Effect of Vesicle-to-Micelle Transition on the Interactions of

Jul 12, 2016 - The addition of NaC causes phospholipid vesicles to transit into phospholipid/NaC mixed vesicles and phospholipid/NaC mixed micelles...
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Effect of Vesicle-to-Micelle Transition on the Interactions of Phospholipid/Sodium Cholate Mixed Systems with Curcumin in Aqueous Solution Sha Zhang and Xiaoyong Wang* School of Chemistry and Molecular Engineering, East China University of Science and Technology, Shanghai 200237, China

ABSTRACT: The role of vesicle-to-micelle transition has been investigated in the interactions of phospholipid vesicles, phospholipid/sodium cholate (NaC) mixed vesicles, and phospholipid/NaC mixed micelles with curcumin in aqueous solution. The addition of NaC causes phospholipid vesicles to transit into phospholipid/NaC mixed vesicles and phospholipid/NaC mixed micelles. Turbidity measurement reveals that the presence of curcumin increases the NaC concentration for the solubilization of phospholipid vesicles, which indicates that the bound curcumin tends to suppress the vesicle-to-micelle transition. The pyrene polarity index and curcumin fluorescence anisotropy measurements suggest that phospholipid/NaC mixed micelles have a more compact structure than that of phospholipid vesicles and phospholipid/NaC mixed vesicles. Curcumin associated with phospholipid vesicles, phospholipid/NaC mixed vesicles, and phospholipid/NaC mixed micelles often results in higher intensities of absorption and fluorescence than those of free curcumin. However, phospholipid/NaC mixed vesicles lead to the highest values of absorption and fluorescence intensities, binding constant, and radical-scavenging capacity with curcumin. The different structures in the phospholipid bilayer of phospholipid/NaC mixed vesicles and the hydrophobic part of phospholipid/NaC mixed micelles where curcumin located are discussed to explain the interaction behaviors of phospholipid/NaC mixed systems with curcumin.

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INTRODUCTION Drug carriers are widely used to enhance drug aqueous solubility, maintain drug stability, and improve drug bioavailability. 1 However, before reaching the intended sites, introduction of drug-containing carriers is often accompanied by a change in the structures of the carriers, which may be brought about by their contact with biomolecules such as enzymes, proteins, and biosurfactants in the human body.2,3 Therefore, knowledge of the interaction mechanisms of both initial and structure-changed carriers with drugs has important value for controlling the benefits of drug carriers. As a polyphenol compound extracted from the rhizome of turmeric, curcumin has become a research focus in recent years due to its low intrinsic toxicity but diverse biological and pharmacological effects, including antioxidant, anticancer, and anti-inflammatory activities.4−6 Chemically, curcumin is a diferuloyl methane molecule containing two ferulic acid residues linked by a methylene bridge. The health-promoting activities of curcumin are related to the hydroxyl groups of the © 2016 American Chemical Society

benzene rings, double bonds in the alkene part, and the central β-diketone moiety.4 However, animal and human studies on the absorption, distribution, metabolism, and excretion of curcumin disclosed that curcumin undergoes rapid intestinal metabolism, which is the root cause of the low bioavailability of curcumin.2,7 Previous work has demonstrated that liposomes are one of the effective carriers for resolving the problem of curcumin.8−10 Liposomes are phospholipid vesicles with the hydrophobic chains of the phospholipids forming the bilayer and the polar headgroups of the phospholipids oriented toward the extravesicular solution and inner cavity. Curcumin can associate with the phospholipid bilayer of liposomes by hydrophobic interactions and hydrogen bonding, which may greatly improve the solubility, stability, and various bioactivities of curcumin.11 The liposome formulation is easily destabilized under Received: March 9, 2016 Revised: July 10, 2016 Published: July 12, 2016 7392

DOI: 10.1021/acs.jpcb.6b02492 J. Phys. Chem. B 2016, 120, 7392−7400

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The Journal of Physical Chemistry B

hydration of the thin phospholipid film with distilled water (pH 6.5) by first stirring for 30 min at room temperature and then incubating for 120 min at 60 °C, samples of 600 μM phospholipid vesicles with desired amounts of curcumin (0− 12.5 μM) were obtained. The samples of phospholipid vesicles with and without curcumin have a constant pH of ∼6.4. To ensure interpreparation comparability, the samples of phospholipid vesicles were checked by dynamic light scattering, and only the samples with a polydispersity index value smaller than 0.25 were used. Solubilization of Phospholipid Vesicles. Turbidity measurements were used to determine the phase boundary of the solubilization of phospholipid vesicles with and without 10 μM curcumin. After sodium cholate (NaC) was added, the mixed solution was equilibrated for at least 30 min at room temperature until its optical density became constant before further analysis. Compared to the pH values of the initial phospholipid vesicles, the samples of phospholipid/NaC mixed solutions with and without curcumin have increased pH values (7−7.4), depending on the amount of added NaC. The turbidity of phospholipid/NaC mixed solutions was measured by measuring the absorbance at 500 nm (A500) on a Shimadzu UV-1800 spectrometer at 25 °C. If the sample contains curcumin, the absorbance of curcumin with the same concentration in water at 500 nm is subtracted from the A500 value for calculating the sample turbidity. From the turbidity curve as a function of the NaC concentration, three samples of 600 μM phospholipid vesicles mixed with 0, 10, and 30 mM NaC were selected, corresponding to phospholipid vesicles, phospholipid/NaC mixed vesicles, and phospholipid/NaC mixed micelles, respectively. To compare the samples of curcumin with phospholipid/ NaC mixed systems and to determine the binding constants for curcumin with the three carriers, samples of curcumin dissolved in water were prepared. As curcumin is poorly soluble in water,25 a stock solution of curcumin in ethanol was prepared and diluted with distilled water. This is a common way to prepare curcumin samples.26−28 The ethanol content in free curcumin solutions is negligible. The obtained sample of curcumin in water is a clear solution, without curcumin precipitates even after centrifugation at 12 000 rpm. TEM Analysis. Samples of curcumin with phospholipid vesicles, phospholipid/NaC mixed vesicles, and phospholipid/ NaC mixed micelles were deposited onto carbon-coated copper grids. After 1−2 min, the films were negatively stained with a solution of 2% (v/v) phosphotungstic acid for 20 s, and the excess phosphotungstic acid was removed using filter paper. The dried copper grid was imaged on a JEOL model JEM 1400 TEM at an operating voltage of 200 kV. Steady-State Fluorescence Measurements. Steady-state fluorescence measurements were performed with a Shimadzu RF-5301 spectrofluorophotometer at 25 °C. The micropolarity of phospholipid vesicles, phospholipid/NaC mixed vesicles, and phospholipid/NaC mixed micelles was estimated from measurement of the pyrene polarity index (I1/I3). I1/I3 is the ratio of the intensities of the first and third vibronic peaks in the fluorescence emission spectrum due to pyrene. Pyrene was excited at a wavelength of 335 nm, and the fluorescence spectra were scanned over the spectral range of 350−500 nm. The steady-state fluorescence spectra of 10 μM curcumin in water, phospholipid vesicles, phospholipid/NaC mixed vesicles, and phospholipid/NaC mixed micelles were taken from 430 to 600 nm, with an excitation wavelength of 423 nm.

physiological conditions by bile salts,12,13 which are a group of biosurfactant molecules produced in the liver and distributed widely in the gastrointestinal tract. Bile salts have a rigid steroid backbone with methyl groups on the convex hydrophobic surface and hydroxyl groups on the concave hydrophilic surface.14,15 The interactions of bile salts with lipids play a significant role in the digestion and absorption of dietary lipids.16 Similarly, bile salts can induce the solubilization of liposomes through the penetration of their molecules into the phospholipid bilayer to form mixed vesicles of phospholipids and bile salts, which subsequently transform into small phospholipid/bile salt mixed micelles.17,18 The selfassembled micelles of bile salts are also good carriers for many bioactive molecules.19,20 Patra et al. found that the formation of mixed vesicles of phospholipids and bile salts can increase the fluorescence of curcumin owing to the enhanced viscosity of liposomes on addition of bile salts.21 Dongowski et al. found that bile salt micelles and phospholipid/bile salt mixed micelles decrease the absorption of quinine in vivo and explained this result in terms of the decreased thermodynamic activity of quinine after its incorporation into micellar systems.22 Their work revealed that the absorption of quinine depends not only on the quantity of carriers but also on the different interactions of quinine with the phospholipid and bile salt. However, there is no report so far showing the quantitative estimation of the role of vesicle-to-micelle transition from phospholipid vesicles via phospholipid/bile salt mixed vesicles to phospholipid/bile salt mixed micelles in their interaction behaviors with curcumin. In the present work, sodium chlolate (NaC) is selected as a representative bile salt to solubilize phospholipid vesicles. Turbidity, transmission electron microscopy (TEM), and pyrene polarity index measurements have been employed to investigate the phase boundary and structure changes associated with vesicle-to-micelle transition. Following the study of changes in the absorption and fluorescence properties of curcumin, the interaction mechanisms of phospholipid vesicles, phospholipid/NaC mixed vesicles, and phospholipid/ NaC mixed micelles with curcumin were studied. Further, the free-radical-scavenging activity of curcumin was also investigated.

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MATERIALS AND METHODS Materials. Phospholipid, sodium cholate (NaC), curcumin, pyrene, and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were purchased from Sigma-Aldrich Chemical Company. As provided by the supplier, phospholipid from dried egg yolk has the following fatty acid content: 33% C16:0 (palmitic), 13% C18:0 (stearic), 31% C18:1 (oleic), 15% 18:2 (linoleic), and other fatty acids as minor contributors. The phospholipid has an average of 60% phosphatidylcholine, and the remaining 40% consists of mostly phosphatidylethanolamine plus other phospholipids as well as traces of triglycerides and cholesterol. The water used in the experiments was double distilled, and the other chemicals were of analytical reagent grade. Preparation of Phospholipid Vesicles. The thin-film hydration method was used to prepare phospholipid vesicles.23,24 The phospholipid and different amounts of curcumin were dissolved in a mixture of methanol and chloroform in a volume ratio of 1:2 in a round-bottom flask. The organic solvents were evaporated at 0.09 MPa on a rotary evaporator for 30 min at 43 °C. The obtained dried thin phospholipid film was further maintained under vacuum at 0.09 MPa for 30 min to remove solvent traces. After complete 7393

DOI: 10.1021/acs.jpcb.6b02492 J. Phys. Chem. B 2016, 120, 7392−7400

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The Journal of Physical Chemistry B The fluorescence anisotropy (r) value of 10 μM curcumin in water, phospholipid vesicles, phospholipid/NaC mixed vesicles, and phospholipid/NaC mixed micelles was determined by measuring the fluorescence of curcumin using the fluorescence polarization technique. The fluorescence intensities of curcumin were obtained at 0−0, 0−90, 90−0, and 90−90 angle settings of the excitation and emission polarization accessories at 25 °C. The value of r was calculated as follows29 r = (I − G × I⊥)/(I + 2G × I⊥)

DPPH solution and a curcumin sample, and Acontrol is the absorption of DPPH solution and double distilled water. Statistical Analysis. Data are presented as means ± standard deviations. For all measurements, a minimum of three to four replicates were taken for data analysis.

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RESULTS AND DISCUSSION The addition of surfactant to phospholipid vesicles can result in phase transition of the vesicles into mixed micelles, which is called “solubilization”.13,17 The phase boundary of solubilization of phospholipid vesicles is studied through the changes in the turbidity of phospholipid/NaC mixed solutions as a function of NaC concentration. As shown in Figure 1, whether

(1)

where I∥ and I⊥ are the fluorescence intensities of the emitted light polarized parallel and perpendicular to the exciting light, respectively, and G is the grating correction factor, which is the ratio of the sensitivities of the instrument for vertically and horizontally polarized light. Ultraviolet−Visible (UV−Vis) Absorption Spectra Measurement. The absorption spectra of 10 μM curcumin in water, phospholipid vesicles, phospholipid/NaC mixed vesicles, and phospholipid/NaC mixed micelles were recorded at 350−525 nm using a Shimadzu UV-1800 spectrophotometer at 25 °C. The binding constants (Kb) of phospholipid vesicles, phospholipid/NaC mixed vesicles, and phospholipid/NaC mixed micelles with curcumin were determined on the basis of the change in the maximum absorption of curcumin at different concentrations according to the Benesi−Hildebrand equation.26,27 1 1 1 1 = + × A − A0 a a·Kb Ccurcumin

(2)

where A0 corresponds to the maximum absorption of free curcumin; A is the recorded maximum absorption of curcumin in phospholipid vesicles, phospholipid/NaC mixed vesicles, and phospholipid/NaC mixed micelles; Ccurcumin is the curcumin concentration, ranging from 2 to 12.5 μM; and a is a constant equal to ΔεCcarrierl, where Δε corresponds to the change in the molar extinction coefficient at the wavelength of the study, Ccarrier is the concentration of the carrier (phospholipid vesicles, phospholipid/NaC mixed vesicles, or phospholipid/NaC mixed micelles), and l is the optical path length. Binding constant Kb can be estimated from the ratio of the intercept, 1/a, to the slope, 1/(a·Kb), of the double reciprocal plot of 1/(A − A0) versus 1/Ccurcumin. DPPH-Scavenging Capacity Measurement. The radicalscavenging activity of curcumin in phospholipid vesicles, phospholipid/NaC mixed vesicles, and phospholipid/NaC mixed micelles was examined according to the DPPH method.30 An ethanolic solution of 300 μM DPPH was mixed with the sample of each curcumin formulation. The maximal absorption of DPPH at 517 nm was monitored for 120 min on a Shimadzu UV-1800 spectrophotometer at 25 °C. The percentage of DPPH-scavenging capacity of curcumin was calculated according to the following equation, and the influence of the mixed solution was subtracted DPPH scavenging capacity (%) ⎛ A sample − A blank ⎞ = ⎜1 − ⎟ × 100% Acontrol ⎝ ⎠

Figure 1. Turbidity of 600 μM phospholipid vesicles with and without 10 μM curcumin as a function of NaC concentration. The turbidity is reported as the absorbance of a phospholipid/NaC mixed solution at 500 nm, which is subtracted for the absorbance of 10 μM curcumin in water for the sample containing curcumin. Attached TEM pictures: (a) curcumin phospholipid vesicles, (b) curcumin phospholipid/NaC mixed vesicles at 10 mM NaC, (c) curcumin phospholipid/NaC mixed micelles at 30 mM NaC.

curcumin is added or not, the variations in the turbidity curves against NaC concentration reveal that phospholipid vesicles can be solubilized by the addition of NaC. Lichtenberg et al. previously proposed a three-stage model for surfactant-induced solubilization of phospholipid vesicles.31,32 However, the observed nonlinearity both below and above the maximum in the two turbidity curves suggests that solubilization of phospholipid vesicles involves more than three stages, related to the ratio of the phospholipid to NaC, in agreement with recent studies on mixed lipid/bile salt systems.13,17,18 The Hbonding and ionic interactions of the hydrophilic headgroup of NaC with the headgroup of the phospholipid and the subsequent force between the convex hydrophobic surface of NaC and the hydrophobic phospholipid acyl chains will cause

(3)

where Ablank is the absorption of a mixed solution of a curcumin sample and ethanol, Asample is the absorption of a mixture of 7394

DOI: 10.1021/acs.jpcb.6b02492 J. Phys. Chem. B 2016, 120, 7392−7400

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varying portion and the almost-horizontal portion at high concentration in the pyrene polarity index, I1/I3, plot in Figure 2. The increased critical micelle concentration of NaC in the

NaC molecules to gradually incorporate into the phospholipid bilayer to form phospholipid/NaC mixed vesicles.33 The resulting size growth of the phospholipid/NaC mixed vesicles can be responsible for the initial increasing part of the turbidity curve.34 After the phospholipid bilayer is saturated with NaC molecules, the dramatic decrease in the turbidity curve indicates that larger phospholipid/NaC mixed vesicles start to disintegrate and fragment into smaller phospholipid/NaC mixed micelles. During this transformation process, various intermediate structures of phospholipid/NaC mixed aggregates, such as nonspherical and thread-like aggregates, were found by other researchers.35,36 The final leveling part of the turbidity curve suggests that all phospholipids are presented as a part of phospholipid/NaC mixed micelles after complete destruction of the phospholipid/NaC mixed vesicles. Meanwhile, the TEM photomicrographs in Figure 1 give direct evidence for the solubilization of phospholipid vesicles with curcumin, induced by the addition of NaC. Using static and dynamic light scattering, Cevc et al. found that the vesicleto-micelle transition in a phospholipid/NaC mixed system was significantly influenced by the starting size and morphology of the phospholipid vesicles.37 Both phospholipid vesicles and phospholipid/NaC mixed vesicles appear as spherical vesicular structures with a bilayer. The particle size of curcumin phospholipid vesicles (∼350 nm) estimated from the TEM picture is close to our previous results obtained using dynamic light scattering.23 When 10 mM NaC is added, curcumin phospholipid mixed vesicles present bigger particle sizes of about 600 nm. A significant size growth is often observed in the lipid/surfactant systems via a “disproportionation mechanism”,32 which consists of a series of repartitioning processes through which the larger vesicles in the population grow in size by absorbing the lipid initially contained in the smaller vesicles in the population. The smallest particle size of curcumin phospholipid/NaC mixed micelles (∼70 nm) at 30 mM NaC suggests that mixed micelles constitute the most stable aggregates in the mixture of phospholipids with surfactants above a certain amount of surfactant. Nevertheless, it should be pointed out that the value of ∼70 nm is incompatible with a spherical micellar structure. With the help of cryo-TEM, Walter et al. identified large nonspheroidal and small spheroidal phospholipid/NaC mixed micelles, depending on the ratio of phospholipid to NaC.35 Maza et al. previously determined an average particle size of 52 nm for the mixed micelles of phospholipid with sodium dodecyl sulfate,38 which is close to but still smaller than that of our curcumin phospholipid/NaC mixed micelles. It is known that NaC has a molecular structure much different from that of the classical surfactant sodium dodecyl sulfate. Curcumin phospholipid/NaC mixed micelles possibly have big disk-like structure due to the rigid steroid backbone of NaC, as discovered in the case of pure bile salt micelles.39 In addition, the polydispersity index value of curcumin phospholipid/NaC mixed micelles is about 0.24, determined by dynamic light scattering. This relatively large polydispersity index value indicates the high polydispersity of phospholipid/NaC mixed micelles, which is also one special feature of bile salt micelles. It is notable that the maximum turbidity appears at 15 mM NaC for the solubilization of phospholipid vesicles but at 20 mM NaC for curcumin phospholipid vesicles. The critical micelle concentrations are 14.8 and 18.5 mM for NaC alone and NaC with 10 μM curcumin, respectively, determined from the intercept between the linear extrapolations of the rapidly

Figure 2. Variation of pyrene polarity ratio I1/I3 in water and phospholipid vesicles with and without 10 μM curcumin as a function of NaC.

presence of curcumin is consistent with our previous observation in a mixture of NaC with another polyphenol compound.20 The result of higher NaC concentrations at maximum turbidity compared to the critical micelle concentration of NaC suggests that surfactant micelles are formed only after the surfactant concentration increases above the critical micelle concentration, which are able to dissolve the phospholipid bilayer saturated by the surfactant. However, the different NaC concentrations of maximum turbidity for the two phospholipid/NaC mixed systems reveals that bound curcumin tends to suppress the solubilization of phospholipid vesicles. Barry et al. proposed by solid-state NMR that curcumin is anchored inside the phospholipid bilayer oriented along phospholipid acyl chains,11 through hydrogen bonding of the −OH groups of the phenolic rings of curcumin with the headgroup of the phospholipid and hydrophobic interactions of the aromatic rings of curcumin with the phospholipid acyl chains. The bound curcumin is expected to increase the packing density of the phospholipid bilayer and thus provide protection for the phospholipid vesicles against NaC solubilization. Meanwhile, NaC and curcumin may compete for the same initial binding sites in the phospholipid bilayer. Therefore, more NaC is needed to achieve the same NaC binding level under such “competitive-pressure” conditions. Other investigators previously reported enhanced stability of lipid membranes upon binding with cholesterol and carotenoids.40,41 Whereas cholesterol can form complexes with bile salts and thus diminish the bilayer solubilizing ability of the latter, curcumin and carotenoids bring “more lipid” into the bilayer, which requires extra solubilizer molecules before bilayer destruction. Figure 2 presents the variation of pyrene polarity index I1/I3 in phospholipid vesicles with and without 10 μM curcumin as a function of NaC concentration. As the NaC concentration increases from 0 to 40 mM, the I1/I3 values decrease gradually, irrespective of curcumin addition. This observation reveals that phospholipid/NaC mixed micelles tend to have a lower 7395

DOI: 10.1021/acs.jpcb.6b02492 J. Phys. Chem. B 2016, 120, 7392−7400

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0.22 to 0.34, which are greater than those for free curcumin. The increased anisotropy of curcumin is a manifestation of the transfer of curcumin from the bulk water to phospholipid vesicles, phospholipid/NaC mixed vesicles, and phospholipid/ NaC mixed micelles, where it experiences a high extent of rotation restriction. Curcumin was shown to be located in the bilayer of phospholipid vesicles via hydrophobic interactions and hydrogen bonding.11,20,21 When curcumin is bound to classical surfactant micelles, it is known to lie close to the polar headgroups in the palisade layer. Although the palisade layer does not exist in bile salt micelles, phospholipid/NaC mixed micelles still have hydrophobic as well as hydrophilic parts, which are made up of the hydrophobic and hydrophilic groups of phospholipid and NaC, respectively. Curcumin is expected to mainly bind in the inner hydrophobic part of phospholipid/ NaC mixed micelles, which is constituted of convex hydrophobic surfaces of NaC molecules and a small amount of phospholipid acyl chains. Meanwhile, the r values of curcumin present the same descending trend with increasing curcumin concentration in the three kinds of carriers. The lower r values at a higher curcumin concentration give an indication that the bound curcumin disturbs the packing of phospholipid vesicles, phospholipid/ NaC mixed vesicles, and phospholipid/NaC mixed micelles with loose and disordered structures. The change in the fluorescence anisotropy of curcumin is consistent with the above result of pyrene polarity index I1/I3 and the previously reported reduction in the phase transition temperature of liposomes with increasing curcumin concentration.24 Compared to those in phospholipid vesicles and phospholipid/NaC mixed vesicles, the higher r values of curcumin in phospholipid/ NaC mixed micelles may be attributed to the compact packing in phospholipid/NaC mixed micelles due to the high amount of NaC with a rigid molecular structure. This is the same reason for the relatively less change in the r values of curcumin in phospholipid/NaC mixed micelles. The observed smaller r values in phospholipid vesicles and phospholipid/NaC mixed vesicles can be attributed to the smaller amounts of NaC molecules. However, the significantly linear decrease in the r values throughout the investigated curcumin concentrations in the systems of phospholipid vesicles and phospholipid/NaC mixed vesicles reveals that the addition of curcumin disturbs the packing of vesicular systems to a larger extent. Finally, it should be noted that the r values of curcumin are the average fluorescence anisotropies of curcumin because there are possibly more than one kind of binding location for curcumin associated with each carrier. Especially, the observed greater nonlinearity of the r versus Ccurcumin curve for curcumin associated with phospholipid/NaC mixed micelles reveals the significant heterogeneity of the hydrophobic part of phospholipid/NaC mixed micelles. Figure 4 shows the spectra of UV−vis absorption and steadystate fluorescence of 10 μM curcumin in water, phospholipid vesicles, phospholipid/NaC mixed vesicles, and phospholipid/ NaC mixed micelles. Curcumin in water has an absorption peak at 430 nm and a shoulder at 360 nm, which are assigned to the π−π* excitation of the conjugated diferuloyl structure and feruloyl unit of curcumin, respectively.42,43 Meanwhile, curcumin in water has a broad but weak fluorescence spectrum with a peak at 540 nm owing to the high polarity of the medium. These observations are similar to the absorption and fluorescence features of curcumin in aqueous buffers.44 However, when curcumin associates with phospholipid vesicles,

micropolarity than phospholipid vesicles and phospholipid/ NaC mixed vesicles. At NaC concentrations below 20 mM, the values of I1/I3 in blank phospholipid vesicles and phospholipid/ NaC mixed vesicles are much smaller than the values measured in water, which may be ascribed to the hydrophobic microenvironments in phospholipid vesicles and phospholipid/NaC mixed vesicles. However, after phospholipid/NaC mixed micelles are completely formed above 20 mM NaC, nearly identical I1/I3 values are observed for phospholipid/NaC mixed micelles and pure NaC micelles, indicating similar micropolarities for the two kinds of micelles. It is also noted that the changes in the I1/I3 values are somewhat different in phospholipid vesicles with and without curcumin. On the one hand, I1/I3 exhibits relatively higher values in curcumin phospholipid vesicles throughout the investigated concentrations of NaC. On the other hand, at 5−15 mM NaC, the I1/I3 values in curcumin phospholipid vesicles show a relatively greater change than those in phospholipid vesicles without curcumin, which is consistent with the steeper change in the initial increasing part of the turbidity curve of curcumin phospholipid vesicles in Figure 1. These differences could have resulted from the bound curcumin disturbing the microstructure of its carriers, although curcumin may protect phospholipid vesicles against NaC solubilization. Fluorescence anisotropy (r) is an experimental measure of the fluorescence depolarization mainly caused by rotational diffusion of the fluorophore during the excited lifetime. Usually, a higher r value reveals greater restriction of rotation of the fluorophore during the excited lifetime. The binding of curcumin with different carrier systems can be revealed by the r value of curcumin, determined through the fluorescence polarization technique. Figure 3 shows the variation of the r

Figure 3. Variation of r values of curcumin in water, phospholipid vesicles, phospholipid/NaC mixed vesicles, and phospholipid/NaC mixed micelles.

values of curcumin with increasing curcumin concentration in water, phospholipid vesicles, phospholipid/NaC mixed vesicles, and phospholipid/NaC mixed micelles. Curcumin in water has a small and nearly unchanged r value (∼0.14) as the curcumin concentration increases from 1 to 15 μM, which may provide evidence for the dissolved state of free curcumin in water. The r values of curcumin in the three kinds of carriers range from 7396

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polarity than phospholipid/NaC mixed vesicles, the high amount of rigid NaC molecules leads to the compact structure of phospholipid/NaC mixed micelles. The compact packing in the hydrophobic part of phospholipid/NaC mixed micelles, indicated by the data of curcumin anisotropy, may restrict the interaction of phospholipid/NaC mixed micelles with curcumin. Besides the pronounced absorption peak at 430 nm, curcumin bound with the three carriers is observed to give a big absorption shoulder at 360 nm, which is even higher than that for curcumin in water. The absorption peak at 430 nm indicates that curcumin is located in the hydrophobic part of phospholipid vesicles, phospholipid/NaC mixed vesicles, and phospholipid/NaC mixed micelles. The absorption shoulder of curcumin at 360 nm is often used as an indicator for the interaction between curcumin and water. The absorption spectra of curcumin are further deconvoluted using the multipeak Gaussian fitting method with the help of Origin 8.5 software. The integrated absorbance ratios of the peak at 360 nm with the peak at 430 nm are 0.33, 0.38, 0.66, and 0.46 for curcumin in water, phospholipid vesicles, phospholipid/ NaC mixed vesicles, and phospholipid/NaC mixed micelles, respectively. On the basis of this preliminary result, it is not possible to know the amount of curcumin in different binding locations in the three carriers. The big absorption shoulder of curcumin at 360 nm suggests that some curcumin molecules are bound in sites with a relatively high polarity (even in contact with small amounts of water) in the hydrophobic part of the three carriers, which is consistent with the above anisotropy result. As shown in Figure 5, when the curcumin concentration increases from 2 to 12.5 μM, the absorption intensity of curcumin increases gradually in water, phospholipid vesicles, phospholipid/NaC mixed vesicles, and phospholipid/NaC mixed micelles, indicating similar concentration-dependent absorptions of curcumin in different carriers. The excellent linearity of the maximum absorption of free curcumin (A0) with curcumin concentration indicates that there are no curcumin aggregates and precipitates in the samples of free curcumin. The binding constants of phospholipid vesicles, phospholipid/ NaC mixed vesicles, and phospholipid/NaC mixed micelles with curcumin are determined according to the Benesi− Hildebrand method.26,27 From the ratio of the intercept to the slope of the linear plot of 1/(A − A0) versus 1/Ccurcumin, the acquired Kb values are (4.15 ± 0.12) × 105, (7.91 ± 0.07) × 105, and (3.19 ± 0.17) × 105 M−1 for the binding of phospholipid vesicles, phospholipid/NaC mixed vesicles, and phospholipid/NaC mixed micelles with curcumin, respectively. These Kb values are in the same range as the reported data for the binding of liposomes and surfactant micelles with curcumin.27,28,46 The strongest binding affinity for phospholipid/NaC mixed vesicles with curcumin is consistent with the result from the absorbance and fluorescence spectra of curcumin. Compared to the hydrophobicity and size of the bilayer of phospholipid vesicles, the incorporation of NaC molecules with high hydrophobicity makes phospholipid/NaC mixed vesicles have a more hydrophobic and larger bilayer, which favor the binding of curcumin with phospholipid/NaC mixed vesicles. However, in small phospholipid/NaC mixed micelles, compact packing of rigid NaC molecules in the hydrophobic part of the phospholipid/NaC mixed micelles weakens the binding of curcumin. It should be noted that the fitting plots of 1/(A − A0) versus 1/Ccurcumin based on the

Figure 4. Spectra of absorption and fluorescence of 10 μM curcumin in water, phospholipid vesicles, phospholipid/NaC mixed vesicles, and phospholipid/NaC mixed micelles. The absorbance of the reference sample without curcumin is subtracted for the absorption curves of curcumin samples.

phospholipid/NaC mixed vesicles, and phospholipid/NaC mixed micelles, both the absorption and fluorescence intensities of curcumin generally increase compared to those in water, together with clear blue shifts in the absorption and fluorescence peaks. It is known that the absorption and fluorescence spectra of curcumin can be enhanced together with blue shifts when solvents are changed from polar to nonpolar.45 The smaller I1/I3 values in phospholipid vesicles, phospholipid/NaC mixed vesicles, and phospholipid/NaC mixed micelles shown above compared to those in water may reveal the nonpolar microenvironments of the three carriers. The enhancements in the absorption and fluorescence intensities of curcumin as well as in their blue shifts in the three carriers are in the following order: phospholipid/NaC mixed vesicles > phospholipid vesicles > phospholipid/NaC mixed micelles. Curcumin is bound in the bilayer of phospholipid vesicles via hydrophobic interactions of the aromatic rings of curcumin with phospholipid acyl chains and via hydrogen bonding of the −OH groups of the phenolic rings of curcumin with the headgroup of the phospholipid.11 When phospholipid vesicles are transformed into phospholipid/NaC mixed vesicles, induced by a small amount of NaC, the penetrated NaC molecules do not destroy the structure of the phospholipid bilayer. However, the hydrophobic convex surface of NaC may increase the hydrophobicity of the bilayer of phospholipid/NaC mixed vesicles, leading to enhancements in the absorption and fluorescence intensities of bound curcumin. When the addition of high amounts of NaC leads to complete solubilization of phospholipid vesicles into phospholipid/NaC mixed micelles, curcumin is considered to bind in the hydrophobic part of the phospholipid/NaC mixed micelles.20,21 Although the I 1 /I 3 result for pyrene suggests that phospholipid/NaC mixed micelles provide a lower micro7397

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The DPPH method is employed to further test the interactions of phospholipid vesicles, phospholipid/NaC mixed vesicles, and phospholipid/NaC mixed micelles with curcumin. Upon reduction by an antioxidant, the characteristic maximum absorption of free-radical DPPH at 517 nm decreases proportionally to the increase in amount of nonradical diphenylpicrylhydrazine. Usually, curcumin exhibits a strong radical-scavenging ability owing to the donation of H from the keto−enol group of curcumin.47 Our previous work has demonstrated that curcumin associated with phospholipid vesicles and surfactant aggregates has a higher DPPHscavenging activity than that of free curcumin.23,28 Figure 6

Figure 6. Variation of the DPPH-scavenging capacity of 10 μM curcumin in phospholipid vesicles, phospholipid/NaC mixed vesicles, and phospholipid/NaC mixed micelles as a function of time.

shows curves of the DPPH-scavenging capacity of curcumin in phospholipid vesicles, phospholipid/NaC mixed vesicles, and phospholipid/NaC mixed micelles as a function of time. All curves of the DPPH-scavenging capacity of curcumin have two increasing steps as a function of time, which is consistent with that in the reported DPPH study.48 The DPPH-scavenging capacity of curcumin often shows an abrupt increase immediately after mixing DPPH with curcumin samples, followed by a much more gradual change. The first fastincreasing step results from the transfer of the most labile H atoms of curcumin to DPPH, and the following slow-increasing step features the residual H-donating ability of the antioxidant degradation products. Usually, only the fast step is subjected to kinetic analysis for the H-atom-transfer reaction between DPPH and the antioxidant. However, the almost identical slopes of the initial increase in the three DPPH curves indicate that the reaction rate for the reaction of curcumin with DPPH is kinetically fast and is nearly unaffected by the reaction medium. However, during the experiment, curcumin in phospholipid/NaC mixed vesicles generally has a higher DPPH-scavenging capacity than that in the other two carriers. Until 120 min after curcumin and DPPH were mixed, the values of the DPPH-scavenging capacity of curcumin were 49.1, 54.1, and 43.5% in phospholipid vesicles, phospholipid/NaC mixed vesicles, and phospholipid/NaC mixed micelles, respectively. The reduction of DPPH by curcumin is only possible when the two compounds are in contact. Thus,

Figure 5. Absorption spectra of curcumin in water, phospholipid vesicles, phospholipid/NaC mixed vesicles, and phospholipid/NaC mixed micelles at different concentrations of curcumin (from bottom to top): 2, 2.5, 3, 3.5, 4, 5, 7.5, 10, and 12.5 μM curcumin. (Insets) Linear plot of A0 vs Ccurcumin for free curcumin in water, and linear plots of 1/(A − A0) vs 1/Ccurcumin for determination of binding constants.

Benesi−Hildebrand equation do not show very good linearity. This indicates that there exists more than one kind of binding site for curcumin in the three carriers, which is also related to the amount of bound curcumin. 7398

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phospholipid/NaC mixed vesicles may serve as the most suitable medium for the reaction of curcumin with DPPH, where the keto−enol group of curcumin has the strongest Hdonating ability to reduce the DPPH radical.

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CONCLUSIONS

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AUTHOR INFORMATION

Although curcumin tends to suppress the NaC-induced solubilization of phospholipid vesicles into phospholipid/NaC mixed micelles, the vesicle-to-micelle transition significantly affects the interaction behaviors of phospholipid vesicles, phospholipid/NaC mixed vesicles, and phospholipid/NaC mixed micelles with curcumin. Compared to phospholipid vesicles and phospholipid/NaC mixed micelles, phospholipid/ NaC mixed vesicles with small amounts of NaC make curcumin bound in the bilayer yield a higher absorption and fluorescence, a larger binding constant, and better radical-scavenging ability. In contrast, the addition of high amounts of NaC causes the formation of phospholipid/NaC mixed micelles with a compact structure of the hydrophobic part, disfavoring the binding of curcumin. Although the Benesi−Hildebrand equation gives the total or average values of the binding constants for curcumin with phospholipid/NaC mixtures, the current spectral data of curcumin also reveals that there is more than one kind of binding site in the hydrophobic part of phospholipid/NaC mixtures. Detailed information about the exact binding sites with different binding kinetics may be obtained by fluorescence quenching and fluorescence lifetime studies of curcumin. The present work reveals that the changed structures of phospholipid/NaC mixtures during the solubilization of phospholipid vesicles greatly influence their interaction characteristics with curcumin, and this is worthy of further investigation in an attempt to effectively control the physicochemical properties and bioactivities of drugs.

Corresponding Author

*E-mail: [email protected]. Tel: 86-21-64252012. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is supported by the National Natural Science Foundation of China (21573071) and the Fundamental Research Funds for the Central Universities (Grant WK1213003).

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REFERENCES

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