Article pubs.acs.org/JPCB
Modulation of the Photophysical Properties of Curcumin in Nonionic Surfactant (Tween-20) Forming Micelles and Niosomes: A Comparative Study of Different Microenvironments Sarthak Mandal, Chiranjib Banerjee, Surajit Ghosh, Jagannath Kuchlyan, and Nilmoni Sarkar* Department of Chemistry, Indian Institute of Technology, Kharagpur 721302, WB, India ABSTRACT: The modulation of the photophysical properties of curcumin inside two different types of microenvironments provided by nonionic surfactant forming micelles and vesicles (niosomes) has been investigated using steady state and time-resolved fluorescence spectroscopy. The formation of small unilamellar Tween-20/cholesterol niosomes with narrow size distribution has been successfully demonstrated by means of dynamic light scattering (DLS) and transmission electron microscopy (TEM) techniques. Our results indicate that niosomes are a better possible delivery system than the conventional surfactants forming normal micelles to suppress the level of degradation of curcumin. The enhanced fluorescence intensity along with the significant blue-shift in the emission maxima of curcumin upon encapsulation into the hydrophobic microenvironments of micelles and niosomes is a consequence of the reduced interaction of curcumin with the water molecules. We found that the more rigid and confined microenvironment of niosomes enhances the steady state fluorescence intensity along with the fluorescence lifetime of curcumin more than in micelles. The rigidity of the niosome membrane which arises basically due to the presence of cholesterol molecules increases the level of interaction between curcumin and the oxoethylene units of Tween-20 molecules. It is also possible for the hydroxyl groups of the cholesterol moieties to form intermolecular hydrogen bonds with curcumin to perturb nonradiative deactivation mechanism through excited state intramolecular hydrogen atom transfer (ESIHT).
1. INTRODUCTION Curcumin, the major component of a naturally occurring yellow-orange pigment named turmeric (curcuminoids), has received a lot of attention in the scientific community due to its effective medicinal activities documented in the literature over the past few decades.1−3 The medicinal benefits of curcumin as an anticancer, anti-inflammatory, antioxidant, antimicrobial, antiamyloid, anti-Alzheimer, anticystic fibrosis, and wound healing properties have been shown in a large number of recent studies.4−9 It is therefore the subject of major interest in the various fields of research including chemistry, biology, medicine, and pharmacology. The poor aqueous solubility and lack of bioavailability are the two major difficulties that prevent its medicinal application as an effective therapeutic agent.10 In addition to that, curcumin undergoes rapid degradation in the aqueous solution of neutral and alkaline pH.11,12 Lin et al.11 studied the degradation kinetics of curcumin under various pH conditions and analyzed the degradation products using HPLC and other experimental methods. As per their report, the degradation of curcumin occurs mostly due to the deprotonation of curcumin. Jagannathan et al.13 have recently proposed that at high temperature the solubility and bioavailability of curcumin in aqueous solution are significantly enhanced due to the breakage of the intermolecular hydrogen bond by thermal energy. The methods of encapsulation of curcumin in various self-assembled organized bioactive systems such as micelles, vesicles, proteins, © XXXX American Chemical Society
and cyclodextrins are shown to be highly effective in increasing the solubility, stability, and bioavailability of curcumin.14−28 Recently, the drug delivery systems composed of pharmaceutically acceptable components are attracting interest to researchers because of their biocompatibility and biodegradability.29 In this study we therefore aim to formulate niosomes, a nonionic nontoxic surfactant, TW-20, and cholesterol forming vesicles to study the modulation of the photophysical properties of curcumin upon encapsulation into the niosomal membrane. In solution, curcumin predominantly exists as the keto−enol tautomeric form with a strong intramolecular hydrogen bond in the ground state as shown in Scheme 1.30,31 Upon photoexcitation curcumin exhibits excited state intramolecular hydrogen atom (or proton) transfer (ESIHT or ESIPT) process.32−39 It has been proposed that the labile hydrogen of curcumin which is involved in the ESIHT process plays an important roles in mediating or at least is correlated with its medicinal properties.40 Along with the ESIHT process, solvation dynamics is another major photophysical process involved in the excited state of curcumin.35−39 Using ultrafast fluorescence upconversion spectroscopy, Adhikary et al.19,35 have shown that the time constant of the ESIHT process of Received: April 15, 2013 Revised: May 13, 2013
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due to the presence of two different types of microenvironments, namely, the hydrophobic niosomal bilayer and the hydrophilic core filled with water. Very recently, Bhattacharyya et al.45 have studied proton transfer dynamics of pyranine in TX-100 and cholesterol forming niosomes in presence of different concentrations of NaCl. However, in their experimental studies the niosomes were bigger (∼1000 nm) in size which are obviously less rigid and more hydrated than the small sized niosomes (∼100 nm). Their results indicate the retardation of the excited state intermolecular proton transfer (ESPT) in niosome solution. The proton transfer process is further retarded upon addition of NaCl. In an earlier study, we have shown how niosomes containing PEG6000 in the bilayer affect the photophysical properties of a potent excited state intramolecular proton transfer (ESIPT) chromophore, 1′hydroxy-2′-acetonaphthone.47 In the present work we have employed two different types of microenvironments, micelles and vesicles formed from a common nonionic surfactant, TW-20, to compare the modulation of the photophysical properties of curcumin upon encapsulation. The formation of small unilamellar TW-20/ cholesterol (in 2:1 mole ratio) niosomes with almost uniform size distribution has been confirmed from dynamic light scattering and transmission electron microscopy measurements. Our results suggest that the presence of cholesterol moiety in the hydrophobic region of the niosome membrane largely affects the photophysics and photodynamics of curcumin which is reflected in the steady-state and time-resolved fluorescence studies. By comparing the results, we have shown that the more rigid and confined environment provided by niosomes are more effective than normal micelles at solubilizing and stabilizing curcumin.
Scheme 1. Chemical Structure of the Keto−Enol Form of Curcumin with Strong Intramolecular Hydrogen Bond Involved in ESIHT Process
curcumin in the conventional surfactants (SDS/CTAB/TX100) forming micellar systems ranges from 50 to 80 ps. Ghosh et al.36 have studied the ultrafast dynamics in the excited state of curcumin in different solvents of varying polarity and hydrogen bonding ability using subpicosecond fluorescence upconversion and transient absorption spectroscopic techniques. Their study reveals that both the hydrogen bond donating and accepting ability of the neat solvents largely affect the hydrogen atom transfer process of curcumin through the perturbation of the existing intramolecular hydrogen bond. In polar solvents, the intramolecular hydrogen bonding is perturbed through dipole−dipole interaction, resulting in a reduced nonradiative deactivation mechanism. However, in protic solvents, although the intramolecular hydrogen bonding is hampered, a new nonradiative deactivation pathway is generated through the formation of intermolecular hydrogen bonding with the solvent molecules. Huppert and co-workers37,38 have recently demonstrated the nonradiative process by the polar protic solvent-controlled proton transfer of curcumin in a wide range of temperatures. They have also investigated the effect of the hydrogen bond accepting ability of the mild base on the PT process of curcumin in methanol and ethanol.38 Das et al.39 have studied the spectral relaxation of curcumin in binary mixtures of water and toluene in the picoseconds time scale. Their results indicate that modulation of the external hydrogen bonding network by the judicious selection of binary solvent mixture plays an important role on the dynamics of ESIHT process of curcumin. The solvent mediated nonradiative deactivation pathways as well as the dynamics of ESIHT are found to be significantly modulated upon encapsulation of the probe molecules into the hydrophobic nanocavities of micelles, pseudomicelles, mixed micelles, vesicles, proteins, and cyclodextrins.16−22,41−44 The proton transfer dynamics in these confined systems plays an important role in many biological processes. The water molecules present in the hydration layer of the micelles and vesicles are responsible for controlling the photophysical processes in such systems. The nonionic surfactants forming vesicles called niosomes can impart desirable nanostructures to provide better protection of the bound guests from the interaction with water.45−51 Niosomes are similar in structure and properties to that of phospholipids forming vesicles (liposomes). In recent years niosomes have been extensively applied in drug delivery as an alternative to liposomes because of their biodegradability, biocompatibility, and low toxicity.52−54 Moreover, niosomes offer a number of basic advantages over liposomes such as high stability, ease of preparation, and relatively low cost of surfactants which make these systems attractive for many chemical, biological, and industrial applications. Niosomes are very effective in solubilizing both hydrophobic and hydrophilic drug molecules
2. EXPERIMENTAL SECTION 2.1. Materials. Curcumin (purity ∼80%) was purchased from Sigma-Aldrich and used as received without further purification. Using high purity curcumin (≥98.5%), Petrich and co-workers have shown that the presence of other curcuminoids (∼20%) negligibly affects the photophysics of curcumin.19 Tween-20 (TW-20) and Tween-80 (TW-80) were also purchased from Sigma-Aldrich and used as received. Cholesterol obtained from SRL was used as received. The chemical structures of the surfactants and cholesterol are presented in Scheme 2. 2.2. Instrumentation. Steady-state UV−vis absorption and fluorescence emission spectra of the samples were recorded on a Shimadzu (model UV 2450) UV−vis spectrophotometer and an Hitachi (model F-7000) spectrofluorometer, respectively. The time-resolved emission spectra were recorded using a timecorrelated single photon counting (TCSPC) picosecond spectrometer. The detailed experimental setup of this instrument has been described in our previous publication.55 In brief, a picoseconds diode laser at 408 nm (IBH, UK, Nanoled) was used as light source, and the signal was detected in magic angle (54.7°) polarization using a Hamamastsu MCP PMT (3809U). The typical instrument response function for the picosecond diode laser at 408 nm is ∼90 ps in our system. The decays were analyzed using IBH DAS-6 decay analysis software. The fluorescence quantum yields of curcumin in the aqueous micellar and vesicular solution were determined using coumarin 153 with absolute quantum yield 0.56 in acetonitrile at 25 °C as secondary standard. The following equation was used for calculation: B
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formation, stability, and rigidity of the niosomal membrane, which in turn affect the binding and location of the drug molecules, strongly depend upon the concentration of cholesterol. In the earlier investigations it has been suggested that Tween-20 molecules can form niosomes in the presence of a minimum concentration of cholesterol.57 Cholesterol is a hydrophobic molecule which increases the stability and rigidity of the niosomal membrane and therefore located in the hydrophobic core of the bilayer (Scheme 3).56 Recently, much
Scheme 2. Chemical Structure of TW-20, TW-80, and Cholesterol
Scheme 3. Schematic Representation of Unilamellar Vesicles with the Bilayer Formed by a Nonionic Surfactant Tween-20 and Cholesterol
ΦS A (Abs)R nS2 = S ΦR AR (Abs)S nR 2
(1)
where Φ represents quantum yield, Abs represents absorbance, A represents area under the fluorescence curve, and n is refractive index of the medium. The subscripts S and R denote the corresponding parameters for the sample and reference, respectively. 2.3. Preparation of Niosome. Small unilamellar vesicles were obtained from nonionic surfactant/cholesterol aqueous dispersion by means of the film method as reported in the literature.56 The methods of preparation of niosomes from nonionic surfactants have been extensively reviewed in a number of articles.56−58 In our present work, TW-20 and cholesterol (molar ratio = 2:1) were dissolved in a CHCl3/ CH3OH (3:1 v/v) mixture in a round-bottomed flask. The concentration of TW 20 was kept ∼1.25 mM, which is remarkably above the cmc. After the evaporation of the solvents, the dried film was hydrated by the addition of 5 mL of double distilled Milli-Q water. The dispersion was then sonicated for 20 min at 60 °C using a sonicator. Finally, the solution was centrifuged to remove free surfactants and larger vesicles. 2.4. Structural Characterization of Niosomes. Transmission electron microscopy (TEM) analysis was carried out for the structural analysis of niosomes by using a JEOL model JEM 2010 transmission electron microscope at an operating voltage 200 kV. TEM images of the niosomes have been taken by using 1 wt % aqueous solution of uranyl acetate as staining agent. Primarily dynamic light scattering (DLS) measurements were carried out to determine the intensity−size distribution of the niosomes in aqueous solution using a Malvern Nano ZS instrument employing a 4 mW He−Ne laser (λ = 632 nm).
interest has been aroused in studying the effects of cholesterol on the structure of lipid bilayer of liposomes both theoretically and experimentally.59,60 Figures 1A and 1B represent the intensity−size distribution histogram of nonionic surfactant, Tween-20, forming normal micelles and cholesterol encapsulated vesicles (niosomes), respectively, as obtained from dynamic light scattering measurements at 25 °C . The average diameter of normal TW-20 micelles is found to be ∼8 nm, while the niosomes formed from TW-20 and cholesterol is found to be ∼100 nm in size. The obtained size of TW-20 micelles is well correlated with the size (∼7.2 nm) reported by Dosche et al.61 using dynamic light scattering and fluorescence correlation spectroscopy techniques. Moreover, the sizes of Tween micelles are independent of concentration (10−4−10−2 M) and also temperature in consistent with the literature report.61 The observation of a narrow intensity−size distribution in the DLS measurement further indicates almost uniform distribution of niosomes in the solution. To get a direct evidence for the formation of niosomes, we have performed TEM measurements. It further provides insight into the microstructural properties of niosomes. Figures 1C to 1G show the TEM images of niosomes with the size distribution ranges from 80 to 200 nm. The average diameter of the niosomes obtained from these images is found to be well correlated with that obtained from DLS measurements. The presence of a bilayer is clearly observed from high-resolution TEM image of a single niosome shown in Figure 1G. The images have been taken using 1 wt % of uranyl acetate as
3. RESULTS AND DISCUSSION 3.1. Structural Characterization of Niosomes. Since the size, shape, and structural properties of niosomes are highly sensitive to the methods of preparation and the concentration of surfactants, it is very necessary to establish the structural features of the niosomes that have been used for our photophysical studies.56−58 It is of great interest because the C
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Figure 1. (A) and (B) represent the DLS intensity−size distribution histogram of TW-20 micelles and TW-20/cholesterol forming niosomes formed in aqueous solution, respectively. Parts (C) to (G) show the TEM images of Tween-20/cholesterol (mole ratio 2:1) niosome formed in aqueous solution through the solvent evaporation method. (Staining agent is uranyl acetate (1 wt %).) The arrows indicate the presence of a niosomal bilayer.
staining agent. In Figures 1C and 1D, the size of few niosomes apparently looks smaller as they are far from the ocular. 3.2. Spectroscopic Studies of Curcumin in Micelles and Niosomes. 3.2.1. Steady-State UV−vis Absorption Studies. The changes in the spectral features of curcumin in micelles and niosomes have been investigated in a systematic way to examine the influence of varying the microenvironments on its photophysical properties. Both the TW-20 and TW-80 micelles have been used in order to understand if there is any significant effect of increasing chain length of the hydrophobic tail part of the surfactants on the binding dynamics of curcumin. The UV−vis absorption spectra of curcumin in the aqueous solution of increasing concentration of surfactants TW-20 and TW-80 have been given in Figures 2a and 2b, respectively, while the absorption spectrum in niosome is given in the inset of Figure 2b. As we can see that with increasing concentration of surfactant in the aqueous solution of curcumin (∼10 μM) the absorption intensity increases, suggesting an increase in the solubilization of curcumin inside the micellar
assemblies. In neutral water, curcumin exhibits a broad absorption band at ∼430 nm with a shoulder at ∼355 nm which are assigned to the π−π* transitions involved in the conjugated curcumin and the feruloyl unit, respectively. The peak at 355 nm appears in the aqueous solution as a result of the strong interaction of water molecules with curcumin.16,20,62 The shoulder peak at 355 nm completely disappears with the appearance of an intense peak at 424 nm above the cmc (0.050 and 0.012 mM for TW-20 and TW-80, respectively) of the surfactants. Such changes in the absorption spectra in the presence of surfactants may serve as an indication of lower level of interaction of curcumin with water. In Tween surfactants forming micelles the vibronic structure of the absorption spectra of curcumin exhibits maxima at 424 nm with a shoulder peak at 445 nm. The absorption spectrum of curcumin has been found to be more structured when it is encapsulated into the bilayer of niosomes as can also be seen in the fluorescence excitation spectra of curcumin in niosome in Figure 3.19 This is due to the change in the micropolarity and rigidity of the D
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Figure 2. UV−vis absorption spectra of curcumin with increasing concentration of nonionic surfactants (0 to 0.30 mM) (a) Tween-20 and (b) Tween-80. Changes in the absorption spectra of curcumin in micelles with increasing time are shown in parts (c) and (d). The insets show the extent of degradation of curcumin in terms of the decrease in absorbance at 430 nm with increasing time interval.
ent absorption spectra of curcumin in TW-20 and TW-80 micelles have been given in Figures 2c and 2d to show the extent of degradation in a confined microenvironment. The reduced level of degradation of curcumin upon encapsulation into the micellar assemblies is a clear manifestation of decrease in interaction with water. The lower level of degradation of curcumin has also been observed upon binding with the hydrophobic nanocavities of proteins and cyclodextrins.16,18 Harada et al.18 studied the formation of inclusion complexes of curcumin with various cyclodextrins and indicated that the level of suppression of degradation in α-CD and β-CD is substantially less than that observed for γ-CD, suggesting relatively weak interaction of curcumin with cyclodextrins particularly with the former CDs. The same group has also demonstrated the utilization of an anionic surfactant, SDS, forming micellar assemblies to deliver curcumin from the cyclodextrins cavity sites to the micellar environments and thereby observed an increase in fluorescence intensity with enhanced quantum yield. The greater the extent of interaction of curcumin with water, the greater will be the extent of degradation. The degradation level of curcumin may be further suppressed through the encapsulation into small niosomes as they provide better protection to the bound guest from the interaction of water. 3.2.2. Steady-State Fluorescence Studies. In the aqueous solution, curcumin exhibits a weak fluorescence with a redshifted broad emission spectrum centered at 550 nm. However, with increasing concentration of surfactant, there occurs a significant enhancement of fluorescence intensity along with the blue-shift in the emission maxima (Figures 4a and 4b). Moreover, the fluorescence quantum yield increased to a
Figure 3. Fluorescence excitation spectrum of curcumin in niosomes and emission spectra of curcumin in niosomes and micelles.
niosomal membrane compared to the hydration layer of micelles. Moreover, the accessibility of water in niosomal bilayer is less than that in the palisade layer of the nonionic micelles due to the presence of hydrophobic cholesterol molecules in the niosome membrane. As we mentioned earlier, in aqueous solution curcumin undergoes a high level of degradation within the time period of 30 min, which is reflected in the rapid decrease of the absorption intensity of curcumin. In aqueous phosphate buffer (pH 7.4) solution, the level of degradation of curcumin is reported to be more than 80% after 1 h.18 Our results indicate that nonionic surfactants forming micellar assemblies significantly reduce the level of degradation (extent of degradation
