Organic Additive, 5-Methylsalicylic Acid Induces Spontaneous

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Organic Additive, 5-Methyl Salicylic Acid, Induces Spontaneous Structural Transformation of Aqueous Pluronic Triblock Copolymer Solution: A Spectroscopic Investigation of Interaction of Curcumin with Pluronic Micellar and Vesicular aggregates Surajit Ghosh, Jagannath Kuchlyan, Debasis Banik, Niloy Kundu, Arpita Roy, Chiranjib Banerjee, and Nilmoni Sarkar J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/jp507378w • Publication Date (Web): 05 Sep 2014 Downloaded from http://pubs.acs.org on September 9, 2014

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The Journal of Physical Chemistry

Organic

Additive,

5-Methyl

Salicylic

Acid,

Induces

Spontaneous

Structural

Transformation of Aqueous Pluronic Triblock Copolymer Solution: A Spectroscopic Investigation of Interaction of Curcumin with Pluronic Micellar and Vesicular aggregates

Surajit Ghosh, Jagannath Kuchlyan, Debasis Banik, Niloy Kundu, Arpita Roy, Chiranjib Banerjee and Nilmoni Sarkar* Department of Chemistry, Indian Institute of Technology, Kharagpur 721302, WB, India E-mail: [email protected] Fax: 91-3222-255303

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Abstract This article presents the interaction of curcumin in the microenvironments provided by aggregation of pluronic triblock copolymer, P123 into micellar and vesicular assemblies. The formation of vesicles using triblock copolymer, P123 and 5-methyl salicylic acid (5 mS) has been successfully characterized by optical spectroscopy, light scattering measurement, steady state anisotropy ( ) and eventually microscopic techniques. Besides, to make a comparative study between the polymeric micelles, we have also investigated the photophysical changes of curcumin in F127 triblock copolymer micelles having variation in polyethylene oxide (PPO) and polypropylene oxide (PEO) unit of polymer chain to that of P123. Time-dependent UV-vis measurement suggests that these polymer micelles are able to stabilize poorly water-soluble curcumin, by suppressing the degradation rate in micellar nanocavity. However, experimental observations suggest that P123 micelles are more efficient than F127, to perturb excited state intramolecular proton transfer (ESIPT)-related nonradiative decay of curcumin. We also observed that rigid and confined microenvironment of P123/5 mS vesicles enhance emission intensity and lifetime of curcumin more compared to P123 micelles. All the observation suggests that modulation of photophysics of curcumin is responsible due to its interaction with polyethylene oxide or poly propylene oxide unit of triblock copolymer.

Keywords: ESIHT, Triblock Copolymer, Curcumin, Encapsulation, Picosecond Time resolved Spectroscopy.


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1. Introduction In recent times, amphiphilic triblock copolymers have attracted increasing attention to their potential use in biological, chemical, medicinal or industrial applications.1-3 A wide range of different Pluronic triblock copolymers of variable molecular weights and having tunable properties are commercially available. The central part of these polymers consist of hydrophobic poly (propylene oxide), PPO block and terminal contain hydrophilic poly (ethylene oxide), PEO moieties. The difference within hydrophobicity between PPO and PEO unit allows it to form versatile self-assembled organized architecture like micelles, vesicles (polymersomes), etc. depending on the experimental conditions.4-6 Due to nontoxic nature and ability to form organized structures, these model systems are used in medicinal chemistry as vehicles of nucleic acid delivery,7 and also used for catalytic reaction media,8 templates for nanoparticle synthesis9,10 etc. Besides, several dynamical and photophysical studies have been carried out in different self assemblies of triblock copolymer.11-14 The phase behavior of Pluronic triblock copolymer mainly depends upon the number of PPO and PEO content present in the polymer chain. The micellization of triblock copolymer in aqueous solution depends upon the concentration of polymer and temperature.15 The size or PPO content of triblock copolymer also influences the critical micelle concentration (cmc) or critical micellar temperature (cmt). It has been accounted that with increasing the PPO content a sharp reduction of cmc and cmt of polymer solution is observed in aqueous medium.16 Sometimes surfactant or triblock copolymer micelles are also transformed into vesicle in presence of different additive molecules or other surfactants. The morphology of these aggregates mainly depends on concentration of additives, temperature, pH etc.17-25 Self assembled nanostructures of vesicles contain one or more hydrophobic concentric bilayer (unilamellar or multilamellar) with a hydrophilic center with water molecules. The ratio of hydrophobic and hydrophilic moiety of triblock copolymer controls the architecture of polymer assemblies.26,27 But, the formulation of small unilamellar vesicles is difficult compared to micelles or large vesicles. Small unilamellar polymer vesicles have potential application in nano-, bio-, or medical research due to its high colloidal stability and capability to solubilize both hydrophobic and hydrophilic drug molecules. Kim et al. have reported formation of unilamellar polymer vesicle using triblock copolymer, P85 (PEO26PPO40PEO26) and organic additive, 5-methyl salicylic acid (5 mS) and characterized these vesicles using DLS, SANS, cryo-TEM etc. They also showed the temperature induced transition of polymer vesicle to cylindrical micelles. Besides, pluronic micelles and vesicles are also able to 3 ACS Paragon Plus Environment


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enhance the effect of certain drugs by sensitizing certain biological cells.28 The hydrophobic region of micelle and vesicle or hydrophilic aqueous core of vesicle are able to solubilize the drug molecules, and therefore; it prevents the interaction with other component which increases the stability in blood system. Moreover, these polymers are able to enhance the activity of anticancer drugs like, doxorubicin, carboplatin, curcumin, cisplatin, camptothecin, paclitaxel etc.29–33 The polyphenolic compound, curcumin, 1, 7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene3,5-dione) exhibits a variety of biological properties, has received a great deal of interest. Researchers have extensively investigated medicinal benefits of curcumin at the molecular level including its anti-oxidant, anti-tumor, or anti-inflammatory activities. Due to its non-toxic nature, it is regularly used as curry spice. The central β-diketone moiety, hydroxyl groups of benzene rings and double bonds in the alkene region were responsible to act as essential functions in the medicinal actions of the drug molecules.34 But the difficulties in the practical application of curcumin due to its poor aqueous solubility and reduced bio-availability.35 Besides, it undergoes rapid degradation in neutral and alkaline pH due to the deprotonation.36 To overcome the problems of solubility and lack of bio-availability, several self assemblies in aqueous solution have been developed to encapsulate curcumin.32,37–42 Among this, FDA also permits pluronic block copolymers for medical applications due to their biocompatibility and biodegradability.43 We have used pluronic P123 and F127 (Scheme 1) as block copolymer to form micelles and vesicles in aqueous solution and investigate the stability, interaction, photophysical properties of curcumin in these micellar nano carriers. Several studies have focused on ESIHT or ESIPT process of curcumin upon photo excitation.44–52 Besides, ESIPT, solvation dynamics is also regarded as nonradiative phenomenon of curcumin manifested in the excited state.44–46 Ultrafast fluorescence techniques extensively used by many groups to investigate the excited-state properties of curcumin. In non polar solvent, the ESIPT process seems to be intramolecular because of six membered chelate rings through hydrogen bonding of the cis-enol form (Scheme 1). But in polar solvent, the intramolecular hydrogen bonded chelate ring is perturbed due to dipole-dipole interaction which suppresses the efficiency of nonradiative channel. But another efficient channel stimulates nonradiative relaxation via intermolecular hydrogen bonding involving curcumin and solvent molecules.44 In addition, Das et al.51 have reported ESIHT reactions of curcumin in solvent mixtures of toluene with methanol, chloroform and acetonitrile to modulate the hydrogen bonding network in picoseconds time region. Hence, the proper choice of binary solvent mixtures can play a significant role on ESIHT phenomenon of curcumin. 4 ACS Paragon Plus Environment

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Huppert et al.49 also investigated the temperature dependent nonradiative processes of curcumin in ethanol and 1-propanol. In addition, they also reported the influence of acetate ion (mild base) on the ESIHT process of curcumin in methanol and ethanol.50 The solvent mediated dynamics of ESIHT and non radiative deactivation pathways of curcumin are found to be significantly modulated when it is encapsulated into the hydrophobic nanocavities of different supramolecular assemblies.53-60 Petrich et al.54 pointed that aggregation of conventional amphiphilic molecules, i.e. sodium dodecyl sulfate (SDS), dodecyl trimethylammonium bromide (DTAB), and Triton X-100 (TX-100), into micelles provide substantial stability of curcumin in aqueous medium. It is also observed that when curcumin binds to different organized assemblies; its fluorescence lifetime is increased due to perturbation of ESIHT mediated nonradiative decay.56,62 In continuation of interaction study of drug molecules with biological organized aggregates, here, we try to investigate the stability and photophysics of curcumin in pluronic triblock copolymer forming micelles and pluronic triblock copolymer/5 mS forming vesicles. The formation of vesicles with P123 and 5 mS is successfully characterized by UV-vis technique, DLS study and TEM measurement. Bora and coworkers32 investigated the physical interaction of curcumin with copolymer F127 and F68 using different spectroscopic techniques. In summary, the encapsulation efficiency of curcumin in pluronic micelles is estimated by in vitro study via cytotoxicity measurement or release of drug carrier micelles. Zhai et al. have used mixed micelles composed of P123 and F68 as nano carrier for curcumin to investigate the in vitro cytotoxicity of drug loaded micelles.33 Molecular dynamics simulations have also been carried out in PEO−PPO−PEO polymers to understand the interaction between curcumin and block copolymer.63 Simulation study also reveals that the presence of lipid bilayer, the polymer chain of micelle releases and bind to lipid bilayer. But upon encapsulation of hydrophobic molecules inside the core of micelles influence this process and provide better stability to micelles.64 Therefore, it is interesting to investigate the photophysics of hydrophobic drug molecules in pluronic micelles. In this manuscript, we have investigated the photophysics of a hydrophobic drug molecule; curcumin, in P123 and F127 forming aggregates using fluorescence spectroscopic techniques. A detailed photophysical evaluation between curcumin and pluronic micelles or vesicles is essential to get the information about the dynamics of ESIHT process. The time dependent UV-visible measurement indicates substantial stability of curcumin inside the micelles compared to aqueous buffer solution. Moreover, pluronic micelles are more efficient to perturb the excited state ESHIT process of curcumin than normal micelles 5 ACS Paragon Plus Environment


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formed by conventional surfactant. Besides, our observation suggest with increasing 5 mS concentration in aqueous P123 solution, the microenvironment of polymer aggregates largely affected the photophysics of curcumin that reflects in emission intensity, quantum yield or lifetime measurements. 2. Experimental Part: 2.1. Materials: Curcumin (purity ∼80%) and 5-methyl salicylic acid (5 mS), Pluronic triblock copolymer P123, F127 were obtained from Sigma Aldrich. The number average molecular weight of the polymers (5800 for P123 and 12000 for F127) was used for the calculation of concentration. All these materials are used without further purification. In an earlier paper, utilizing high purity curcumin (≥98.5%, Alexis Biochemicals), Petrich et al. have reported that fluorescence upconversion results obtained from these curcumin are identical. Hence, other curcuminoids (∼20%) present in curcumin negligibly effect its photophysics.54 To prepare aqueous micellar solutions, we have used triply distilled Milli-Q water. The stock solution of curcumin was prepared in methanol. First, appropriate amount of copolymer (P123 and F127) were added to water in a volumetric flask room temperature and stirred using magnetic stirrer for overnight. The solution was kept sometimes for stabilization. The concentration of curcumin in polymer solution was kept at ~10−6 M. The structures of curcumin, 5-methyl salicylic acid (5 mS) and composition of P123 and F127 are depicted in Scheme 1.

2.2. Instrumentation The UV-vis absorption and fluorescence experiments were conducted in Shimadzu (model no. UV-2450) spectrophotometer and a Hitachi (model no. F–7000) spectrofluorimeter, respectively. The emission quantum yield of curcumin in was analyzed using the UV-vis and fluorescence spectra of coumarin-153 (C-153) in acetonitrile at 298 K as reference46,65 using following equation66

Φ = × × 1 Φ


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where Φ, Abs, n and A represents quantum yield, absorbance, refractive index of the medium and area under fluorescence spectra. The subscripts S and R refer to the corresponding parameters for the sample and reference, respectively. The time resolved fluorescence decays were monitored using a time correlated single photon counting (TCSPC) pico-second setup. The detailed experimental procedure for this instrument is accounted in our earlier publication.67 In brief, a pico-second diode laser of 408 nm (IBH, UK, Nanoled) used as light source, and emission was collected in magic angle (54.7o) using a Hamamatsu MCP PMT (3809U). The obtained decays were analyzed by IBH DAS-6 decay analysis software. In temperature dependent measurement circulating water bath was used to maintain the temperature. We have also determined the average lifetime ( ) using the following equation:66 = 2

where and represents decay time constant and its relative contribution, respectively. 2.3. Preparation and structural characterization of P123/5-methyl salicylic acid (5 mS) aggregates: Polymer containing nano aggregates were prepared by using required amount of triblock copolymer, P123 with varying concentration of 5-methyl salicylic acid (5 mS). First, filtration of Milli-Q water was done to remove dust particles by using Syringe Filter (0.2 µm). The 5 methyl salicylic acid (5 mS) content in aqueous 5 mM P123 solution is expressed as =

[5 − ℎ!" "#$!"#$ $#% 5 & ] 3 [()"!, (123]

5-methyl salicylic acid was dissolved in different polymer solution to achieve required R value. The resulting solution mixtures were sonicated using a normal sonicator and kept at room temperature for few days to stabilize the solution mixtures. 2.4. Structural Characterization of P123/5-methyl salicylic acid (5 mS) polymer aggregates: Malvern Nano ZS instrument utilizing a 4 mW He−Ne laser (λ = 632 nm) with fixed detector angle at 173º is used to characterize polymer/5 mS nano aggregates. The hydrodynamic diameter of nano aggregates is calculated using the following equation: %, =

-. / 4 3012

where kB, T, D and η denotes Boltzmann constant, Temperature, diffusion coefficient and viscosity, respectively. 7 ACS Paragon Plus Environment


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The conformation of vesicle formation was done by analyzing morphology by transmission electron microscope (JEOL model JEM 2010). Uranyl acetate (0.1 wt %) in water was used as staining agent to visualize the morphology of polymer aggregates.

3. Results and Discussion: 3.1. Structural Characterization of P123/5-methyl salicylic acid (5 mS) polymer aggregates: The morphological transition between aqueous solution of P123 and 5-methyl salicylic acid (5 mS) depends on the concentration of 5 mS in a fixed polymer concentration. Previously, Raghavan et al.17 showed the influence of 5 mS concentration on the aggregation behavior of cationic surfactant, cetyl trimethylammonium bromide solution. The temperature dependence switching of vesicles to “wormlike micelles” is also characterized using phase behavior, SANS, rheology study. Kim and coworkers21 characterized the self assembled nanostructures of P85 and 5 mS using DLS, cryo-TEM and SANS study. Here, we have used 5 mM aqueous solution of P123 which forms vesicles. According to literature report, temperature dependence switching of vesicles to wormlike micelles is observed at low 5 mS concentrations.17,21 In our study, higher concentration of 5 mS is required to form P123/5 mS vesicles. Therefore, with increasing the temperature, probably the phase transition of vesicle solution is not observed at R=8.0. The phase transition of micelle to vesicle is observed by monitoring the optical density of the solution using UV-vis spectrometer. Micellar solution of aqueous P123 is transparent but with increasing 5 mS concentration, it becomes light bluish colour and a sharp change in optical density value is observed after R=3.0 (Figure S1, Supporting Information). This indicates that the solution contains the aggregates of larger diameter. The structural alterations of P123 micelles owing to the incorporation of 5 mS are determined by observing the size of the nano assembled using DLS measurements. P123 micelles shows average hydrodynamic diameter of ~18 nm with a narrow DLS profile which remains unaltered up to R=2.00. But, when the R value is increased, a dramatic change in size distribution is observed that correlated with the visual observation or optical density measurement results. The size distribution profile of P123 and 5 mS nano assembled with varying 5 mS content are depicted in Figure 1. This observation signifies that progressive incorporation of 5 mS to P123 solution, another peak around ~100 nm is appeared. The dual distribution of intensity-size profiles obtained from DLS measurement clearly indicates presence of free micelles along with polymer vesicles. Although with the increase in concentration of 5 mS, the intensity at low 8 ACS Paragon Plus Environment

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hydrodynamic diameter (~18 nm) range decreases gradually along with regular increase in hydrodynamic diameter in ~100 nm region without interfering the peak position. The microstructure of the P123 micelles and P123/5 mS vesicular aggregates are shown in Figure S2 (Supporting Information). The TEM images indicate that existence of spherical micelles (P123 solution) and vesicles (P123/5 mS solution) in the system. 3.2. Photophysical Properties of curcumin in Polymer micelles and Vesicles: 3.2.1. UV-vis Absorption Studies: Alternation in steady state absorption spectral characteristics of curcumin in different pluronic micellar solution formed by P123 has been investigated with increasing concentration of polymer. Curcumin is almost insoluble in water but its solubility is substantially enhanced in polymer containing aqueous medium. These results also indicate the partition of curcumin from water to block copolymer micelles. The variation of UV−vis profile of curcumin in the aqueous polymer solution at different concentration of P123 is shown in Figure 2. We have also used another triblock copolymer, F127 to understand the effect of hydrophobic PPO and hydrophilic PEO unit of micelles on binding features of curcumin. Similar variation in UV−vis profile of curcumin in aqueous F127 solution at different concentration is observed (Figure S3, Supporting Information). In neutral water, curcumin shows broad absorbance spectra having band maxima around 430 nm with a shoulder band at 355 nm. The absorbance band at 430 nm signifies lowest (π, π*) transitions of conjugated curcumin and the shoulder peak at 355 nm is assigned to the transitions (π–π*) involved in feruloyl unit. These observations are well correlated with the absorption spectra of curcumin in aqueous buffer solution.57,68 In water, the vibronic feature of absorption spectra is disappeared owing to the strong interaction between water and curcumin molecules.6971

In micellar solution of triblock copolymer of P123 and F127, the shoulder peak at ~355 nm is absent and an intense band at ~426 nm is observed. Therefore, the absorption spectra exhibit maxima at ~426 nm and a shoulder peak at ~445 nm. In nonpolar solvent curcumin shows structured emission corresponds to the fully conjugated form of the protonated enol.69,71,72 Hence, this structured absorption spectra indicates lower degree of interaction between curcumin and water molecules in copolymer micelles.

3.2.2. Stability of Curcumin in Polymeric Micelles: 9 ACS Paragon Plus Environment


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It is well documented that in aqueous solution of alkaline or neutral pH, curcumin undergoes rapid degradation.57,59 To get an idea about the degradation rate in polymeric micelle of P123, we have monitored the UV-vis profiles of curcumin in aqueous micellar solution as a function of time. In present study, UV-vis spectra of curcumin were monitored for 12 hours and changes in spectra are given in Figure 3. As depicted in Figure 3, it is clear that binding of curcumin into the hydrophobic environment of polymeric micelles efficiently decreases the rate of degradation. Similarly, F127 micelle is also able to stabilize curcumin as observed from time dependent UVvis measurement (Figure S4, Supporting Information). According to Harada et al., curcumin displays eminent degradation rate in 30 min and its absorption intensity decreases about 50% of initial value.57 In addition to aqueous phosphate buffer solution, they also studied the stability of curcumin in aqueous solution of α-cyclodextrin, β-cyclodextrin and γ-cyclodextrin. The suppression of degradation rate of curcumin into different biomimicking nanocavities is owing to decrease the interaction with water molecules. The insets of Figure 3 and Figure S4 (Supporting Information) clearly indicate that in 5 mM P123 and F127 micellar solution curcumin degrades less than ~7% after 12 hr. In aqueous solution, curcumin exists as keto-enol tautomeric form.73 But in alkaline or neutral medium, due to deprotonation of enol group, it undergoes rapid degradation and leads to formation of trans-6-(4′-hydroxy-3′-methoxyphenyl)-2,4-dioxo-5hexanal, vanillin, ferulic acid, and feruloylmethane.74 In aqueous solution, shoulder band at 355 nm in UV-vis spectra indicates the interaction between curcumin and water molecules. But in micellar solution this band is absent. This observation suggests that in micelle, the watercurcumin interaction decreases as the molecule is encapsulated in hydrophobic core of the polymer micelles. As a consequence, the rate of degradation of curcumin decreases in micelle significantly than aqueous solution. The suppression of degradation rate of curcumin upon partition into the restricted environments like, proteins, micelles, vesicles etc. was already accounted in literature.56,58,59,61 Comparisons with the previous observations indicate that polymeric micelles composed of P123 and F127 are also efficient systems for carrier of curcumin.

3.2.3. Steady-state Fluorescence studies: The results of interaction of curcumin as a function of concentration of triblock copolymer, P123 and F127 are reflected in the emission spectra profiles (Figure S5, Supporting Information). In aqueous solution, curcumin is almost non-fluorescence. However, sequential addition of triblock 10 ACS Paragon Plus Environment

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copolymer in aqueous solution of curcumin leads to the blue shift in the emission profile with significant enhancement in emission intensity and after certain concentration of triblock copolymer; it reaches saturation (Figure S6, Supporting Information). These results clearly indicate the solubilization and partition of the curcumin in polymeric micellar aggregates from aqueous phase. The quantum yield values (Φ) of curcumin also increase with increase in polymer concentration and reaches saturation after a certain concentration (Figure 4). In ~0.05 mM solution of P123, the fluorescence intensity is very low and quantum yield of curcumin is ~0.010. With increase in concentration of P123, significant increase in emission intensity is observed and quantum yield reaches a limiting value of ~0.049. Similarly, in aqueous solution of F127, the emission intensity reaches saturation at ~1.02 mM concentration and the observed quantum yield is ~0.042. Moreover, variations in emission intensity and Φ values also help us to judge the cmc of pluronic triblock copolymer. The calculated cmc of P123 and F127 triblock copolymer in aqueous solution at 298 K is around ~0.056 mM and ~0.560 mM which are well correlated with literature value.16 It is well documented that photophysics of curcumin is predominately depends with polarity and hydrogen bonding capability of the surrounding environments. So, these results indicate that hydrophobic poly (propylene oxide) (PPO) block and terminal hydrophilic poly (ethylene oxide) (PEO) blocks of triblock copolymer play an important in modulation of photophysics of curcumin. The emission intensity of curcumin again further increased with addition of 5 mS in P123 solution (Figure S7, Supporting Information) without any alternation in peak maxima. The interesting observation is that incorporation of curcumin in vesicle bilayer leads to increase in quantum yield significantly. In polymer micelles (5 mM concentration), the maxima Φ value of curcumin is determined in the range of 0.058-0.062. But in P123/5 mS vesicles, it becomes 0.160, which is almost 3 times compared to micelles. The bilayer region of polymer vesicles offers more rigid and confined microenvironment around the curcumin molecules; therefore intramolecular hydrogen bond mediated nonradiative processes reduced significantly. As a result, the enhanced Φ value of curcumin in polymer vesicle is due to substantial perturbation of ESIHT processes. 3.2.4. Determination of Partition Coefficient of Curcumin: To use polymeric micelles as delivery agent for curcumin, it is necessary to obtain the quantitative estimation about the inclusion of drug in micelle of amphiphilic molecules. One 11 ACS Paragon Plus Environment


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such possibility is to determine the partition coefficient in curcumin-polymer systems. Partition coefficient of a solute between micellar solution and aqueous solution is defined as:75 78 : /[()"!] 7 45 = 9 5 7< 6 7 : /[=] 9 6

where 79 , 78 and 7< represent total concentration of curcumin, concentrations of curcumin in polymer and water, respectively. We have estimated the partition coefficient and free energy change by using variation of fluorescence spectra of curcumin in water at various concentrations of P123 and F127. Experimentally, the partition coefficient of curcumin in polymer micelles are calculated using the following equation: 75,76 [A] >∞ − > 1 =1+ 6 >? − > 45 [()"!]

where > , >? , and >∞ are the fluorescence intensities of curcumin in absence of polymer, at

intermediate concentration of polymer and at the saturation, respectively. 45 is the partition coefficient of curcumin between polymeric micelle and the bulk water. We have obtained linear plots of >∞ − > /(>? − > ) versus the [polymer]KL in both triblock copolymer, P123 and F127 (Figure 5). The partition coefficients of curcumin in these triblock copolymers, P123 and F127 were estimated using the slope of the respective straight line plot and obtained values are ~7.58×105 (for P123) and ~1.71×105 (for F127), respectively. Previously, we have calculated the partition coefficient (Kp) of curcumin between water and 1butyl-3-methylimidazolium octyl sulfate ([C4mim][C8SO4]) containing micelles.61The estimated Kp value is 8.59 × 103. Similarly, Kp value is also calculated in zwitterionic N-hexadecyl-N,Ndimethylammonio-1-propanesulfonate (SB-16) micelles which is 15 ×104.62 Therefore, the 45 values in P123 and F127 polymer micelles are quite high than normal micelles.61,62 The pluronic triblock copolymer micelles, the central hydrophobic core is formed by PPO unit and corona region by comparatively hydrophilic PEO unit. Curcumin is almost water insoluble; therefore, it strongly interacts with PEO and PPO unit of triblock copolymer. As a consequence, the favorable interaction between curcumin and polymer micelles leads to higher partition of drug in micelles. The corresponding change in free energy values for the partition of curcumin in copolymer micelles are also calculated using the following equation: ∆N = −/"45 7 12 ACS Paragon Plus Environment

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The calculated ∆G values are −33.54 kJmolKL and −29.85 kJmolKL for P123 and F127 solution respectively. The 45 and ∆G values indicate that interactions of curcumin with P123 micelles are greater than F127 micelles. 3.2.5. Determination of Steady State Anisotropy (UV ): The steady state anisotropy (r ) measurements also provide selective information concerning the probe location and binding in microheterogenous media.66,77 Here, we have monitor the change of anisotropy of curcumin with the addition of polymer, P123 and F127. Such study also helps us to get a qualitative idea on the rigidity and stability of the microenvironments provided by various polymeric aggregates. The variation of r values of curcumin in the aqueous solutions with increase in concentration of P123 and F127 has been shown in Figure S8 (Supporting Information). The gradual increase of P123 or F127 solution to aqueous solution of curcumin, the anisotropy value gradually increases followed by saturation at higher concentration. This increment in anisotropy value is due to the encapsulation of drug molecule in polymeric micelles. Anisotropy studies reflect how micellar environments impose rotational restriction on probe molecules upon binding to micelles. The higher value indicates better restriction imposed on the motion of curcumin molecules in micelles. The anisotropy value of curcumin in ~0.005 mM polymeric solution is around ~0.05 while in presence of higher concentration of polymer, this value is increased to ~0.30–0.31. High value of curcumin was reported when it bound to proteins.78 These observations clearly indicate that curcumin molecules preferentially incorporate into the hydrophobic core of polymeric micelles. Among these two polymers, the trends in increase in anisotropy value are quite different. In P123 micelle, the anisotropy value reaches saturation after ~0.06 mM whereas ~0.60 mM F127 solution is required to reach the saturation.

3.2.6. Polarity of the Microenvironment: The polarity of probe binding site in different biomimicking microenvironments are estimated using spectral characteristics of probe molecule in different solvent or in solvent mixture of known polarity.79 We are interested to determine the polarity sense by curcumin when encapsulated in polymeric micelles. So, the fluorescence behavior of curcumin is monitored in dioxane-water mixture at different composition. We have used solvent polarity parameter, W9 30 , of dioxane-water composition reported in literature.80

Then, a correlation graph was obtained from the plot of emission maxima (Y\Z Z[ ) of the curcumin against the solvent polarity parameter (W9 (30)) of dioxane-water mixture and 13 ACS Paragon Plus Environment


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represented in Figure 6. We have assumed that this relation is also applicable for micelle. Now, curcumin shows emission maxima at~504 nm in polymer micelle which is almost same for P123 and F127 or P123/5 mS vesicle. By comprising this fluorescence maximum, it is estimated that W9 30 value is about ~44.75 4$" )" KL . The micropolarity value of curcumin in the vicinity of its interaction site within the polymeric micellar environment suggests a reduced polarity in comparison to aqueous solution. 3.2.7. Time-Resolved Fluorescence Studies: The time resolved emission decays of curcumin in aqueous solution with varying polymer concentration are monitored at their respective emission maxima. Although there are reports available on steady state fluorescence measurement of curcumin in polymer aggregates, but details analysis of photophysical properties of curcumin on the basis of lifetime measurement are rare.32 Figure 7 represents the emission decays of curcumin in P123 solution at different concentration. These decays are found to be biexponential in nature and in picoseconds time scale. For the comparative study between the polymeric micelles, the time resolved decays of curcumin is also collected in F127 solution (Figure S9, Supporting Information). To predict the influence of solvent polarity on ESIHT processes of curcumin, emission decays are monitored in different solvent extensively.44-49,51,81,82 These studies mainly concentrated on ESIPT or ESIHT and solvation processes of curcumin. The very short life time of curcumin in nonpolar solvent is assigned to nonradiative ESIHT process through unperturbed six membered chelate ring of the cis-enol moiety and this process occurs between few hundreds of femtosecond. Although, as mentioned earlier, in polar protic medium because of perturbation of intramolecular hydrogen-bond, intermolecular ESIPT with solvent molecules along with electronic solvation are anticipated to be the major nonradiative process involved in the relaxation of the S1 state of the curcumin.44 In micellar assemblies of conventional surfactants, the intramolecular hydrogen bond is also perturbed due to interaction with the head group region of the surfactant.54,56 In this article, the average time constant of fluorescence decay of curcumin in polymeric micellar solution of P123 and F127 are ∼281 ps with the component of 173 ps, and 603 ps, in 0.320

mM P123) and ∼193 ps (with the component of ~130 ps and ~526 ps, in 1.020 mM F127) at 298

K, respectively. In our earlier paper, we have showed that in Tween surfactant containing both micelles (Tween 20 and Tween 80) the average lifetime of curcumin is ~120 ps at 293 K.56 Therefore, the obtained lifetime components of curcumin in polymer micelles (P123 and F127) 14 ACS Paragon Plus Environment

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are longer than that nonionic micelles (Tween 20 and Tween 80) of conventional surfactant. The longer lifetime can be rationalized due to the interaction between curcumin and lone pair oxygen of oxyethylene moieties. Based on the above discussion, it is clear that the fluorescence lifetime values of curcumin are quite high in polymeric micelles than conventional nonionic micelles. But, in the present study, another interesting observation is that the time constant of fluorescence decays in P123 micelles is quite higher than F123 micelles. This can be explained on the basis of hydrophobicity of polymeric micelles. In P123 micelle, the number of hydrophobic polypropylene oxide unit is more than hydrophilic polyethylene unit, whereas in F127, the order is reverse.43 So, P123 micelles are more stable in comparison with F127 micelles. Due to this higher stability, P123 micelles are very efficient to increase the lifetime of curcumin through the perturbation of ESIPT or ESIHT mediated nonradiative decay channel than F127 micelles. Therefore, the rigid polymeric microenvironment also enhances the interaction of curcumin with the polyethylene oxide or polypropylene oxide units of polymer micelles. The disruption of the intramolecular hydrogen bond in curcumin also results in decrease the ESIHT rate in polymeric micelles than in normal micelles. The change in lifetime values of curcumin on increasing polymer concentration helps us to obtain the estimation about the radiative and nonradiative processes through encapsulation of probe molecules in micellar environment. The radiative rate constant (-f ) and nonradiative rate constant (-gf ) were calculated using quantum yield (Φ) and average lifetimes ( ) with the help of the following equations:66 -f =

h 8 < >

-gf =

1 − -f 9 < >

The calculated results are shown in Table 1. As shown in Table 1, the -gf values decrease with

increase in concentration of polymer. Moreover, the decrease of -gf values of curcumin is higher

in P123 solution than F127 solution. The -gf values of curcumin in aqueous P123 (0.321 mM) and F127 (1.020 mM) solutions are respectively ~3.385×10k KL and ~4.963×10k KL .

In present study, we have also noticed that when curcumin molecules incorporate in polymer vesicles, the lifetime value again increased (Table S1, Supporting Information). The variation of 15 ACS Paragon Plus Environment


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Page 16 of 34

time resolved decays of curcumin in P123 and P123/5 mS aggregates are shown in Figure 8. The lifetime component of curcumin in vesicles becomes ~259 ps (68 %) and ~731 ps (32 %) with an average lifetime of ~410 ps at 298 K. As, the P123/5 mS vesicles are more hydrophobic than micelles; the further increase in emission intensity and fluorescence lifetime of curcumin are observed with increasing concentration of 5 mS in P123 solution. In both aggregates, the lifetime components are in picoseconds range. Most interestingly, the component of short lifetime decreases and longer lifetime component increases significantly in vesicles than micelles. Fluorescence up conversion measurement of curcumin in surfactant micelles suggests that long lifetime component of bi-exponential fitting of time resolved decay is due to ESIPT process.54 Due to the limited time resolution of our set up ( nU noU (ns) (pVu vKp) (pVu vKp)


0.076 0.095 0.117 0.144 0.175 0.236 0.251 0.263 0.268 0.281 0.049 0.053 0.061 0.069 0.075 0.103 0.140 0.167 0.174 0.181 0.193

0.132 0.179 0.188 0.208 0.206 0.178 0.175 0.171 0.179 0.174 0.102 0.151 0.279 0.319 0.400 0.369 0.278 0.240 0.236 0.227 0.218

13.026 10.347 8.359 6.736 5.508 4.059 3.809 3.631 3.552 3.385 20.306 18.717 16.114 14.174 12.933 9.340 6.865 5.745 5.511 5.298 4.963

Page 29 of 34

Table 2: Fluorescence Quantum Yields (Φ), Lifetimes (τf), Radiative (kr) and Nonradiative Rate (knr) Constants of Curcumin in Polymeric Micelle and Vesicles as a Function of Temperature

lp (qp ) (ns) 0.167 (0.75) 0.158 (0.77) 0.148 (0.77) 0.140 (0.78) 0.147 (0.80) 0.137 (0.81) 0.129 (0.81) 0.125 (0.82) 0.259 (0.68) 0.241 (0.68) 0.222 (0.68) 0.201 (0.71)

System

Temp. Quantum (K) Yield (Φ) 298 0.062 5 mM 303 0.058 Aqueous 308 0.053 P123 313 0.048 Solution 298 0.057 5 mM 303 0.052 aqueous 308 0.047 F127 313 0.044 solution 298 0.161 P123 & 5 303 0.154 mS 308 0.132 Vesicle 313 0.112 (R=8.00) * Experimental error ~5%

lr (qr ) (ns) 0.655 (0.25) 0.634 (0.23) 0.598 (0.23) 0.573 (0.22) 0.600 (0.20) 0.573 (0.19) 0.548 (0.19) 0.525 (0.18) 0.731 (0.32) 0.685 (0.32) 0.642 (0.32) 0.608 (0.29)

(ns) 0.289 0.267 0.252 0.235 0.238 0.220 0.209 0.197 0.410 0.383 0.356 0.319

nU (10 vKp ) 0.215 0.217 0.210 0.204 0.239 0.236 0.225 0.223 0.393 0.402 0.371 0.351 9

noU (109 vKp ) 3.245 3.528 3.758 4.051 3.963 4.309 4.560 4.853 2.046 2.209 2.339 2.784

Figures:

R=7.00 Peak 1

Peak 2

R=5.00

Intensity (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


R=4.00

R=2.00

R=0.50

R=0.00

10

100

1000

Size (diameter, nm)

Figure 1: Size-Intensity plot of P123/5mS at a fixed concentration of P123 (5 mM) with varying

concentration of 5mS.



0.30

0.321 mM

Absorbance

0.24 0.18

0 mM 0.12 0.06 0.00 320

360

400

440

480

520

Wavelength (nm)

Figure 2: Absorption spectra of Curcumin in presence of increasing concentration of pluronic

triblock copolymer P123.

0 hour

(a) 5 mM P123

12 hour

% curcumin

100 95 90 85 80 75

Absorbance (a.u. )

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 34

0

3

6

9

12

15

Time (Hour)

360

400

440

480

520

560

Wavelength (nm)

Figure 3: Changes in the absorption spectra of curcumin in P123 micelles with increasing time.

The insets show the extent of degradation of curcumin in terms of the decrease in absorbance at 430 nm with increasing time interval.


Page 31 of 34

0.04

0.05

(a)

cmc

(b) Quantum Yield (Φ )

Quantum Yield (Φ )

0.05

cmc

0.03

0.02

0.01

0.04 0.03 0.02 0.01 0.00

0.00

0.07

0.14

0.21

0.28

0.0

0.35

0.2

0.4

0.6

0.8

1.0

Concentration (mM)

Concentration (mM)

Figure 4: Variation of Quantum yield (Φ) with increasing concentration of (a) P123 and (b)

F127.

5

9

7 6

(b)

(a)

Experimenta data Linear fit

4

Experimental data Linear fit

(Iα -I0)/(It-I0)

8

(Iα -I0)/(It-I0)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


5 4 3

3

2

2 1

1 0 1000

2000

3000

4000

5000

100

6000

200

300

400

500

600

Water/[F127]

[Water]/[P123]

Figure 5: Plot of >w − > / >? − > versus (a) ([Water]/[P123]) (b) ([Water]/[F127]).


700


Emission Maxima (λ max, nm)

580

Experimental data Linear fit

560 540 520 500

Micropolarity of Pluronic Triblock Copolymer Micelle

480 460 35

40

45

50

55

60

65

-1

ET (30), Kcal mol

Figure 6: Variation of fluorescence maxima of curcumin in dioxane-water mixture and polymer

micelle with ET (30).

Prompt 0.005 mM 0.030 mM 0.020 mM 0.050 mM 0.150 mM 0.254 mM 0.276 mM 0.320 mM

(a) P123 1000

Counts

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 34

100

10 4

5

6

7

8

9

Time (ns)

Figure 7: Time-resolved fluorescence decay of Curcumin in aqueous solutions of (a) P123, (b)

F127 with increasing Concentration (Y\[? = 408 .)


Page 33 of 34

Prompt P123 P123 and 5 mS vesicle

1000

Counts

100

10 5

6

7

8

9

Time (ns)

Figure 8: Time resolved decays of curcumin in P123 micelles and P123/5 mS vesicles.

2.00

P123 F127 P123/5 mS vesicle

1.75 1.50

ln knr

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1.25 1.00 0.75 0.50 -3

3.2x10

-3

3.2x10

-3

3.3x10

-3

3.3x10

-3

3.4x10

1/T (K-1)

Figure 9: Arrhenius plots of ln(knr) of curcumin versus 1/T in 5 mM P123, F127 and P123/5 mS

vesicle solution.



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Table of Content Only:


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