Unique Influence of Cholesterol on Modifying the Aggregation

Article pubs.acs.org/JPCB

Unique Influence of Cholesterol on Modifying the Aggregation Behavior of Surfactant Assemblies: Investigation of Photophysical and Dynamical Properties of 2,2′-Bipyridine-3,3′-diol, BP(OH)2 in Surfactant Micelles, and Surfactant/Cholesterol Forming Vesicles Surajit Ghosh, Jagannath Kuchlyan, Subhajit Roychowdhury, Debasis Banik, Niloy Kundu, Arpita Roy, and Nilmoni Sarkar* Department of Chemistry, Indian Institute of Technology, Kharagpur 721302, West Bengal, India S Supporting Information *

ABSTRACT: The binding and rotational properties of an excited-state intramolecular proton transfer (ESIPT) fluorophore, 2,2′-bipyridine-3,3′-diol, BP(OH)2 has been investigated in alkyltrimethylammonium bromide containing (CnTAB, n = 12, 14, and 16) micelles and alkyltrimethylammonium bromide/ cholesterol (CnTAB (n = 14 and 16)/cholesterol) forming vesicles using fluorescence-based spectroscopy techniques. The formation of thermodynamically stable unilamellar self-assemblies of alkyltrimethylammonium bromide/cholesterol are characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM) measurements. Individually, aqueous solutions of all these alkyltrimethylammonium bromide form micelles after certain surfactant concentration (critical micelle concentration, cmc) of surfactant, whereas cholesterol molecules are insoluble in water. But with the variation of the cholesterol-to-surfactant molar ratio (Q = [cholesterol]/[surfactant]), uniform distribution of vesicular aggregates in aqueous solution can be obtained. The micelle-to-vesicle transition of surfactant solution upon addition of cholesterol also influences the steady state emission profile, fluorescence lifetime, and rotational dynamics of BP(OH)2 molecule. The diketo tautomer of BP(OH)2 molecule gets stabilized as the concentration of surfactant increases in aqueous solution. Fluorescence lifetime and rotational time constant of the BP(OH)2 molecule are also influenced by the variation of alkyl chain length of surfactant molecule. The emission quantum yield (Φ) is also found to be sensitive with surfactant concentration, variation in chain length of surfactants, and it saturates after the cmc of surfactants. The rigid and restricted microenvironment of vesicle bilayer enhance the lifetime and also rotational relaxation of BP(OH)2 significantly. The rotational behavior of BP(OH)2 in surfactant/cholesterol self-assemblies is also explained by using analytical parameters related to time-resolved anisotropy following two-step process and wobbling in a cone models.

1. INTRODUCTION

The alternative of phospholipids in preparation of vesicles is the hydrated mixture of cationic and anionic surfactants, mixture of cholesterol, or other organic additives with surfactant molecules, etc.10−20 It is also reported that certain block copolymer, polypeptides are also able to organize into vesicular-like structures.21−24 In recent years, the nonionic surfactant-forming vesicle termed as niosome has been extensively used in drug delivery due to its low toxicity, biodegradability, and biocompatibility. Moreover, the basic advantages of niosomes over liposomes are its simple preparation techniques, better stability, low price of desired surfactants, etc. These unique properties make fascinating use of niosome in industrial, chemical, and biological applications.16,17,25,26 Recently, Ventosa et al.20,27 prepared a nanoscopic vesicle, composed of cholesterol and quaternary

Liposomes (vesicles) are mainly made with the phospholipids and one of the widely studied nano aggregates since the 1960s.1,2 In vesicles/liposomes, the aqueous volume is enclosed by one or more concentric lipid bilayer. It received a lot of attention from researchers due to their unique structural features, which enable the solubilization of hydrophobic molecules and hydrophilic molecules within their bilayer and polar core filled with water, respectively. Hence, liposomes are used as nano carrier for protection and delivery for different drug molecules.3−7 However, their potential use as a drug carrier is hindered due to lack of physical and chemical stability, degradation possibility by the hydrolysis of phospholipids, etc. Besides, phospholipids are also expensive, and it requires a rigorous procedure for preparation of vesicles that are not easy.8,9 Therefore, researchers are interested in preparing vesicles without using lipids and successfully they are able to substitute lipids by using different surfactant molecules.10−15 © 2014 American Chemical Society

Received: April 22, 2014 Revised: July 17, 2014 Published: July 18, 2014 9329

dx.doi.org/10.1021/jp503938b | J. Phys. Chem. B 2014, 118, 9329−9340

The Journal of Physical Chemistry B

Article

Scheme 1a

a

Single and double proton transfer of DE tautomer leads to the formation of monoketo tautomer (MK) and diketo tautomer (DK), respectively.

years, the interesting characteristics of ESIPT is utilized in sensing, laser dyes, photoswitches, and other electronic devices.45−50 The response to solvent polarity and the potential of hydrogen-bonding ability of these molecules help researchers to gain useful information regarding various confined environment and biomolecules.51−54 Therefore, the application of proton transfer molecules is also extended in analyzing conformation and binding-site polarity of protein molecules. Among various ESIPT compounds, 2,2′-bipyridine-3,3′-diol have some significant interest, owing to involvement of double proton transfer reactions.55 This unique feature is also utilized to serve BP(OH)2 as an example for DNA base pairs to examine the tautomerization phenomenon in DNA.56 Keeping this in mind, we are interested in investigating the photophysical and dynamical properties of 2,2′-bipyridine-3,3′diol, (BP(OH)2) in cationic surfactant micelles and surfactant/ cholesterol vesicles. BP(OH)2, a planar aromatic molecule, shows excited state intramolecular double proton transfer (ESIDPT) upon photoexcitation. This molecule undergoes ESIDPT reaction either in a concerted or in a stepwise mechanism, and three possible tautomers exist: dienol tautomer (DE), monoketo tautomer (MK), and diketo tautomer (DK). The single and double proton transfer of DE tautomer leads to the formation of monoketo tautomer (MK) and diketo tautomer (DK), respectively, as shown in Scheme 1.55 There are several experimental and theoretical investigations regarding the proton transfer process of BP(OH)2 molecules.55,57−62 In the stepwise reaction, the first step [i.e., formation of monoketo tautomer (MK*) in the excited state] is reported to be ultrafast (∼100 fs timescale or less). Then, second proton transfers from monoketo tautomer (MK*) results in formation of emissive diketo tautomer (DK*) in a ∼10 ps time regime. In accordance

surfactant, cetyl ammonium bromide (CTAB), and termed this system as quatsomes. They have also shown that these systems are stable for long periods and a rise in temperature or dilution do not change the morphology of these vesicles. Several reports are available regarding the photophysical and dynamical aspect of different probe molecules in surfactant forming vesicles. Bhattacharyya and co-workers28 have investigated proton transfer dynamics of pyranine in niosome with variation of NaCl concentration. Previously, we have also investigated the photophysical study of 1′-hydroxy-2′-acetonaphthone (HAN) and solvation dynamics study using coumarin 153 and coumarin 480 as probe molecules in PEG6000/Tween-80based niosome.29,30 The photophysical property and stability of curcumin is also investigated in Tween-20 and cholesterolforming niosomes. This result indicates that the nano assemblies of niosome are efficient compared to micelles to control intramolecular hydrogen bond mediated fast nonradiative processes of curcumin.16 Although, the binding or photophysical study of proton transfer chromophores in cationic surfactant and cholesterol containing vesicles (i.e., quatsomes) are rare in literature. Excited-state intramolecular proton transfer (ESIPT) phenomenon is a crucial photochemical reaction in the field of chemistry and molecular biology.31−34 In the ESIPT process, a hydroxyl or amino proton from the donor site is transferred to the acceptor site, such as a nitrogen atom or a carbonyl oxygen in the photoexcited state.35 Therefore, these chromophores possess a large Stokes shifted emission spectra of the tautomer compared to the normal fluorophores, which is the fingerprint of ESIPT phenomenon. Molecules exhibit ESIPT phenomenon that includes imidazoles, benzoxazoles, benzothiazoles, anthraquinones, flavones, pyranine, napthols, etc. and their derivatives.32,35−44 In recent 9330



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Scheme 2. Structures

with transient absorption and femtosecond fluorescence upconversion measurements, in concerted mechanism, the double proton transfer of BP(OH)2 occurs within an ∼100 fs timescale.63−65 Besides, absorption and fluorescence spectra of BP(OH)2 are sensitive in hydrophilic and hydrophobic microenvironments. In steady-state absorption spectra, it shows unique features (the band around 400 nm), which can be utilized as water sensors in surfactant assemblies as well as biological systems. Considering these unique features of the BP(OH)2 molecule, a growing interest has been observed in the study of the ESIDPT phenomenon in neat solvents, binary mixtures, and also in different restricted environments, like micelles, cyclodextrins, proteins, bile salt aggregates, etc.66−74 Due to high stability of organized assemblies and also its applicability as drug delivery vehicles, in the present study, effort has been made to get better insight on the binding and dynamics of BP(OH)2 molecules in two different microenvironments: various cationic surfactant micelles having different chain length and surfactant/cholesterol-forming vesicles. Although there have been few studies on the modulation of photophysical properties of a proton transfer molecule, BP(OH)2, in conventional micellar systems, no such studies have been performed in conventional surfactantforming vesicle aggregates.66,69 Fluorescence anisotropy studies designate the influence of cholesterol concentration on the hydrophobicity and microviscosity of quatsomes (i.e., vesicle bilayer).

2.2. Steady State and Time Resolved Measurements. The UV−visible absorption of BP(OH)2 molecules were recorded with a Shimadzu spectrophotometer, model no. UV2450. The fluorescence spectra were monitored by Hitachi (model no. F-7000) spectrofluorimeter using an excitation wavelength at 336 nm. The quantum yield of BP(OH)2 was calculated using the following equation75 and anthracene (λabs = 350 nm, ΦR = 0.27) in ethanol76 at 298 K as reference. ΦS A (Abs)R nS2 = S ΦR AR (Abs)S nR2

(1)

where the subscripts R and S signify the reference and sample, respectively. Abs, A, Φ, and n represent absorbance, the area under the fluorescence curve, quantum yield, and refractive index of the medium, respectively. The time-resolved fluorescence measurements were recorded using a time-correlated single photon counting (TCSPC) picosecond spectrophotometer. The experimental setup is depicted in a previous publication.77 Briefly, a picosecond diode (IBH, Nanoled, 336 nm) was used as the excitation source, and the emission decays were collected with magic-angle (54.7°) polarization, using a Hamamatsu MCP PMT (3809U). The instrument response function is ∼100 ps in our set up. The lifetime decays were analyzed using the following equation by IBH DAS-6 decay analysis software:

I (t ) =

2. EXPERIMENTAL SECTION 2.1. Materials and Solution Preparation. 2,2′-Bipyridine-3,3′-diol [BP(OH)2], dodecyltrimethylammonium bromide (DTAB), tetradecyltrimethylammonium bromide (TTAB), and hexadecyltrimethylammonium bromide (CTAB) were obtained from Sigma-Aldrich and used as received without further purification. Cholesterol was obtained from Sisco Research Laboratories Pvt. Ltd. (SRL), India. For preparation of micellar and vesicular solution, triply distilled Milli-Q water was used. The stock solution of BP(OH)2 was prepared in methanol. The stock solution of micelles (stirring with a magnetic stirrer) was prepared with an appropriate amount of surfactant in water in a volumetric flask and kept overnight for stabilization. The concentration of BP(OH)2 was maintained at ∼10−6 M. The structures of these materials are shown in Scheme 2.

∑ aie−t/τ

i

i

(2)

The average lifetime (τav) is determined using the following equation:75 ⟨τav⟩ =

∑ aiτi i

(3)

where ai and τi represent the pre-exponential factor and lifetime of the ith component, respectively. The anisotropy measurements were executed using the same TCSPC setup with a motorized polarizer on the emission side and a 375 nm laser as the excitation source. The emission decays for parallel I∥(t) and perpendicular I⊥(t) polarizations were collected alternatively by vertically polarized excitation light until a certain peak difference between I∥(t) and I⊥(t) decays were reached. The analysis of the anisotropy decays was 9331



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Figure 1. DLS intensity-size distribution histogram of supramolecular assemblies present in surfactant/cholesterol mixtures in water at different Q values. (a) CTAB/cholesterol and (b) TTAB/cholesterol.

Figure 2. TEM images of vesicles composed of (i) CTAB/cholesterol, (a and b) Q = 1.0 and (ii) TTAB/cholesterol, (c and d) Q = 1.0.

200 kV. An aqueous solution of uranyl acetate (0.1 wt %) was used as a staining agent. 2.5. Viscosity (η) Measurements. The bulk viscosities of TTAB/cholesterol and CTAB/cholesterol systems at different Q values were measured using a Brookfield DV-II+ Pro viscometer at 298 K. We prepared the solution of surfactant/ cholesterol systems of required Q values and kept it for one week. Then, 1 mL of each solution was used for the determination of bulk viscosity of the surfactant/cholesterol system.

performed using IBH DAS, version 6, and decay analysis software. 2.3. Preparation of Cholesterol/Surfactant Aggregates by Sonication. Initially, dust particles in Milli-Q water were removed by filtration through a syringe filter (0.2 μm). Calculated amount of cholesterol and surfactant (TTAB and CTAB) were weighted and dissolved in Milli-Q water in different glass bottles. The resulting solutions were sonicated at 298 K, using an ultrasonicator (processor SONOPROS PR-250 MP, Oscar Ultrasonics Pvt. Ltd.) with a titanium probe working at a frequency of ∼20 ± 3 kHz for 15 min. The solutions were kept at room temperature for 7 days to stabilize the systems. 2.4. Structural Characterization of Cholesterol/Surfactant Aggregates. Initially, dynamic light scattering (DLS) experiments were performed to get the estimation about the size distribution of surfactant and cholesterol nanoaggregates in aqueous solution using a Malvern Nano ZS instrument, employing a 4 mW He−Ne laser (λ = 632 nm) with a fixed detector angle at 173°. For surfactant micelles and vesicles, the dispersion medium was water. Therefore, in a DLS measurement, we have used water (refractive index = 1.33) as a dispersion medium, and the collected scattering intensity was analyzed by instrumental software to get the hydrodynamic diameter (dh) of particles. It is defined as

dh =

kBT 3πηD

3. RESULTS AND DISCUSSION 3.1. Structural Characterization of Cholesterol/Surfactant Vesicles. Morphology and size of cholesterol-containing surfactant vesicles mainly depend on the concentration of surfactant and the method of preparation. Therefore, it is necessary to check the morphology of vesicles for photophysical characterization of probe molecules. Ventosa et al.20,27 have characterized CTAB and cholesterol-containing vesicles using DLS, Cryo-TEM measurement, and also molecular simulation study at different Q values. Cholesterol is a hydrophobic molecule and insoluble in water, whereas CTAB forms micelles in water. Therefore, cholesterol molecules located in the hydrophobic core of the vesicle bilayer provide rigidity and stability to vesicle membranes. We have also performed DLS measurement of CTAB/cholesterol and TTAB/cholesterol mixtures at different Q values to distinguish the structural transformation from micelle to vesicle with increasing cholesterol concentration. The intensity−size distribution profiles of normal micelles and vesicles composed of CTAB and TTAB with increasing cholesterol concentration (different Q values) are shown in Figure 1 (panels a and b respectively), as observed from the DLS experiment at 298 K.

(4)

where kB, T, D, and η denote Boltzmann constant, temperature, diffusion coefficient, and viscosity, respectively. To confirm the vesicle formation, the transmission electron microscopy (TEM) experiment was executed for the morphological analysis by utilizing a JEOL model JEM 2010 transmission electron microscope with an operating voltage of 9332



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Figure 3. UV−vis absorption spectra of BP(OH)2 in aqueous solution with increasing concentration of (a) DTAB, (b) TTAB, and (c) CTAB.

water sensor in surfactant assemblies and in other biological systems, although no distinct difference in absorption spectra of BP(OH)2 molecules is observed with the variation of an alkyl chain of a surfactant molecule. The steady state emission spectra of BP(OH)2 at different concentration of aqueous surfactant solutions is monitored with an excitation source as 336 nm. The emission profiles of BP(OH)2 in the presence of surfactant are shown in Figure S1 of the Supporting Information. Upon gradual addition of DTAB, TTAB, and CTAB in the aqueous solution of BP(OH)2, significant enhancement in emission intensity with a gradual red shift in emission maxima is observed. This observation signifies the extent of partition of probe molecules into micellar aggregates. In water, the BP(OH)2 molecule shows maxima around ∼466 nm, whereas in 80 mM DTAB, TTAB, and CTAB solutions, the emission maxima is around ∼487, ∼489, and ∼491 nm, respectively. Therefore, it is noteworthy to mention that the alkyl chain length of surfactant influences the position of emission maxima. The extent of spectral shift enhances in increments of the hydrophobic part of the surfactant molecules and follows the order CTAB > TTAB > DTAB. This spectral shift in emission spectra of BP(OH)2 on encapsulation in the hydrophobic environment are in good agreement with previous literature.66,70 This large Stokesshifted emission spectra is owing to the double proton transferred DK tautomer. The change in intensity along with the spectral position of the emission spectra confirm that BP(OH)2 molecules experienced more hydrophobic environment in micelles compared to water. As the absorption and fluorescence spectra of BP(OH)2 are sensitive in hydrophilic and hydrophobic microenvironments, therefore, we can use it as an environmentally sensitive probe. We have also calculated the quantum yield (Φ) of BP(OH)2 at different concentrations of surfactants using eq 1 and depicted in Figure 4. Initially, the quantum yield value increases with increasing surfactant concentration and saturates at higher concentration. The saturation in Φ values appears at very low concentrations of CTAB and TTAB solutions, whereas it requires a higher concentration in the case of DTAB, as the critical micelle concentration (cmc) of DTAB is quite higher than CTAB or TTAB.80 As we mentioned earlier, with the addition of cholesterol in aqueous solution of surfactant, micelles to vesicles transition is observed. Therefore, it is necessary to investigate the spectral changes of BP(OH)2 molecules in the surfactant/cholesterol mixture. The fluorescence spectra of BP(OH)2 molecules in quatsomes at different Q values are shown in Figure 5. The gradual red shift in emission with increasing cholesterol content is indicating location of probe molecules in the hydrophobic bilayer region of vesicles. Very little shift in emission maxima is

The mean diameter of CTAB and TTAB micelles are in the range of ∼1 nm. Due to the presence of free surfactant molecules or larger aggregates, a minor population in larger diameter range is also observed in the intensity−size profile. The progressive incorporation of cholesterol to micellar solutions of CTAB and TTAB, an intense peak in the range of ∼100 nm is observed in the intensity−size distribution profile of DLS measurement. The narrow distribution profile in the DLS measurement further suggests an almost consistent dispersion of vesicles in the medium. At equimolar concentration of surfactant and cholesterol, the disperse system contains spherical and unilamellar vesicles. Therefore, to confirm the formation of spherical vesicles, we have executed TEM measurements at Q = 1.0 for the system, CTAB/cholesterol, and TTAB/cholesterol. The TEM micrographs of aqueous CTAB/cholesterol, and TTAB/cholesterol mixtures at Q = 1.0 have been depicted in Figure 2 (panels a−d, respectively). Aqueous uranyl acetate (0.1 wt %) is used as the staining agent to visualize the morphology of aggregates. The TEM images distinctly designate the formation of spherical vesicle with an average size of ∼100 nm. These TEM images of the vesicle also support the results that are obtained from DLS measurement studies. The micrographs also indicate that there are few vesicles present in the system of a size less than ∼100 nm. 3.2. Influence of Structure and Concentration of Surfactant and Surfactant/Cholesterol Mixtures. 3.2.1. Steady-State Absorption and Fluorescence Studies. The UV−vis spectra of BP(OH)2 in aqueous solution with an increase in concentration of surfactants (DTAB, TTAB, and CTAB) are shown in Figure 3. In water, the diketo tautomer of BP(OH)2 is stabilized and a specific absorption band in 400 nm region observed, whereas the dienol tautomer of BP(OH)2 absorbs at ∼340 nm.23,68,78,79 In accordance with the previous observation, an absorption band around ∼400−450 nm region found only in aqueous solution is due to the stabilization of the DK tautomer of BP(OH)2 in the ground state, and the absorbance peak around ∼340 nm signifies the transition between the lowest (π, π*) state. With the addition of surfactant to the aqueous solution of BP(OH)2, absorbance increases at ∼340 nm, while the reduction in optical density in the region of ∼400−450 nm is observed. An increase in absorbance around ∼340 nm is an indication of lowering the availability of water molecules toward the BP(OH)2 molecules encapsulated in micelles. The suppression of low energy absorption band at ∼400 nm with addition of a surfactant can also be assigned as the perturbation of hydrogen-bonded complexes between water and diketo tautomers of BP(OH)2 in the ground state upon confinement. Therefore, this unique absorption band can be utilized as the 9333



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BP(OH)2 in water are ∼0.58 ns, which is well-correlated with the literature report.66,79 But gradual addition of surfactant in aqueous solution leads to biexponential decay with a longer lifetime component (Table S1 of the Supporting Information). The fast component is due to intramolecular hydrogen-bonded DK tautomer that is stabilized by water molecules. The longer lifetime component is assigned to BP(OH)2 molecules encapsulated in micelles. The solubility of BP(OH)2 molecules is very less in water. Therefore, preferential incorporation into hydrophobic micellar aggregates restricts the water accessibility around the BP(OH)2 molecules. The enhancement of fluorescence lifetime of BP(OH)2 molecules is due to the hindrance of water-mediated nonradiative decay processes. Moreover, the gradual increment of relative contribution of the longer lifetime component is observed as the concentration of surfactant increases. But, at a higher concentration of surfactant, it becomes a single exponential with a longer lifetime component. These observations indicate that the emissive diketo tautomer of BP(OH)2 is stabilized in the presence of surfactant assemblies. At a particular concentration of surfactant, the longer lifetime component is greater in aqueous CTAB solution compared to DTAB and TTAB solutions. The variation of fluorescence decays of BP(OH)2 in 80 mM DTAB, TTAB, and CTAB solutions are shown in Figure 6. So, it is found that, the chain length of surfactant molecules can play an effective role in modifying the photophysical properties of probe molecules.

Figure 4. Variation of quantum yield of BP(OH)2 in aqueous solution with increasing surfactant concentration.

observed up to Q = 0.5, whereas at Q = 1.0, it shifts by ∼5−6 nm in the red region. The distribution of vesicles becomes uniform at higher cholesterol content, which induces a more hydrophobic and rigid environment surrounding the BP(OH)2 molecules. The excitation spectra in water, micelles, and vesicles are given in Figure S2 of the Supporting Information. The band around ∼400 nm is also absent in vesicles like micelles, which also suggests the incorporation of BP(OH)2 in vesicle bilayers. Although like micelles, no distinct difference in fluorescence spectra of BP(OH)2 molecules in TTAB/ cholesterol and CTAB/cholesterol solutions is observed. Therefore, it can be concluded that the microenvironment around probe molecules in both solutions are almost similar. Besides, vesicles can mimic various biological membranes, and BP(OH)2 can act as the model DNA base pair;56 so, in the future, detailed investigation can provide various structural information regarding the penetration of DNA in vesicle bilayer by monitoring the excitation and emission spectra of BP(OH)2, as it is sensitive to the water environment. 3.2.2. Time-Resolved Fluorescence Studies. The emission decays of BP(OH)2 in three surfactant solutions at different concentration and cholesterol-containing surfactant vesicles (at different Q values) are monitored at the corresponding peak maxima, using an excitation wavelength of 336 nm. This section can allow us to get better insight about the partition of BP(OH)2 molecules in hydrophobic micelles or vesicle bilayers. The overlay of emission decays of BP(OH)2 in different micellar solutions are represented in Figure S3 of the Supporting Information. In aqueous solution, the decay fits with a single exponential function. The time constants of

Figure 6. Variation fluorescence lifetimes of BP(OH)2 in 80 mM DTAB, TTAB, and CTAB micelles.

Figure 5. Steady state fluorescence spectra of BP(OH)2 in 30 mM (a) CTAB and (b) TTAB with an increase in cholesterol concentration. 9334



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Different physical properties, such as micellar size, critical micelle concentration (cmc), morphology of aggregates, aggregation number, and hydrophobicity can vary, depending on the chain length of surfactant molecules. The solvation process of water molecules also depends on the alkyl chain length of surfactant molecules as well as on the nature of the molecule. Solvation dynamics and reorientional measurement of probe molecules in different organized assemblies with variation of chain length and charge of surfactant molecule has been studied extensively by various groups.81−84 In our study, the increase in alkyl chain length of alkyl trimethylammonium bromide molecule without affecting the headgroup region also influenced the fluorescence lifetime of the probe molecule. Time-resolved fluorescence decays showed that the micropolarity experienced by BP(OH)2 in various micellar surfaces are also different. Since the ESIPT process of BP(OH)2 occurs on an ultrafast timescale63−65 and due to limited time-resolution of our TCSPC set up (∼100 ps), the fluorescence lifetime components that we observe are solely for the DK tautomer in the excited state located in the different microenvironments. Therefore, the radiative (kr) and nonradiative (knr) rate constant can provide valuable information to explain the excited-state binding dynamics of BP(OH)2 in micellar assemblies. Therefore, we have calculated average lifetime using eq 3 and the radiative and nonradiative decay rate constants are determined using the equations:

kr = k nr =

Φ ⟨τav⟩ 1 − kr ⟨τav⟩

Table 1. Time-Resolved Fluorescence Decay Parameters of BP(OH)2 in Surfactant/Cholesterol System at Different Q Values (λext = 336 nm) system TTAB/cholesterol

CTAB/ cholesterol

a

Q value

τ1 (ns)

a1

τ2 (ns)

a2

(ns)

0 0.05 0.10 0.30 0.50 1.00 0

1.38 1.65 1.79 1.84 1.91

0.26 0.60 0.66 0.51 0.61

1.90 2.15 2.53 3.15 3.34 3.42 2.08

1.00 0.74 0.40 0.34 0.49 0.39 1.00

1.90 1.95 2.00 2.25 2.58 2.50 2.08

0.05 0.10 0.30 0.50 1.00

1.63 1.67 1.81 1.82 1.85

0.20 0.36 0.61 0.53 0.50

2.24 2.37 2.96 3.21 3.24

0.80 0.64 0.39 0.47 0.50

2.12 2.12 2.26 2.47 2.55

Experimental error is ∼5%.

the microenvironment experienced by BP(OH)2 in TTAB- and CTAB-containing vesicles are almost the same. 3.2.3. Time-Resolved Anisotropy Studies. Steady state UV− vis absorption and fluorescence measurements can provide qualitative ideas about the binding site of dye molecules in organized assemblies. But from the time-resolved anisotropy measurement, we can get a quantitative estimation of the reorientation dynamics of organic dye molecules in organized assemblies. It is defined by the following equation:75

(5)

r (t ) = (6)

Where Φ and τav represent emission quantum yields and average lifetimes of BP(OH)2, respectively. The measured parameters are given in Table S1 of the Supporting Information. Variation of the nonradiative rate constant with the increase in surfactant concentration for DTAB, TTAB, and CTAB is depicted in Figure S4 of the Supporting Information. It clearly indicates that in aqueous solution, the knr value of BP(OH)2 is very high, but with addition of DTAB, TTAB, and CTAB, it decreases significantly. The rate of decrease in knr values is higher in CTAB micelles, and it follows the trend as CTAB > TTAB > DTAB. The incorporation of the BP(OH)2 molecule in a small unilamellar vesicle results in further increase in fluorescence lifetime (Table 1). The fluorescence lifetime decays of TTAB and CTAB micellar solutions with increasing cholesterol concentration are shown in Figure 7. Although, in 30 mM TTAB and CTAB solution, emission decays are single exponential in nature, but with addition of cholesterol, another lifetime component arises (i.e., emission decays become biexponential in nature). This observation clearly signifies the heterogeneity experienced by the BP(OH)2 molecule in the vesicular solution containing cholesterol. In cholesterol containing TTAB and CTAB vesicles (Q = 1.00), the average lifetime of BP(OH)2 molecules is ∼2.50 and ∼2.55 ns, respectively. This result indicates that quatsomes being very effective to perturb intramolecular hydrogen bond mediated fast nonradiative deactivation of BP(OH)2. Although in micellar solution of TTAB and CTAB, the lifetime values of BP(OH)2 differ considerably, but after formation of the vesicle, the lifetime values are almost the same. This distinctly suggests that

I (t ) − I⊥(t ) I (t ) + 2GI⊥(t )

(7)

I∥(t) and I⊥(t) are emission decays polarized vertically and horizontally to the polarization of the excitation light, respectively. G is the correction factor. The G factor is calculated using horizontally polarized excitation light. Then, the emission polarizer is fixed vertically and horizontally, respectively. For horizontally polarized excitation light, the vertical component (IHV) and the horizontal component (IHH) of the emission are collected through the emission monochromator. The ratio of IHV and IHH designates the G value of the system (i.e., G = IHV/IHH). For our TCSPC setup, the G value is 0.6. To investigate the influence of variation in chain length of surfactant molecule on the rotational motion, the rotational relaxation parameters for BP(OH)2 are collected in 50 mM DTAB, TTAB, and CTAB solutions and listed in Table S2 of the Supporting Information. The anisotropy decays are biexponential, and the average rotational time constant is estimated using the following equation:75 ⟨τr⟩ = aslow τslow + a fastτfast

(8)

where τslow and τfast are the slow and fast components of the decay time of the BP(OH) 2 , a slow and a fast are the corresponding relative magnitude of these components, and designates the average rotational relaxation time of BP(OH)2. The representative anisotropy decay profiles are also shown in Figure S5 of the Supporting Information. Now, values of BP(OH)2 vary with an increase in the alkyl chain length of the surfactant molecule. However, the variation is much less between the DTAB and TTAB micellar solution compared to the TTAB and CTAB micelle. The interesting 9335



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Figure 7. Time-resolved fluorescence decay of BP(OH)2 in 30 mM solutions of (a) TTAB and (b) CTAB with increasing cholesterol concentration (λex = 336 nm).

Figure 8. Anisotropy decays of BP(OH)2 in water and 30 mM (a) TTAB and (b) CTAB solutions with increasing cholesterol concentration (λex = 375 nm).

observation is that the relative contribution of the slow component enhances as the alkyl chain length of surfactant molecule increases. For example, ongoing from DTAB to TTAB micelles, the value increases ∼27%, whereas between TTAB to CTAB micellar solutions an ∼46% increase is observed. This phenomenon shows that the rotational motion of BP(OH)2 becomes slower as the hydrophobic chain length of surfactant molecule increases. It is obvious that with an increase in chain length of surfactant molecules, the hydrophobic association makes micelles more close-packed, which enhances the microviscosity of the core of micelles.85,86 As a consequence, the microviscosity and closed-packing of micelles are responsible for this variation of rotational time of the BP(OH)2 molecules in different surfactant assemblies. In order to gain a better understanding of rotational motion of BP(OH)2 upon different cholesterol concentration in surfactant solution, we have executed the time-resolved fluorescence anisotropy measurement. The gradual increase in rotational time of BP(OH)2 with increase in concentration of cholesterol in aqueous solution of both TTAB and CTAB micelles indicates the decrease in the hydration behavior surrounding the probe molecules due to the transition of surfactant micelles to vesicles. Figure 8 represents the changes of anisotropy decays of BP(OH)2 in aqueous TTAB/ cholesterol and CTAB/cholesterol solutions. In all solution, the decays are fitted with a biexponential function and the corresponding values are given in Table 2. As the solubility of BP(OH)2 in water is much less, it is preferentially located in the bilayer of vesicles. Therefore, the increased hydrophobicity and rigidity of the confined microenvironment around the BP(OH)2 restricts its motion, and gradually, the anisotropy decays

Table 2. Dynamic Parameters of Fluorescence Anisotropy of BP(OH)2 in Aqueous Surfactant/Cholesterol Mixture at Different Q Values (λex = 375 nm) system TTAB/ cholesterol

CTAB/ cholesterol

*

Q value

τfast(afast) (ns)

τslow(aslow) (ns)

(ns)

viscosity (cP)

0.00

0.21 (0.91)

0.91 (0.09)

0.27

0.94

0.05 0.10 0.30 0.50 1.00 0.00

0.18 0.24 0.24 0.25 0.20 0.21

(0.85) (0.93) (0.94) (0.90) (0.75) (0.88)

1.39 1.77 2.16 2.34 2.68 1.18

(0.15) (0.07) (0.06) (0.10) (0.25) (0.12)

0.36 0.35 0.36 0.46 0.82 0.33

0.96 1.08 1.15 1.20 1.45 0.95

0.05 0.10 0.30 0.50 1.00

0.24 0.25 0.30 0.28 0.23

(0.88) (0.91) (0.95) (0.90) (0.80)

1.33 1.62 2.71 3.22 3.50

(0.12) (0.09) (0.05) (0.10) (0.20)

0.37 0.37 0.42 0.57 0.88

0.97 1.12 1.20 1.30 1.60

Experimental error of ∼6%.

become slower with an increase in cholesterol content. In 30 mM TTAB and CTAB solutions, the average rotational time constant is about ∼0.27 and ∼0.33 ns, respectively. But in the vesicle solutions (Q = 1.00) of TTAB and CTAB containing cholesterol, the average rotational time constant increases to ∼0.82 and ∼0.89 ns, respectively. It is interesting to mention that the contribution of slow component increases significantly in vesicle solution at Q = 1.00 compared to both micelles. But, τr values obtained in TTAB/cholesterol and CTAB/cholesterol vesicles are nearly identical. As mentioned earlier, in structural 9336



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Table 3. Analytical Rotational Parameters Obtained from the Anisotropy Decays of BP(OH)2 in 30 mM TTAB and CTAB Solution in the Presence of Different Amounts of Cholesterol system TTAB-cholesterol

CTAB-cholesterol

Q value 0.05 0.10 0.30 0.50 1.00 0.05 0.10 0.30 0.50 1.00

radius (rh) (nm) 50.00 54.00 55.50 59.00 60.00 50.00 54.50 55.50 59.50 63.00

τM (ns) 10.99 17.99 20.00 25.08 31.87 12.34 18.45 20.88 27.87 40.71

× × × × × × × × × ×

4

10 104 104 104 104 104 104 104 104 104

τe (ns)

τD (ns)

DW × 10−9 (s−1)

DL × 10 (cm2 s−1)

θ0 (deg)

S

ηmic (cP)

0.21 0.28 0.27 0.28 0.22 0.29 0.30 0.34 0.31 0.24

1.39 1.77 2.16 2.34 2.68 1.33 1.62 2.71 3.22 3.50

1.45 1.46 1.58 1.28 1.06 1.17 1.17 1.10 1.42 1.12

29.98 27.45 23.77 24.79 22.39 31.33 30.56 18.94 18.32 17.64

59.01 67.82 69.24 63.68 57.83 61.66 61.66 65.04 70.69 55.08

0.39 0.26 0.24 0.32 0.50 0.35 0.35 0.30 0.22 0.45

9.43 9.17 9.43 12.05 21.49 9.70 9.70 11.01 14.94 23.32

characterizations of vesicles and steady state fluorescence measurement studies the size of vesicles, polarity, and rigidity of vesicle bilayers are also mostly identical. Therefore, we can say that the binding dynamics of probe molecules into supramolecular aggregates can provide structural features of organized systems. The biexponential scenario of anisotropy decays of BP(OH)2 molecules can be explained in the surfactant/cholesterol medium following reorientional motions between the probe molecule and vesicle. An accurate appreciation about the motion can be obtained by applying two-step and wobbling in a cone models. These models help us to estimate respective parameters correlating the motion of probe molecule in these vesicular media, presuming the fast motion and slow motion are separable. The anisotropy decay is related with the wobbling motion of probe molecules, lateral or translational diffusion along the surface, and overall rotational motion of vesicles. In accordance with the two-step model, the slow component of rotational relaxation time (τslow) is associated with lateral diffusion of the probe (τD) along the inner surface of the vesicle and overall rotational motion of vesicles (τM). The wobbling in a cone model describes the fast component of rotational relaxation time involving the internal motion of probe (τe) correlating cone angle (θ0) and wobbling diffusion coefficient (DW). All the analytical rotational parameters obtained from anisotropy decays of BP(OH)2 molecules are tabulated in Table 3, and the useful equations are given in the Supporting Information.87 As described in Table 3, the overall rotation motion, τM is very high in vesicles compared with the value of τslow and τfast values of BP(OH)2 molecules. Therefore, the contribution of overall rotational motion of vesicles to anisotropy decay is very low. Moreover, τD values are similar to τslow, indicating that the translational diffusion of BP(OH)2 is effectively represented by the τslow values. The lateral diffusion coefficient, DL, of BP(OH)2 molecules decreases with an increase in cholesterol concentration in micelle solution. This can be accounted for by an increase in rigidity and compactness of vesicles with cholesterol content. In CTAB/cholesterol and TTAB/cholesterol systems, the order parameter, S values are between 0.35 and 0.50. This observation clearly indicates the incorporation of BP(OH)2 molecules inside the vesicle bilayers, as its value range from “0” (unrestricted motion) to “1” (restricted motion). Now, the microviscosity (ηmic) inside the pseudophase of vesicle bilayers have been determined using the well-established Stokes−Einstein-Debye equation:88−90

⟨τr⟩ =

ηmicV (9)

kT

where V is the volume of the probe molecule and and T represent average rotational time and absolute temperature, respectively. The volume of BP(OH)2 was taken as 157 Å3, which was calculated using Edward’s volume increment method.71,91 The variation of bulk viscosity of the medium is shown in Figure S6 of the Supporting Information. The results assured that the microviscosity obtained in vesicle solutions are quite larger than bulk viscosity. Cholesterol molecules are water insoluble; therefore, it must be located in the bilayer region of vesicles. The strong interaction between the ammonium moiety of the surfactant and cholesterol molecules increase the compactness and rigidity of the vesicle bilayer. As a consequence, the water penetration decreases significantly, which increases the microviscosity of vesicle bilayer. Therefore, the microviscosity of CTAB- and TTAB-containing vesicles (Q = 1.00) increases to ∼23.32 and ∼21.49 cP, respectively.

4. CONCLUSION In the present study, we have utilized a ESIDPT molecule, BP(OH)2, to observe its photophysical changes upon micelleto-vesicle transition in surfactant solution concerning modulation of its ground- and excited-state physical processes as a function of cholesterol concentration and also utilize this molecule to get information about the other rotational parameters and microviscosity of vesicle bilayers. The spectroscopic alternation in UV−vis and fluorescence signals of BP(OH)2 molecules confirmed the location of it in micelles or bilayers of vesicles. But the nanoaggregate of vesicles (quatsomes) are relatively better than micelles at decreasing the interaction of BP(OH)2 with water molecules. The observed increase in rotational time in higher cholesterol concentration confirms the increment of microviscosity around the fluorophore induced by close packing between surfactant and cholesterol molecules. Therefore, the increase in hydrophobicity and the structural changes of vesicle bilayers are successfully followed by this molecule. Since, due to unique structural features of the vesicle, ESIPT molecules entrapped in nanoassemblies of vesicle resemble different biological phenomenon like host−guest or substrate−enzyme interaction; therefore, modulation of their ground- and excited-state properties have tremendous applications regarding the drug carrier or encapsulating vehicles for different drug molecules. 9337



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ASSOCIATED CONTENT

S Supporting Information *

A useful equation to calculate rotational parameters, emission spectra with increasing surfactant concentration, excitation spectra with an increase in cholesterol concentration, timeresolved decays with increasing surfactant concentration, anisotropy in different surfactant solution, viscosity of surfactant-cholesterol solution at different Q values, lifetime values with increasing surfactant concentration, and timeresolved anisotropy in different surfactant solutions. 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: 91-3222-255303. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS N.S. is thankful to the Department of Science and Technology, Government of India, for a generous research grant. S.G. and A.R. are thankful to CSIR for research fellowships. J.K. is thankful to UGC for a fellowship. D.B. and N.K. are thankful to IIT Kharagpur for research fellowships.

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