 0, 1, 2, 3, 4, and 5 M NaCl, (b) with increasing temperature: curves 1 to 5 represent 10, 20, 30, 40, and 50 °C temperature.

spectra of niosome-bound NHM as a function of NaCl concentration. With increasing concentration of NaCl, the absorbance of the neutral band (λabs ∼ 348 nm) of NHM is enhanced with concomitant reduction of absorbance of the cationic band (λabs ∼ 370 nm) (Figure 2a). This observation can be adequately described by favorable stabilization of the cation ⇌ neutral prototropic equilibrium of NHM toward the neutral species with added NaCl within the niosomeencapsulated state. The modification of the absorption spectra of niosomebound NHM with increasing temperature is depicted in Figure 2b. The absorption profile of niosome-bound NHM with increasing temperature is modulated in a reverse pattern compared to that with added salt (NaCl); that is, the characteristic absorbance of the neutral species at λabs ∼ 348 nm decreases with increasing absorbance of the cationic species

3. RESULTS AND DISCUSSION 3.1. Characterization of Niosome: DLS and Confocal Fluorescence Microscopy Studies. Figure 1a displays the size distribution (intensity versus hydrodynamic diameter) of the niosome particles at 25 °C as obtained from DLS B

DOI: 10.1021/acs.jpcb.6b02168 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry B at λabs ∼ 370 nm with rise of temperature. This suggests preferential stabilization of the cationic species of NHM over the neutral counterpart with increasing temperature within niosome. 3.3. Emission Spectroscopic Study. The emission profile of NHM in aqueous buffer (Figure 3a) is characterized by a

signatures for the cationic (λem ∼ 450 nm) and neutral (λem ∼ 380 nm) species of NHM. 3.3.1. Effect of NaCl. The cation ⇌ neutral prototropic equilibrium of NHM is found to be markedly modified with addition of NaCl to the niosome-encapsulated state, resulting in prominent reduction of the cationic fluorescence intensity (λem ∼ 450 nm) with simultaneous enhancement of the neutral band intensity (λem ∼ 380 nm) (Figure 3c). This finding evinces that the excited-state proton transfer reaction of NHM is increasingly favored toward the neutral species with increasing ionic strength of the medium (addition of NaCl).28−30 This observation bears reasonable resemblance of the photophysical properties of NHM to that in a varying composition of 1,4dioxane/water solvent mixture (Figure S1 of the SI), which shows that a gradual decrease of medium polarity (increasing volume percentage of 1,4-dioxane in the solvent mixture) accompanies a progressive reduction of emission intensity of the cationic band (λem ∼ 450 nm) coupled with the enhancement of the neutral band intensity (λem ∼ 380 nm) (Figure S1 of the SI). This provides a clear signature for increasing the degree of stabilization of the neutral species of NHM over the cationic species with decreasing medium polarity.28−30 Thus, a rational resemblance of the fluorescence spectral properties of NHM within niosome dictates that the drug molecules are encapsulated within a more hydrophobic environment inside niosome compared to that in aqueous buffer. Niosome is composed of cholesterol and the nonionic surfactant TX100. Here, cholesterol is strongly hydrophobic because of the possession of hydrocarbon rings and a sole hydroxyl moiety (Scheme S1 of the SI).26 The surfactant (TX100) comprising a hydrophobic aromatic nucleus, a single hydroxyl moiety, and ether oxygen atoms (Scheme S1 of the SI) is also considerably hydrophobic.26 This argument underpins the notion that the considerably hydrophobic interior of niosome will result in depletion of the number of water molecules surrounding the bound probe as compared with that in the bulk aqueous phase.26,31 This rationale can be invoked to substantiate the observation stated above (Figure 3 a and b), that is, favorable stabilization of the neutral species of NHM over the cationic counterpart within niosome in comparison to that in bulk water. Drawing on this, it can be comprehensively argued that, following addition of the strong electrolyte (NaCl), the water molecules within niosome will experience strong electrostatic attraction by Na+ and Cl−, leading to hydration of the ions with consequent decrease of the number of relatively free water molecules within the niosome structure. Such further lowering of the number of available water molecules in the presence of NaCl should accompany further reduction of micropolarity at the drug binding site within niosome. Thus, the preferential stabilization (destabilization) of the neutral (cationic) species of NHM with added NaCl in the presence of niosome (Figure 3c) can be understandably rationalized on the basis of the above discussion.26,31 Recently, this line of interpretations has been explicitly applied by Bhattacharyya et al.26 in a study of intermolecular proton transfer in niosome. Figure 3d depicts the variation of the steady-state fluorescence anisotropy (r) of NHM with added NaCl in the presence of niosome. First, the higher anisotropy value of NHM entrapped in niosome (∼0.051) compared to that in aqueous buffer (∼0.002)28 signifies the imposition of motional restriction on the drug molecules inside niosome compared to that in aqueous buffer. As exemplified by Figure 3d, an increase

Figure 3. Resolved emission spectra (λex = 340 nm) of NHM (∼2.0 μM) in (a) aqueous buffer and (b) niosome. The solid black lines denote the experimental spectra, the blue dashed lines designate the resolved bands into the individual Gaussian components, and the red dotted lines denote the simulated spectra based on the resolved bands. (c) Emission spectra (λex = 340 nm) of niosome-bound NHM (∼2.0 μM) in the presence of various concentrations of NaCl. Curves 1 to 6 represent 0, 1, 2, 3, 4, 5, and 6 M NaCl, respectively. (d) Variation of steady-state fluorescence anisotropy (r) of niosome-encapsulated NHM as a function of added NaCl concentration. Each data point is an average of 20 individual measurements. The solid line is only for a visual guide to the pattern of variation.

broad, structureless band at λem ∼ 450 nm, which is attributed to the cationic species of NHM in analogy to reported literature.10,28−30 The remarkable modulation of the emission profile of NHM following interaction with niosome is illustrated in Figure 3b, which reflects significant quenching of the cationic fluorescence intensity coupled with a slight blueshift (∼4 nm) and a concomitant appearance of a new band at λem ∼ 380 nm. Based on the well-established literature reports, the emission band at λem ∼ 380 nm is ascribed to the neutral species of NHM.10,28−30 These observations point out a considerable lowering of micropolarity in the immediate vicinity of NHM within the niosome-bound state as compared with the bulk aqueous buffer phase. This is reflected in the relative stabilization of the neutral species of NHM (λem ∼ 380 nm) over the cationic counterpart (λem ∼ 450 nm) and a slight blue-shift of the cationic emission wavelength (Figure 3 a and b). The emission spectra of NHM in aqueous buffer and niosome environments have been deconvoluted into individual Gaussian curves (parts a and b of Figure 3) in order to enable a distinct visual perusal of the emission band features. The deconvoluted profile exhibits the presence of only one band centered at ∼450 nm in aqueous buffer, thereby confirming the presence of the cationic species alone. In the presence of niosome, however, the resolved band features reveal clear C

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The Journal of Physical Chemistry B in the fluorescence anisotropy of NHM with increasing NaCl concentration indicates that the drug molecules experience an increasing degree of rigidity with increasing concentration of NaCl, and hence aids further support to our spectroscopic results. 3.3.2. Effect of Temperature. Figure 4a represents the effect of increasing temperature on the prototropic transformation of

leads to the observation of differential ultraslow components of solvation dynamics in niosome.33 However, it is worthwhile to note that the overall solvent relaxation dynamics in such a complex microheterogeneous assembly may be governed by several factors, including the energy of the hydrogen bonds, the orientational dynamics, the dipole moments of the constituent components, and so forth.33,38,39 Considering such enormous complexities regarding the heterogeneity within the niosome structure, it is argued that hydrated oxyehtylene chains are present in a relatively large amount in the proximity of the niosome-encapsulated dye, and it is the cumulative relaxation dynamics of the microenvironment of the bound dye which gives rise to such complicated ultraslow dynamics of hydration in niosome.33 Thus, it may appear logical to think that the hydration structure surrounding the drug within the niosome microheterogeneous environment will be significantly modulated by extrinsic factors (such as addition of salt or rise of temperature), which in turn gets reflected through the environment-sensitive photophysical properties of the embedded drug NHM. With added salt (NaCl, a strong electrolyte), the number of available water molecules in the vicinity of the bound drug molecules is effectively reduced following strong electrostatic attraction of water molecules by the Na+ and Cl− ions, thus leading to preferential stabilization of the neutral species of NHM over the cationic counterpart. On the contrary, the results are typically reversed with increasing temperature, which accompanies a greater degree of penetration of water molecules to the hydration layer of niosome and hence an effective enhancement of micropolarity surrounding the drug environment embedded inside niosome, which leads to a preferential stabilization of the cationic species of NHM (this aspect has been further elaborated in a forthcoming section relating to estimation of the relative polarity of the niosomeencapsulated drug microenvironment; Section 3.4, Figure 6). However, it must be remembered that a quantitative correlation and one-to-one mapping of the present findings with the results of solvation dynamics reported in the literature33 might not be apt in this context, given the markedly different chemical structures of the dyes involved in the individual investigations (it is also noteworthy that the solvation (or hydration) dynamics within an organized assembly is usually explored in terms of the dynamics of solvent (or water) relaxation as sensed by the bound dye molecules in their close proximity). Thus, an attempt toward quantitative correlation of results involving structurally different dye molecules could be misleading24,38,39 and hence avoided herein. 3.3.3. Effect of βCD on Niosome-Bound NHM. The absorption profile of niosome-bound NHM upon interaction with βCD does not reflect the proper interaction scenario due to extensive broadening of the spectral profiles in the complex microheterogeneous environment (figure not given).23,40 However, the emission profile of niosome-bound NHM displays more dramatic modification due to addition of βCD. As seen in Figure 5a, the addition of βCD to niosome-bound NHM leads to gradual decrease of the neutral emission intensity (λem ∼ 380 nm) together with a regular intensity enhancement of the cationic emission (λem ∼ 450 nm) coupled with a red-shift of ∼4 nm. Thus, the βCD-induced spectral changes of the photophysical properties of niosome-encapsulated NHM occur in a qualitatively reverse pattern with respect to those noted in the course of NHM−niosome interaction (Section 3.3). At the outset, these results point out an apparent increase of micropolarity in the immediate vicinity of NHM

Figure 4. Modulation of emission spectra (λex = 340 nm) of niosomebound NHM at various temperatures ([NHM] ∼ 2.0 μM). Curves 1 to 6 represent 10, 20, 30, 40, 50, and 60 °C temperature. (b) Variation of the steady-state fluorescence anisotropy (r) of niosome-bound NHM with rising temperature. Each data point is an average of 20 individual measurements. The solid line is only for a visual guide to the pattern of variation.

niosome-bound NHM: progressive enhancement of the cationic fluorescence intensity (λ em ∼ 450 nm) with concomitant reduction of the neutral band (λem ∼ 380 nm). Driven by the discussion in the previous section, this result can be argued to reflect the increase of polarity at the drug binding site within the niosome microheterogeneous environment with rise of temperature. A greater degree of penetration of water molecules to the hydration layer of niosome with increasing temperature (and hence the accompanying reduction in rigidity around the microenvironment of bound NHM) may be invoked to rationalize the findings.31−35 Figure 4b shows the variation of the steady-state fluorescence anisotropy (r) of NHM in niosome as a function of temperature. The decrease of fluorescence anisotropy provides the signature of the lowering of the motional constraint of NHM within niosome with increasing temperature. Recently, it has been argued that the multilamellar vesicle structure of niosome is comprised of a thick oxyethylene−water shell in its bilayer headgroup region.33 It is also reported that the poly(ethylene oxide) (PEO) moiety exhibits a constrained icelike structure consisting of a highly dense network of water molecules36,37 which are bound to the PEO groups in the niosome headgroup region and hence believed to be responsible for slow hydration dynamics (the dynamics of solvation within niosome has been shown to be 3 orders of magnitude slower compared to that in bulk water using a coumarin probe).33 The water molecules in the bilayer headgroup region are believed to be hydrogen bonded, giving rise to the slow hydration dynamics.33 However, the layered structure of niosome is intrinsically very complex, and the PEO moieties in the headgroup are capable of creating a clustering of water molecules which could remain multiply hydrogen bonded among themselves (accounting for the close packed compact structure), whereas the reduced rigidity of PEO moieties toward the extremes leads to the absence of such clustering and hence a considerable lowering of the density of hydrogen bonded water.33 Such varied rigidity (firmness) of the water layer within the microheterogeneous niosome environment D

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450 nm) species of NHM with solvent polarity (on ET(30) scale42) has been processed to construct a calibration curve. The equivalent parameter FNeutral/FCationic is then calculated for the modulations of the emission properties of niosome-bound NHM under various experimental conditions under investigation, e.g., (i) in the presence of the added salt, NaCl, (ii) at different temperatures, and (iii) in the presence of added βCD, followed by interpolation of the calibration line to extract the micropolarity surrounding NHM. As seen from Figure 6, the Figure 5. (a) Variation in emission spectral profiles of niosome-bound NHM (λex = 340 nm, [NHM] ∼ 2.0 μM) with incremental addition of βCD. Curves 1 to 7 represent [βCD] = 0, 0.15, 0.23, 0.3, 0.4, 0.6, and 0.8 mM. (b) Variation of steady-state fluorescence anisotropy of niosome-bound NHM with added βCD. Each data point is an average of 20 individual measurements. The solid line is only for a visual guide to the pattern of variation.

following addition of βCD. These results appear to open up two possible windows: (i) A substantially strong NHM-βCD interaction leading to scavenging of the niosome-bound drug by βCD (ii) Interaction of βCD with niosome, leading to the rupture of the structural integrity of the latter, resulting in liberation of the niosome-entrapped NHM molecules to the bulk. Therefore, the NHM-βCD interaction has been explored through modulation of the absorption and emission properties of NHM with addition of βCD in an aqueous buffer medium. No discernible change in the photophysical properties of NHM upon addition of βCD (figure not shown) within the range of concentration used for the following experiments consequently negates significant interaction of NHM with βCD.23,40 This finding provides a crucial diagnostic basis for the assignment of the spectral modulation of niosome-bound NHM upon interaction with βCD to have originated from the interaction between βCD and niosome only. Cumulatively, it can be argued that βCD disrupts the structure of niosome by host:guest interaction with the constituent components (TX100 and cholesterol) of niosome, leading to release of NHM to the aqueous buffer phase.23,40 The lowering of fluorescence anisotropy (Figure 5b) of niosome-bound drug following interaction with βCD corroborates the aforementioned inference in the sense that βCD-induced release of the niosome-entrapped drug should accompany release of the motional constraint imposed on the drug molecules within the niosome. A further elaborate characterization of the niosome:βCD interaction has been undertaken by isothermal titration calorimetry (ITC) and discussed in a forthcoming section. 3.4. Polarity of the NHM Microenvironment. The polarity of the microenvironment in the immediate vicinity around NHM within niosome under various environmental conditions has been elucidated here. For this purpose, an analysis has been undertaken by comparing the emission characteristics of NHM inside niosome with the emission spectral properties of NHM in varying compositions of the 1,4dioxane/water solvent mixture.28,29,41 The modulation of the fluorescence spectra of NHM in various compositions of 1,4dioxane/water solvent mixtures of known polarity has been discussed in Section 3.3.1 and in Figure S1 in the SI. The ratiometric variation of the fluorescence intensities of the neutral (FNeutral at λem = 380 nm) and cationic (FCation at λem =

Figure 6. Micropolarity values of NHM in various environments (in niosome at 10 °C, red circle; in niosome at 25 °C, blue open triangle; in niosome at 60 °C, magenta open square; in niosome with added 5 M NaCl, purple filled triangle; in niosome with added βCD, green open triangle). log(FCation/FNeutral) versus ET(30) (in kcal mol−1) constitutes the calibration curve. FCation and FNeutral denote the fluorescence intensities of the cationic (λem = 450 nm) and neutral (λem = 380 nm) species of NHM, respectively, in the reference 1,4dioxane−water solvent mixture.

estimated micropolarity values of NHM in niosome undergo a reduction in polarity (ET(30) = 51.76 kcal mol−1) compared to that in aqueous buffer (ET(30) = 63.1 kcal mol−1),42 thereby substantiating the inference that NHM senses a reduced polarity inside niosome compared to that in bulk aqueous buffer. The enhanced polarity of the microenvironment surrounding NHM within the niosome-encapsulated state with rising temperature is also aptly rationalized from this analysis, e.g., ET(30) = 51.36 kcal mol−1 at 10 °C versus 53.5 kcal mol−1 at 60 °C. Further, the lowering of polarity around NHM with added NaCl is also categorically established (ET(30) = 49.36 kcal mol−1 in the presence of 5 M NaCl). The enhanced polarity of the NHM binding site within niosome in the presence of βCD is also demonstrated from this analysis; ET(30) = 54.5 kcal mol−1 in the presence of 0.85 mM βCD (Figure 6). The perturbation of the integrated niosome structure upon addition of βCD, leading to release of the bound drug to more polar aqueous medium, can be argued to tenably account for this observation. 3.5. Time-Resolved Fluorescence Decay. The timeresolved fluorescence decay transients of NHM in niosome with incremental addition of the strong electrolytic salt (NaCl), rising temperature, and increasing βCD concentration are displayed in Figure 7. In aqueous buffer, NHM exhibits a single exponential decay (λex = 375 nm, λem = 450 nm), having the characteristic lifetime (τ = 21.62 ns) of the cationic species of NHM (Figure 7a).28−30,41 The fluorescence decay of the cationic species of NHM has been collected at λem = 450 nm following photoexcitation at λex = 375 nm, which in turn provides a commensurate platform for direct comparison of the time-resolved data with those obtained in steady-state measurements (λabs ∼ 375 nm and λem ∼ 450 nm for the cationic species of NHM; Figures 2 and 3).28−30,41 The data collected in E

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(τC2 ), strongly corroborating to the steady-state spectral results in the sense that increasing concentration of NaCl accompanies progressive destabilization of the cationic species of NHM within the niosome-encapsulated state. Extending on this, the time-resolved fluorescence decay characteristics of the neutral species of NHM has been explored following photoexcitation at λex = 340 nm and recording the decay at λem = 380 nm (Figure 7b inset). The decay parameters summarized in Table S1 unveil a progressive increase of amplitude (αN2 ) of the characteristic lifetime of the neutral species (τN2 ) with increasing NaCl concentration. Collectively, these observations thus signify the stabilization of the cation ⇌ neutral prototrophic equilibrium of NHM within niosome more toward the neutral species over the cationic counterpart with added NaCl. 3.5.2. Effect of Temperature. The temperature-induced modulation of the fluorescence decay behavior of niosomebound NHM has been explored following similar experimental protocols as described above. Figure 7c represents the fluorescence decay of niosome-encapsulated NHM with rising temperature following photoexcitation at λex = 375 nm and data collection at λem = 450 nm, that is, addressing the fluorescence decay properties typical of the cationic species of NHM. A noticeable increase of the relative amplitude (αC2 ) of the characteristic cationic lifetime (τC2 ) of NHM (Table S2 of the SI) demonstrates a greater degree of stabilization of the cationic species of NHM over the neutral species with rise of temperature. This argument appears to be strongly reinforced by observing the fluorescence decay behavior of the neutral species of NHM within the experimental window of λex = 340 nm and λem = 380 nm (Figure 7c inset). The collected data in Table S2 manifest a regular depletion of the amplitude (αN2 ) of the neutral fluorescence lifetime (τN2 ) of NHM with increasing temperature. Thus, in excellent harmony with the steady-state spectral results (Figure 4), it can be comprehended that the cation ⇌ neutral prototrophic transformation of NHM is preferably favored toward the cationic species over the neutral counterpart, thereby reflecting an enhanced polarity of the NHM microenvironment with rise of temperature (Section 3.3.2). 3.5.3. Effect of βCD. The modulations in the time-resolved fluorescence decay transients of niosome-bound NHM with addition of βCD (λex = 340 nm and λem = 380 nm) are displayed in Figure 7d. The data summarized in Table S3 of the SI reveal significant reduction of the amplitude (αN2 ) of fluorescence decay corresponding to the neutral species (τN2 ) with added βCD. This finding is readily understandable on the basis of the release of niosome-bound NHM to the aqueous buffer phase following disruption of the niosome structure upon interaction with βCD, which in turn should accompany the increase of polarity of the microenvironment surrounding NHM, accounting for a greater degree of stabilization of the cationic species compared to the neutral species. This argument is adequately supported by the observation of the gradually increasing amplitude (αC2 ) of the cationic fluorescence lifetime (τC2 ) of NHM following addition of βCD (Table S3 of the SI). The present findings are found to be in reasonable accord with previous reports based on the interaction of fluorophores with various complex supramolecular assemblies.28−30,41,43 In the absence of any substantial interaction of NHM with βCD (within the concentration region under investigation), the aforesaid observations can be rationalized on the basis of disruption of the niosome architecture due to interaction with

Figure 7. Time-resolved fluorescence decay transients of NHM (∼2.0 μM) (a) in aqueous buffer (λex = 375 nm, λem = 450 nm) and in 1,4dioxane (λex = 340 nm, λem = 380 nm, inset); (b) in niosome in the presence of varying concentrations of NaCl: curves 1 to 6 represent 0, 1, 2, 3, 4, and 5 M NaCl (λex = 375 nm, λem = 450 nm); (c) in niosome with rising temperature: curves 1 to 6 represent 10, 20, 30, 40, 50, and 60 °C (λex = 375 nm, λem = 450 nm); (d) in niosome with added βCD: curves 1 to 5 represent 0, 0.23, 0.38, 0.6, and 0.85 mM βCD (λex = 375 nm, λem = 450 nm). The insets of parts b, c, and d represent the fluorescence decay profiles of niosome-bound NHM with λex = 340 nm and λem = 380 nm under the experimental conditions as specified above. IRF: Instrument response function.

Table S1 of the SI show the intrinsically complex fluorescence decay behavior of NHM in niosome such that a complicated biexponential function is required to properly fit the data. Though not uncommon, the indigenous complexities associated with various microheterogeneous environments render the assignment of the explicit mechanistic model to individual decay components in such a multiexponential decay function a nontrivial task.28−30,41 Nevertheless, we have made deliberate attempts to rationalize the dynamical behavior of NHM in niosome. The fluorescence decay behavior of the neutral species of NHM has been explored by photoexcitation of the sample at λex = 340 nm and monitoring the decay at λem = 380 nm (Figure 7a inset). The data assembled in Table S1 reveal a biexponential decay pattern comprising a fast (τ1) and a longer (τ2) decay component. The longer decay constant (τ2) is attributed to the neutral species of NHM (as characterized by the fluorescence lifetime of the neutral species (τ ∼ 3.08 ns)) in pure 1,4dioxane, in which only the neutral species exists (Figure S1).28−30,41 At the moment, the origin of the ultrafast decay component (τ1 in Table S1) is not precisely understood; however, in tune with recent reports,28−31,41 the asymmetric charge distribution over the molecular framework of NHM coupled with complex or/and unequal hydration within the microheterogeneous environments may be argued to contribute to this effect. 3.5.1. Effect of NaCl. The time-resolved fluorescence decay behavior of the cationic species of niosome-bound NHM with incremental addition of NaCl has been monitored at λex = 375 nm and λem = 450 nm (Figure 7b, Table S1). The data collected in Table S1 show a regular decrease of the amplitude (αC2 ) corresponding to the cationic fluorescence lifetime of NHM F

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The Journal of Physical Chemistry B βCD, resulting in release of the niosome-bound drug to aqueous buffer. 3.6. Rotational Relaxation Dynamics. The time-resolved fluorescence depolarization profiles of NHM under various experimental conditions are illustrated in Figure 8, with the

S4). This can be aptly described based on the rationale of disruption of the integrated niosome structure upon interaction with βCD, resulting in release of the bound drug molecules, which strongly corroborates other experimental findings stated earlier. It is imperative to note in this context that the rotational correlation time of niosome-bound NHM progressively decreases with rising temperature (Figure 8c, Table S4), an observation which is in excellent parity with the pattern of variation of steady-state fluorescence anisotropy (Section 3.3, Figure 4). The postulate of enhanced water penetration to the hydration layer of niosome with rising temperature leading to reduced rigidity surrounding the bound drug is thus further corroborated from time-resolved fluorescence anisotropy results.26,31−35 Again the lack of residual anisotropy (Figure 8c) is justified from the ultrafast rotational correlation time (Table S4), indicating completion of rotational relaxation within the excited-state lifetime of NHM in the concerned microenvironment. However, the anisotropy decay profile of niosome-bound NHM is found to be remarkably modified with addition of salt (Figure 8d), which reveals a biexponential decay behavior comprising an ultrafast and a slow component (Table S4). A marked increase of the average (mean) rotational correlation time (⟨τr⟩ = 2.72 ns, Table S4) manifests the enhanced degree of motional constraints on the niosome-bound drug molecules in the presence of the salt (5 M NaCl). In consensus with numerous literature reports,24,29,44,45 the biexponential anisotropy decay can be adequately analyzed on the basis of the wobbling-in-cone model,44 which postulates the overall anisotropy decay of the drug to have emanated from a complex interplay of three independent motions, namely,24,29,44,45 (i) wobbling of the fluorophore (rw(t)), characterized by a time constant τw, (ii) translational motion of the fluorophore (rD(t)) along the surface of the macromolecular niosome framework characterized by a time constant τD, and (iii) overall rotation of niosome (rn(t)), characterized by a time constant τn. According to the model, the wobbling time constant of NHM in the concerned microheterogeneous environment has been estimated as, τw = 149 ps (Table S4). A generalized order parameter of S = 0.94 reinforces the inference of the restricted motional behavior of NHM. The order parameter provides a physical measure of the degree of spatial restriction on the bound drug molecules within the limit of S = 0 for unrestricted motion and S = 1 for completely restricted motion. This is further corroborated from the small magnitude of the semicone angle, θ = 16.4° (Table S4). According to the model, the fast rotational relaxation component is described as a restricted rotor undergoing orientational diffusion within a potential cone characterized by semiangle θ, however, the rotational diffusion of the cone itself occurs on a slower time scale. The details of the calculations are given in the SI. The wobbling diffusion coefficient is calculated to be Dw = 5.27 × 1011 s−1 (Table S4). 3.7. Thermodynamics of the Interactions: Isothermal Titration Calorimetry. The energetics of the interaction of the drug (NHM) with niosome has been evaluated by isothermal titration calorimetry (ITC) to achieve an in-depth understanding of the molecular forces governing the interaction. The raw data depicting the heat burst curves (appropriately baseline corrected) for titration of niosome

Figure 8. Time-resolved fluorescence depolarization profiles of NHM (∼2.0 μM) under various experimental conditions (λex = 375 nm, λem = 450 nm), e.g., (a) within the niosome-bound state, turquoise open circle (inset: in aqueous buffer, orange open triangle); (b) with addition of 0.85 mM βCD in niosome-bound NHM (magenta open circle); (c) with variation of temperature for niosome-bound NHM (10 °C, green open circle; 50 °C, red filled circle); and (d) with added salt (5 M NaCl) in niosome-bound NHM (purple open circle).

relevant decay parameters being summarized in Table S4 of the SI. The time-resolved anisotropy decay function is expressed as r (t ) =

∑ r0αri exp(−t /τri) i

(1)

where r0 = ∑ir0i describes the inherent depolarization of the fluorophore (also referred to as the fundamental or limiting anisotropy of the fluorophore), and has been defined as the fractional anisotropies that decay with correlation times τri,24 and where αri designates the amplitude of the ith rotationalcorrelation time, τri. That the fluorescence depolarization of NHM within the presently employed experimental window starts below 0.4 (Figure 8) provides a manifestation of the fact that the anisotropy decay consists of ultrafast components in the initial time regime (immediately following photoexcitation) which are not resolved by our instrument.24,28 NHM displays a monoexponential anisotropy decay having a rotational correlation time of τr = 132 ps in aqueous buffer, which is significantly shorter than the excited-state lifetime of NHM (τ = 21.62 ns28). This follows that the fluorescence depolarization of NHM is essentially completed within the excited-state lifetime of NHM, as is further exemplified by the lack of noticeable residual anisotropy (Figure 8a inset). The enhancement of the rotational correlation time of NHM with added niosome (e.g., τr = 340 ps at 25 °C, Table S4) suggests the imposition of motional restrictions on the drug molecules within the niosome-encapsulated state. However, following addition of βCD to niosome-bound NHM, the rotational correlation time is found to be significantly lowered (τr = 235 ps at 25 °C with addition of 0.85 mM βCD, Figure 8b, Table G

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The Journal of Physical Chemistry B

Figure 9. (a) Top: Primary heat burst curves (appropriately corrected for heat of dilution) for isothermal calorimetric titration of niosome with βCD at 10 °C. Bottom: ITC enthalpograms obtained at various temperatures (10 °C, green filled circles; 20 °C, blue filled squares; 25 °C, red open circles). The solid lines designate the best fit lines to the raw data according to a one set of sites binding model. According to the power sign convention of the software associated with our ITC instrument, “upward”/“downward” heat bursts are used to represent an exothermic/endothermic process. The actual thermodynamic signature of a given process is, however, deduced from the sign of enthalpy change (ΔH) obtained from deconvolution of the experimental data (see also Experimental Section). (b) Plot of variation of enthalpy change (ΔH in kJ mol−1) with temperature. The correlation coefficient for the linear fit to the experimental data points is r2 = 0.99.

Table 1. Thermodynamics of (i) NHM−Niosome Interaction and (ii) βCD−Niosome Interaction as Obtained from ITC Measurementsa

a

Temp (°C)

ΔH (kJ mol−1)

25

−5.02 ± 0.2

10 15 20 25

−54.04 ± 2.0 −45.51 ± 1.8 −41.8 ± 1.7 −36.09 ± 1.5

TΔS (kJ mol−1)

ΔG (kJ mol−1)

(i) NHM−Niosome Interaction 30.99 ± 1.5 −36.01 (ii) βCD−Niosome Interaction −32.74 ± 2.0 −21.29 −23.19 ± 1.4 −22.32 −17.24 ± 1.2 −24.56 −10.92 ± 1.0 −25.17

n

Ka (M−1)

0.047 ± 0.01

(2.04 ± 0.08) × 106

0.82 0.97 1.05 1.23

± ± ± ±

0.1 0.1 0.1 0.1

(0.68 (1.11 (2.39 (2.58

± ± ± ±

0.08) 0.08) 0.08) 0.08)

× × × ×

104 104 104 104

The error bars are deduced from four individual measurements.

with NHM at 25 °C are displayed in Figure S2 of the SI. The NHM−niosome interaction is found to be characterized by a strong affinity constant, Ka = (2.04 ± 0.08) × 106 M−1. The overall thermodynamics of the interaction phenomenon is found to be governed by a favorable exothermic enthalpic contribution (ΔH = −5.02 ± 0.2 kJ mol−1) coupled with a strongly positive entropic contribution (TΔS = 30.99 ± 1.24 kJ mol−1), leading to a favorable negative free energy change (ΔG = −36.01 kJ mol−1) dictating the spontaneity of the process (ΔG < 0).46−48 Thus, it is intriguing to note that the free energy change (ΔG) of the process is constituted of an instrumental entropic contribution (strongly positive TΔS) in comparison to an enthalpic contribution. Such an entropydriven thermodynamic signature for the interaction is conventionally described in terms of the release of solvating water molecules to the bulk aqueous phase following the formation of the niosome:NHM complex.48−50 The energetics of the interaction of niosome with added βCD has also been studied by ITC techniques. To this end, an elaborate analysis of the thermodynamics of the interaction has been undertaken at various temperatures. A representative plot of primary heat change data for the niosome:βCD interaction is depicted in Figure 9a, and the as-obtained thermodynamic parameters are summarized in Table 1 (according to the power sign convention of the software associated with our ITC instrument, “upward”/“downward” heat bursts are used to represent an exothermic/endothermic process.25,49,51 The actual thermodynamic signature of a given process is, however, deduced from the sign of the enthalpy change (ΔH) obtained from deconvolution of the experimental data25,49,51). The

concerned interaction process is found to be characterized by favorable enthalpic (ΔH < 0, exothermic) and unfavorable entropic (TΔS< 0) contributions. A net negative free energy change (ΔG < 0) dictates the thermodynamic feasibility of the host:guest supramolecular interaction between niosome and βCD (Table 1). In this context, it is important to note that the niosome:βCD interaction becomes increasingly thermodynamically favored (as exemplified by increasingly negative ΔG, Table 1) with rise of temperature. Concurrently, the affinity constant (Ka) of the interaction also varies proportionately with temperature (Table 1). However, the exothermicity of the process decreases (ΔH becomes gradually less negative, Table 1) with rising temperature. These results evidently unravel the marked dependence of the thermodynamic parameters on temperature and point out the intrinsically complex nature of the molecular forces underlying the overall interaction process. The variation of ΔH as a function of temperature has been exploited according to the standard thermodynamic relationship δ(ΔH) = ΔCp·δT, for the determination of the constant pressure heat capacity change (ΔCp) of the process, which reveals a not-so-common observation of a positive ΔCp = 1.15 kJ mol−1 (Figure 9b). This can be rationally perceived on the basis of burial of polar molecular interfaces following complexation.48,52−57 Typically, it is believed that hydrophobic interaction arises from the large Gibbs energy cost associated with exposure of nonpolar functional moieties to an aqueous medium (that is, solvation of nonpolar groups in water, often designated as hydrophobic hydration).48,52−57 A positive heat capacity change is often regarded as the hallmark of hydrophobic hydration.48,52−57 H

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The Journal of Physical Chemistry B 3.8. Fluorescence Correlation Spectroscopy. In order to gain better insight regarding the microheterogeneity of the niosome membrane system, FCS measurements have been performed which yield valuable information regarding the translational diffusion of a suitable fluorophore inside a small observation volume. Herein, we have compared the translational diffusion coefficients (Dt) of the fluorescent dye Rh6G encapsulated within the niosome membrane under various experimental conditions to that in the bulk water (it could be pertinent to state here that NHM is not a suitable dye for FCS studies within the present experimental window; thus, another dye, namely Rh6G, was used for FCS measurements). The representative FCS traces for Rh6G under different experimental conditions under investigation are displayed in Figure 10, with the relevant data being summarized in Table 2.

hydration structure and compactness within niosome, as manifested through the reduced diffusion coefficient of the dye (Table 2).26

4. SUMMARY The present work demonstrates the interesting modifications of the photophysical properties of a prospective biological photosensitizer NHM within the niosome membrane as a potential host system. Our steady-state and time-resolved spectroscopic results unveil that the photophysics of the drug within the microheterogeneous niosome environments can be effectively controlled by simple means of variation of temperature and extrinsic addition of salt; the focus being rather the contrasting effects of such extrinsic parameters in tuning the spectral properties of the niosome-bound drug. The model of water penetration to the hydration layer of niosome can be invoked to adequately rationalize these findings. Our results also show that βCD can be employed as an efficient host system for the release of the drug encapsulated within niosome. To this end, an extensive characterization of the thermodynamics of the niosome:βCD interaction unveiled an intriguing and not-so-common result in terms of a positive heat capacity change (ΔCp > 0) as the signature for the instrumental role of hydrophobic hydration underlying the interaction process. The variation of important thermodynamic parameters for the niosome:βCD interaction as a function of temperature also yielded critical insight into an in-depth understanding of the overall interaction.

■

Figure 10. Normalized autocorrelation traces of (i) Rh6G in water (green filled circles); (ii) Rh6G in niosome (purple open circles); (iii) Rh6G in niosome in the presence of 2 M NaCl (black open triangles); (iv) Rh6G in niosome in the presence of 3 M NaCl (green open squares); and (v) Rh6G in niosome in the presence of 0.85 mM βCD (inset: magenta open circles). The solid lines designate the respective fitted data.

* Supporting Information

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.6b02168. Schematic structures of NHM, cholesterol, and Triton X100; fluorescence spectra of NHM in 1,4-dioxane/water reference solvent mixture; ITC data for NHM−niosome interaction,; time-resolved fluorescence decay parameters and rotational relaxation dynamical parameters of NHM under various experimental conditions; details of calculation of the wobbling-in-cone rotational parameters; and detailed description of the experimental methods and instrumentation techniques (PDF)

Table 2. Diffusion Coefficients of Rh6G in Various Environments under Investigation at 25 °C

a

Environment

Dta (μm2 s−1)

Water Niosome Niosome + 2 M NaCl Niosome + 3 M NaCl Niosome + 0.85 mM βCD

414.72 25.92 19.73 16.93 36.57

ASSOCIATED CONTENT

S

■

±4%.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (SM); Tel.: +917556692323.

The translational diffusion coefficient of Rh6G in niosome is found to be remarkably lowered (∼16 times) compared to that in bulk water (Table 2). In the presence of 2 M NaCl, the diffusion constant of niosome-bound Rh6G is slightly smaller (∼1.3 times) than that in niosome alone, whereas with an increase of concentration of the added salt to 3 M a further lowering of Dt is noted (∼1.53 times compared to that in niosome alone, Table 2). However, with addition of βCD, the observation is found to be apparently reversed, such as ∼1.41 times enhancement of Dt of niosome-encapsulated Rh6G with addition of 0.85 mM βCD (Table 2). These findings are found to be in reasonable accord with the spectroscopic and thermodynamic results stated earlier. The βCD-induced rupture of the structural integrity of the niosome membrane may be invoked to rationalize the relative enhancement of the diffusion coefficient of the bound dye (Rh6G), whereas the penetration of Na+ and Cl− ions significantly alters the

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS Postdoctoral research fellowship from IISER Bhopal for B.K.P. and doctoral research fellowships from CSIR for N.G. and IISER Bhopal for R.M. are acknowledged. The Central Instrumentation Facility (CIF) of IISER Bhopal is thankfully acknowledged for providing access to the ITC, confocal laser scanning microscopy. and FCS measurements.

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