and Nanoaggregated Chitosan Oligosaccharide Lactate - American

Article pubs.acs.org/JPCC

Two Modes of Associations of Curcumin with Pre- and Nanoaggregated Chitosan Oligosaccharide Lactate: Ionic Strength and Hydrophobic Bile Salt Modulate Partition of Drug and SelfAssembly Process Mazhar Chebl,† Mohamad G. Abiad,‡ Zeinab Moussa,† and Digambara Patra*,† †

Department of Chemistry and ‡Department of Nutrition and Food Science, American University of Beirut, Beirut, Lebanon ABSTRACT: Chitosan oligosaccharide lactate (COL) has been modified to improve water solubility of chitosan, especially for the uses in drug delivery and biomedical applications. The present study reveals that self-assembly of COL in solution forms nanoaggregates of size 10−30 nm with a critical aggregation concentration (cac) of ∼5 μM. Fluorescence quenching of pyrene establishes that one COL chain may form around five independent hydrophobic microdomains during self-assembly in solution that are crucial to drug−polymer contact. Interaction of COL with a representative hydrophobic drug molecule, curcumin, implies two different kinds of binding mechanisms of curcumin with the pre- and nanoaggregated forms of COL, respectively. A strong ground state interaction between curcumin and nanoaggregated COL has been noted with an association constant of 3.91 × 104 L/mol at 298 K. This association has been found to be diffusion controlled, enthalpy driven, and as consequences of hydrophobic effects due to van der Waals interactions. Increase in ionic strength, such as NaCl concentration, in the medium pushes the hydrophobic chain of COL and curcumin out from the solution by marginally lowering the cac and increasing the size (∼30−60 nm) of the nanoaggregate; thus, it also exponentially boosts the partition of curcumin into COL nanoaggregates. However, similar increase in NaCl concentration in the medium discourages contact of curcumin with preaggregated COL, confirming an electrostatic interaction between curcumin and preaggregated form of COL. This is further supported by FT-IR spectra. On the other hand, hydrophobic bile salt surges both the cac and size of nanoaggregates (∼100 nm), indicating bulky and hydrophobic cholate/deoxycholate group cooperatively binds with COL and curcumin for which higher concentration of COL is needed to accommodate bulky size of cholate/deoxycholate and form large nanoaggregates. The present study also reports that water vapor permeability of COL film declines linearly with curcumin concentration under investigation due to blocking of the hydrophilic part of COL by curcumin and hydrophobic nature of curcumin.

1. INTRODUCTION Smart polymers having responsive properties have drawn numerous interest due to their use in nanoscience and nanotechnology.1 Interest in water-soluble polymers is further enhanced for their applicability in stabilizing nanoparticles, drug delivery, tissue engineering, bioelectronics, etc.2 Biopolymers that are nontoxic, biologically compatible, and chemically versatile are of special interest to create a plethora of formulations and scaffolds for utilization in health care.3 Chitosan is one such special polymer which self-assemble by changing the pH;4,5 thus, it has a distinctive combination of properties.6−11 Moreover, chitosan is biodegradable, biocompatible, and bioactive polymer. Therefore, it has gained a wide range of application in drug delivery,12,13 biosensing,14 sorption material,15,16 tissue engineering,17 etc. Chemically chitosan is a polysaccharide and obtained by partial chitin N-deacetylation under alkaline conditions. It is a linear random distribution of N-acetylglucosamine and glucosamine units and more watersoluble than chitin due to the presence of the protonated amine group. The characteristics of chitosan are imposed by the ratio of acetylated and deacetylated units (degree of deacetylation), © 2016 American Chemical Society

their distribution, and the molecular weight of the chitosan chain. Its pKa ranges between 5.5 and 6.5.18 Chitosan has three reactive sites: the amine function of the deacetylated unit and the primary and secondary hydroxyl functions. Interestingly, polymers like chitosan are known to form aggregation in aqueous medium, having a hydrophobic core and a hydrophilic surface in contact with the solvent molecules. This particular characteristic of polymers means aggregates/micelles are excellent carriers for hydrophobic drugs lacking solubility in water19 and helps to deliver drug molecules. In the past 20 years, curcumin, which is bis (4-hydroxy-3methoxyphenyl)-1,6-diene-3,5-dione, has shown effective therapeutic properties as an anti-inflammatory drug,20 chemopreventive,21 chemotherapeutic, antioxidant,22 antiamyloid,23 antiarthritic,24 anti-HIV,25 hepatoprotective,26 antimicrobial27−29 and thrombosuppressive30 agent. Curcumin is also being used in the treatment of cystic fibrosis31 and Alzheimer Received: February 12, 2016 Revised: April 13, 2016 Published: May 4, 2016 11210

DOI: 10.1021/acs.jpcc.6b01486 J. Phys. Chem. C 2016, 120, 11210−11224

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The Journal of Physical Chemistry C disease.32 Recent research work has proven that curcumin owes a strong therapeutic potential against numerous types of cancer.33 However, the challenge of curcumin for medicinal application lies in its poor stability (as it degrades fast in neutral and basic condition as well when exposed to UV/vis radiation34) and poor bioavailability (due to its poor water solubility).35 Interestingly, stability of curcumin can be improved when complexed with metal ions36 and organic molecules (protein, micelles, liposomes).37 There are efforts to improve stability and bioavailability of curcumin using polymer matrix.38 Taking advantage of the positively charged amine groups (below the pKa) or hydrophobic polymeric chain (above the pKa), chitosan can interact with charged electrolyte or hydrophobic molecule to form a polyelectrolyte (below the pKa) or polymeric (above the pKa) complex. These complexes are formed mainly by electrostatic and/or hydrophobic interaction between polymer backbone and the charged/ hydrophobic entity of interacting molecule.39,40 Chitosan oligosaccharide lactate (COL) (see Scheme 1A) is a minor

drug delivery as well as biomedical and nanotechnological applications. In this work, we have investigated self-assembly and aggregation behavior of COL in solution and interaction with curcumin, a representative hydrophobic drug molecule. The association constant, thermodynamic parameters, and aggregation behavior during interaction have been quantified. Results on aggregation behavior have been compared to those obtained by electrical conductivity and pyrene fluorescence. Critical aggregation concentration, binding constant, partition coefficient, etc., of curcumin with pre- and nanoaggregated forms of COL are also evaluated and studied by varying ionic strength and bile salt.

2. MATERIALS AND METHODS 2.1. Materials. COL, curcumin, pyrene, cetylpyridinium bromide (CPB), sodium cholate, sodium deoxycholate, and pyrene were obtained from Sigma-Aldrich and used as received. The solvents used were of HPLC grade and also obtained from Sigma-Aldrich. 2.2. Sample Preparation. A stock solution of COL (2 mg/ mL) was prepared in doubly distilled water. Similarly, stock solution of curucmin and pyrene was made in methanol. The stock solutions of KI, CPB, NaCl, sodium cholate, and sodium deoxycholate were separately prepared in doubly distilled water. Dilutions were made as desired. For fluorescence measurement at different COL concentration, concentration of curcumin was kept constant at 2 μM and that of pyrene was at 1 μM pyrene. It was made sure that the amount of methanol present in the solution was less than 1% (v/v) and did not affect our measurement. For quenching experiments, COL concentration was maintained constant at 100 μM; similarly, curcumin and pyrene concentration was kept at 2 and 1 μM, respectively. The quenchers such as KI and CPB were varied accordingly. For the binding and thermodynamics study, COL was maintained at 100 μM, and curcumin concentration was varied from 0, 2, 5, 10, 15, 20, 40, and 60 to 80 μM. COL fluorescence was monitored at different temperatures. 2.3. Water Vapor Transmission Rate (WVTR). The water vapor permeability of the COL−curcumin films was measured according to ASTM F1249 using a water vapor analyzer model 7002 (Systech Instruments Ltd., UK) at 23 °C and 75 ± 2% RH with 99.9995% purity N2 gas having 0% RH as a carrier at a flow rate of 100 sccm. The data were collected every 15 min until stable saturated state was achieved. The transmission rate of 100 μm thick films was measured using aluminum masks with 5 cm2 area. Data were collected in triplicates. 2.4. Instrumentation. Room temperature absorption spectra were recorded using a SCOV-570 UV−vis−NIR spectrophotometer. Steady state fluorescence measurements were performed by a Jobin Yvon Horiba fluorometer; emission and excitation slits were both set at 5 nm (except in the measurements for pyrene it was kept at 1 nm), equipped with a 100 W xenon lamp and an R-928 detector operating at 950 V. A thermostat was coupled to the fluorometer sample holder, and temperature reading was measured by an external thermometer. Time-resolved fluorescence were done by a Jobin Yvon Horiba fluorometer, using a pulsed diode laser at 282 nm coupled to an R-928 detector operating at 950 V. FTIR spectra were recorded on a FT-IR-Raman spectrometer (Thermo-Nicolet). Morphological characterizations of the aggregate were performed using a scanning electron microscope (SEM), Tescan, Vega 3 LMU with Oxford Edx detector (Inca XmaW20). Briefly, few drops of a chitosan solution were

Scheme 1. Structure of COL (A) and Curcumin (B)

modification form, having a shorter chain and lactate as a counterion, to enhance water solubility and its application in drug delivery, nanotechnology, and biomedicine. Interesting physicochemical properties of COL are directly connected to the intra- and intermolecular associations between the hydrophobic functionalities in aqueous solution within a certain concentration range. However, COL’s behavior in solution and interaction with drug molecule has not been explored. Curcumin is a hydrophobic molecule containing β-diketone group that tautomerizes between its enol and keto structures (see Scheme 1B). It has also two phenolic groups in the two different benzene rings. Though interaction of curcumin with liposomes and micellar system is strong, where it often gets buried into the hydrophobic cavity of liposomes/micelle,41,42 study on polymer−curcumin interaction is limited. Application of curcumin as a molecular probe to study micelle,41 liposomes,42 and heterogeneous systems43 is widely getting realized. It is also being used for fluorescence nanosensing44,45 and synthesis of nanoparticles46,47 along with polymer matrix. Thus, it is vital to understand interaction of curcumin with COL, keeping the applicability of both curcumin and COL in 11211


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The Journal of Physical Chemistry C

Figure 1. (A) UV−vis absorption and fluorescence spectra of COL and curcumin; the absorbance/fluorescence intensity have been normalized with respect to absorbance/fluorescence intensity at the peak position. (B) Fluorescence spectra of COL in different concentration at excitation wavelength 290 nm. Variation of emission maximum (C) and fluorescence intensity (D) of COL with concentration.

deposited on an aluminum stub and coated with carbon conductive adhesive tape.

higher concentration it starts aggregating due to self-assembly/ folding of monomeric unit and/or formation aggregation of many monomeric units. Although micellization is a spontaneous self-assembly, it only occurs above certain monomer concentration known as critical micelle concentration (cmc) or critical aggregation concentration (cac). This specific concentration can be detected by the change in certain physical properties, such as conductivity, surface tension, osmotic pressure, etc., of the solution as monomer concentration increases.48 To understand the nanoaggregation behavior better, electrical conductivity measurement was carried out as a function of COL concentration at room temperature. As can be seen in Figure 2A, the conductivity increased linearly with COL concentration; however, molar conductivity decreased until a particular concentration and remained almost constant afterward (Figure 2B). Since electrical conductivity of water depends on concentration of dissolved salt monomers,49 such linear enhancement of electrical conductivity with COL concentration is expected. The break in molar conductivity vs COL concentration must have originated from micellization/ aggregation of COL.49 The concentration corresponding to breaking point is cac. The estimated value for cac was found to be ∼2−5 μM (Table 1). This value is like the breaking point of two different linear trends observed for fluorescence intensity of COL with concentration (see Figure 1D). Further spectroscopic techniques like fluorescence can be used for determining cmc/cac. Pyrene is one of the most widely used fluorescence probe50,51 to determine cmc/cac. To reconfirm the cac, pyrene was used as a fluorescence probe to monitor nanoaggregation behavior of COL. The fluores-

3. RESULTS AND DISCUSSION 3.1. Self-Assembly and Critical Aggregation Concentration. The backbone of COL is made of N-acetylglucosamine entity that absorbs in the UV domain at around 200 nm and of the nonabsorbent entity, glucosamine. The UV−vis absorption spectrum of COL is shown in Figure 1A, which gave a broad spectrum in the 250−350 nm region with a maximum at ∼290 nm. When excited at 290 nm, the fluorescence emission of 100 μM COL was found to be broad in the 300−500 nm wavelength ranges with a maximum at ∼395 nm. However, at very low concentration (∼600 nM), the emission maxima was located at ∼364 nm (see Figure 1B). The fact is that COL emission is solely due to acetylglucoseamine moiety present in chitosan backbone. Thus, at low concentration no major interaction between the acetylated unit and the surrounding is expected, but at higher concentration this interaction cannot be avoided due to selfassembly/folding process of polymeric chain. For example, with increase in concentration of COL from 600 nM to 200 μM the emission maxima exponentially red-shifted (Figure 1C) and remained constant at ∼400 nm at higher concentration ranges, suggesting there is a change in polarity of surrounding environment of fluorophore (acetylglucoseamine). The fluorescence intensity of COL (Figure 1D) also enhanced continuously with concentration, but the rate of enhancement was higher in the low concentration range than at higher concentration ranges. This is possible when the COL is present in the unfolded/monomeric form at low concentration, and at 11212


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Figure 2. (A) Variation of electrical conductivity with COL concentration; inset shows linear fit before (lower) and after (upper) critical aggregation concentration. (B) Variation of molar conductivity with COL concentration. (C) Fluorescence spectra of pyrene in the presence of different concentration of COL. (D) Plot of fluorescence intensity of pyrene (monomer) at three different emission wavelengths with COL concentration; inset shows variation of 440 nm peak of pyrene with COL concentration.

Table 1. Critical Aggregation Concentration of COL with and without Different Concentrations of NaCl and Bile Salt critical aggregation concentration (cac) (mol/L) [salt] NaCl NaC NaDC

0 mM

0.25 mM −6

5.4 × 10 5.4 × 10−6 5.4 × 10−6

−6

1.5 × 10 9.35 × 10−6 1.7 × 10−4

0.5 mM

1.0 mM −6

1.7 × 10 2.45 × 10−5

5.0 mM −6

2.3 × 10 4.53 × 10−5

−6

2.5 × 10

10.0 mM 2.5 × 10−6

present case we followed the fluorescence spectra of curcumin in different concentration of COL as depicted in Figure 3B. Remarkably, the fluorescence emission maximum of curcumin illustrated a blue-shift with increase in COL concentration, and at very high concentration, >100 μM, it remained constant. The variation of fluorescence maximum of curcumin with COL concentration fitted well with an exponential decay curve (see Figure 3C). Fluorescence emission maximum of curcumin is sensitive to solvent environment,41,53 and blue-shift in wavelength scale signifies a more nonpolar environment in nanoaggregated form compared to monomeric form of COL. At higher concentration the emission maximum of curcumin was found to be similar to that observed in TX 100 micelle.41 Likewise, COL quenched fluorescence intensity of curcumin at low concentration. When the COL concentration reached ∼5 μM, COL began to fold and/or aggregate together so the fluorescence intensity of curcumin started recovering and continued to increase at very high concentration, >100 μM of COL, due to increase in aggregate formation. This proves that curcumin, being a hydrophobic probe molecule, could detect hydrophobic microdomains that exist in higher concentrations

cence intensity of pyrene at three different vibrational peaks (at ∼371, ∼381, and ∼391 nm) displayed in Figure 2C was quenched in low concentration of COL (until ∼5 μM), and then a negligible improvement was observed in higher concentration of COL, >5 μM. Interestingly, the fluorescence intensity at 440 nm of pyrene (inset of Figure 2D) also decreased until 5 μM of COL and then started increasing. Based on these two trends, the cac was estimated to be ∼5 μM (see Figure 2D), which is same as earlier measured value. In case of pyrene, fluorescence intensity ratio of the monomer vibronic peaks is often used to estimate micropolarity in heterogeneous media.52 The I371/I440 depicted in Figure 3A decreased before the cac, then slightly fluctuated, and continued to decrease at higher concentration of COL. This observation is similar to sodium dodecyl surfactant solution where a continuous decrease in I1/I3 values of pyrene was obtained in post critical micelle concentration,52 suggesting growth of compact hydrophobic core of COL aggregates due to increase number of COL monomer units participating in aggregation. Earlier we have also established that curcumin can probe micellization of surfactant molecules.41 Therefore, in the 11213


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Figure 3. (A) Plot of I371/I440 of pyrene vs COL concentration. (B) Fluorescence spectra of curcumin in different concentration of COL at excitation wavelength 425 nm. (C) Variation of emission maximum of curcumin with COL concentration; inset shows variation of fluorescence intensity of curcumin with COL concentration. (D) SEM image of COL nanoaggregates; scale bar = 100 nm.

of COL. The two kinds of fluorescence intensity change of curcumin with COL concentration gave an intersection point which allowed the detection of cac consistent with the other three earlier measurements. 3.2. Size and Degree of Counterions Bound to Nanoaggregates. The size and shape of the nanoaggregates were evaluated by SEM images. The nanoaggregates were morphologically found to be spherical in size with the diameter in the ranges 10−30 nm as shown in Figure 3D. The size of the nanoaggregates is relatively larger than normal surfactant based micelle formation above the cmc, but a polymeric micelle/ aggregation may not form the same assembly process that of surfactant units. On the other hand, a careful look of Figure 2A (inset) implies that variation of electrical conductivity with COL gave a relatively higher and lower slope before and after cac of COL, respectively. It is anticipated that higher mobility of free monomer ions and counterions (lactate) offers higher conductivity below the cac. On the other hand above the cac, a decrease in mobility upon aggregation with a fraction bound lactate ions leads to relative reduction in conductivity.49 To estimate the fraction of bound counterions to aggregated nanostructures, the change in slopes of conductivity vs COL concentration in pre- and post-cac was used.49,54 In our case, the degree of lactate ion dissociation (α) was obtained as the ratio of slopes (k2/k1) of conductance vs [COL] for below (k1) and above (k2) cac. The degree of lactate ions bound to COL aggregates was calculated to be (1 − α), and the value was estimated as 0.67, which is similar to reported values for various other micelles.54

3.3. Distribution of Drug. Fluorescence quenching studies on polymer bound fluorescence probe by external quencher molecule provide important information regarding distribution and microenvironment. In the present case two different wellknown quenchers, one hydrophilic KI55 and another hydrophobic CPB,56 were applied. Because of negatively charged surface, I− prefers to stay in aqueous phase whereas long 16 carbon unit hydrocarbon chain of CPB easily gets incorporated into the hydrophobic domain, but the positive pyridinium moiety of CPB stays exposed to water interfaces.56 The fluorescence quenching of pyrene by KI in the absence and presence of nanoaggregated COL (∼100 μM) was measured using the Stern−Volmer plot55 as ln

I0 = 1 + KSV[KI] I

where I0 and I are the fluorescence intensity in the absence and presence of quencher molecule, respectively, and KSV is the Stern−Volmer quenching constant. The Stern−Volmer plot is shown in Figure 4A. As can be seen from the calculated KSV, the quenching rate slightly decreased (from 102 to 95 L/mol) in the presence of COL, indicating a large part of pyrene remains exposed to the aqueous phase. When pyrene was replaced by curcumin, the quenching rate by KI in the absence and presence was almost similar (with in the error margin, see inset of Figure 4B), but as soon as the KI concentration increased to 1 M, the quenching by KI was higher in the absence compared to the presence of COL. Further, increase in KI exponentially increased quenching of curcumin by KI in the absence of COL whereas in the presence of it did not increase. This is not 11214


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Figure 4. Stern−Volmer plot of pyrene (A) and curcumin (B) quenched by KI in the presence and absence of COL nanoaggregates. Fluorescence spectra of curcumin in the absence (C) and presence (D) of COL nanoaggregates in different concentrations of CPB.

Figure 5. (A) Fluorescence quenching of pyrene and curcumin by COL in preaggregated concentration. (B) Plot of ln(F0/F) vs [CPB]. (C) Fluorescence spectra of COL nanoaggregates in different concentration of curcumin. (D) Excited state lifetime profile of COL nanoaggregates in different concentration of curcumin.

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process can be called nanogels.65,66 The character of multiple numbers of microdomains depends on the concentration of polysaccharide,67 lipophilic group present in the polysaccharide,68 and degree of substitution68 in the polysaccharide chain. Therefore, in the present case each pyrene molecule may bind to such microdomain pockets, and the aggregation number values indeed reflects independent hydrophobic microdomain associated with pyrene, which suggests one polymer chain may form around five independent hydrophobic microdomains in the interior of a self-aggregates. 3.5. Thermodynamic Parameters. In an attempt to determine the thermodynamic parameter on association of curcumin with COL nanoaggregates, the fluorescence intensity of COL (100 μM) was monitored at 278, 283, 288, 293, and 298 K in the presence of different curcumin concentration (2, 5, 10, 15, 20, 40, 60, 80, and 100 μm). The fluorescence intensity of COL at ∼390 nm decreased with increase in concentration as portrayed in Figure 5C for 288 K. A similar trend was observed in all other temperatures. Interestingly, the fluorescence intensity at around 540 nm increased with curcumin concentration. It can be recalled from Figure 1A the UV−vis absorption spectrum of curcumin and fluorescence emission spectrum of COL has a strong overlapping region; thus, fluorescence resonance energy transfer (FRET) from COL nanoaggregates to curcumin is possible which can quench the fluorescence of donor COL and enhance the fluorescence of acceptor (curcumin). However, the excited state lifetime of COL in water at room temperature gave biexponential decay (see Figure 5D) with a short component having a lifetime of 1.86 ns (58%) and long component with a lifetime of 7.92 ns (42%). The average excited state lifetime was calculated as 4.41 ns (see Table 2). In the presence of curcumin the excited state

absurd as higher concentration of KI may help in pushing more fraction of curcumin from aqueous phase to hydrophobic domain of COL. This will be discussed further later on. In the absence of COL, hydrophobic CPB quenched the fluorescence of curcumin in water without affecting the emission maximum (see Figure 4C), which is similar to pyrene reported in the literature.56 However, in the presence of COL, instead of fluorescence quenching an enhancement in fluorescence intensity and a blue-shift in emission maximum of curcumin were observed (see Figure 4D). In contrast, CPB quenched pyrene fluorescence in the presence of COL nanoaggregates, which is in the line of reported literature in heterogeneous system.57 Thus, there must be something different between pyrene and curcumin while interacting with CPB in the presence of nanoaggregated COL. To explore this behavior, the quenching rate (k) for pyrene by COL in preaggregated form (Figure 2D) was compared with that of curcumin (Figure 3C, inset). It was noticed that quenching rate for pyrene was about 2.4-fold higher than that of curcumin (see Figure 5A), implying that pyrene has a very strong interaction with monomeric COL compared to curcumin. Water solubility of pyrene (∼0.135 mg/L) is 2.2 times lower than that of curcumin (∼0.3 mg/L); thus, hydrophobicity of the fluorophore is the main driving force to bring pyrene and COL (monomeric form) much closer compared to curcumin. It is also well-known in the literature that the fluorescence quenching of pyrene by CPB is due to electron transfer reaction from pyrene to pyridinium ion,58 which means in COL nanoaggregates pyridinium ion is not closely located to curcumin to have electron transfer reaction unlike in water. This will happen only when COL nanoaggregates would encourage curcumin to align parallel to hydrophobic chain of CPB similar to reported for curcumin in liposomes;59,60 therefore, as the concentration of CPB increased, curcumin showed a blue-shift in the emission maximum when it gets buried more into hydrophobic tail of CPB. 3.4. Independent Hydrophobic Microdomains f COL. Fluorescence quenching of pyrene by CPB in heterogeneous system like micelle etc.61 has been used to estimate aggregation number. As per the theory by Turro and Yekta62 ln

Table 2. Excited State Lifetime Values of COL in the Presence of Different Concentration of Curcumin [curcumin] 0 5 10 20 40 80

I0 Q = I M

τ1 (B1) in ns 1.86 1.95 1.83 1.99 1.74 1.65

(58%) (58%) (60%) (56%) (60%) (57%)

τ2 (B2) in ns 7.92 7.35 7.76 7.07 8.32 8.32

(42%) (42%) (40%) (44%) (40%) (43%)

τav in ns 4.41 4.20 4.21 4.25 4.37 4.5

where M=

lifetime was not affected appreciably. When FRET mechanism operates, it is expected that excited state lifetime would decrease. In the present case the excited state lifetime variation was negligible. In addition, the quenching mechanism can further be established based on temperature dependence of association constant. Thus, the driving force for the interaction of curcumin with COL was studied by determining the association constant using fluorescence quenching of COL by curcumin. The association constant (Kasso) of curcumin with COL could be evaluated as

C − cac N

and C is the total concentration of polymer, cac is the critical aggregation concentration, and N is the aggregation number. In the present case ln(I0/I) of pyrene vs [CPB] (Figure 5B) was followed in the presence of COL nanoaggregates (100 μM). The estimated N was found to be 0.21. This value is too small compared to micelles.63 However, COL has a polysaccharide backbone along with other functionalities. Polysaccharide backbone is hydrophobic in nature and encourages attachment of a hydrophobic molecule like pyrene and subsequently helps in the self-assembly process. In such polysaccharide backbone polymeric solution, it is widely reported that instead of a single hydrophobic core multiple numbers of microdomains are formed during aggregation of polysaccharide backbone in solution.2,64 Within the boundaries of the exterior lipophobic shell, these microdomains act as physical cross-links. The construction of such a form in this fashion during self-assembly

F0 = 1 + K asso[curcumin] F

or F0 = 1 + KSV[curcumin] = 1 + kdτ0[curcumin] F where F0 and F are fluorescence intensity in the absence and presence of curcumin. KSV is the Stern−Volmer constant, kd is 11216


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Figure 6. (A) Plot of F0/F vs [curcumin] at 298 K. (B) Plot of ln Kasso vs 1/T. (C) Variation of fluorescence intensity of different concentration of COL in the presence of NaCl without curcumin; inset shows the same plot in the presence of curcumin. (D) Variation of fluorescence intensity of curcumin with different concentration of COL in the presence of NaCl.

bimolecular quenching rate constant, and τ0 is the excited state lifetime without quencher molecule. Plot of F0/F vs [curcumin] gave straight line passing through 1 as shown in Figure 6A. The association constant for the equilibrium at 278 K was found to be 9.36 × 104 L/mol. The association constant at different temperature is given in Table 3. The results gave a significant

which is proportional to the sum of the diffusion coefficients for COL and curcumin, was estimated to be 8.9 × 1012 L/(mol s) (average excited lifetime value was used for kd estimation). This value of kd suggests a diffusion-controlled reaction. To estimate other thermodynamic parameters such as change in standard enthalpy (ΔH°) and change in standard entropy (ΔS°), the Van’t Hoff equation was applied as

Table 3. Association Constant and Thermodynamic Parameters of Curcumin Binding with COL Nanoaggregates temp (K) 278 283 288 293 298

Kasso (L/mol)

ΔG° (kJ/mol)

ΔH° (kJ/mol)

ΔS° (J/(K mol))

× × × × ×

−26.55 −26.58 −25.61 −26.02 −26.20

−33.98

−27.0

9.36 8.07 4.42 4.35 3.91

104 104 104 104 104

ln K asso = −

ΔH ° ⎛⎜ 1 ⎞⎟ ΔS° + R ⎝T ⎠ R

where R is the universal gas constant and T is the absolute temperature. Figure 6B depicts a linear plot for the variation of ln Kasso as a function of reciprocal temperature. From this plot, ΔH° and ΔS° of association were determined as −33.98 kJ/ mol and 27.0 J/(K mol), respectively (see Table 3). Similar values of negative ΔH° and ΔS° have been reported for the interaction of curcumin with liposomes69 and proteins.70 These negative values in several protein−ligand interactions have been explained as due to introduction of van der Waals interactions as consequences of hydrophobic effects.71 The standard Gibbs free energy change (ΔG°) was calculated from the binding constant using the equation

decrease in association constant or quenching rate constant with increase in temperature, suggesting a stronger binding at low temperature. The temperature dependence of quenching rate constant further confirms that the quenching mechanism is static rather than dynamic. This means the interaction between COL and curcumin is due to ground state complex formation rather than excited state complex formation ruling out any kind of FRET mechanism (despite overlap between absorption spectrum of curcumin and emission spectrum of COL). Therefore, the increase in fluorescence intensity at ∼540 nm could be as a result of direct excitation of curcumin at 290 nm; this was further established by measuring fluorescence emission of curcumin in these concentrations at excitation wavelength 290 nm (not shown). At room temperature (298 K), the kd,

ΔG° = −RT ln K asso

The ΔG°’s at various temperatures are summarized in Table 3. In the present case it was found that ΔH° for the association is higher than total free energy change, suggesting that the association is enthalpy driven. Similar results have been obtained for the curucmin−protein system.70 3.6. Effect of Ionic Strength in Preaggregated Form of COL. Effect of ionic strength on the interaction of curcumin 11217


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Figure 7. (A) Plot of F0/ΔF vs 1/[COL]; inset shows fa vs [NaCl]. (B) Plot of kSV vs [NaCl]. (C) FT-IR spectra of COL, curcumin, and a mixture of COL and curcumin.

differently interacting with COL. In this case, a modified Stern−Volmer plot73 was used as

with COL was investigated by varying NaCl concentration from 0.25 to 10 mM. As control experiments, the fluorescence spectral change of COL in different concentration of NaCl with and without curcumin has been compared in Figure 6C (see also inset). The fluorescence intensity remained the same with and without curcumin (2.5 μM) for various concentrations of NaCl, indicating curcumin concentration used here has little influence on conformational change of COL. Similarly, the fluorescence intensity of curcumin was monitored for different COL concentration in the presence of NaCl (0.25−10 mM). As evident from Figure 6D, the trend in the presence of NaCl was similar to that of without NaCl. Presence of up to 10 mM of NaCl in the medium marginally lowered the cac in the presence of NaCl as given in Table 1. Marginal decrease of cac in the presence of NaCl is not surprising; it is widely reported in the literature that NaCl reduces cmc of micelle.72 However, unlike surfactant systems, in the present case, an increase in NaCl concentration had little effect on the hydrophobic group. To understand it better, the association in the presence of NaCl in preaggregated form of COL was evaluated. Since fluorescence intensity of COL in preaggregated form was quenched by curcumin in the absence and presence of NaCl, F0/F vs [COL] was plotted, which showed a downward curvature indicating that curcumin is fractionally accessible to the quenching effect of COL in preaggregated form. In other words, curcumin in solution is distributed into two populations

F0 1 1 1 1 = + ΔF fa k SV [chitosan] fa

where F0 and F are curcumin fluorescence intensity in the absence and presence of COL, respectively; ΔF = F0 − F, kSV is the Stern−Volmer constant, and fa is the fraction of the initial fluorescence that is accessible to quencher. A representative modified Stern−Volmer plot in the presence of 0.25 mM is shown in Figure 7A. The fraction of the initial fluorescence slightly decreased in the beginning and later on remained constant with NaCl concentration (inset of Figure 7A). A similar trend was also observed for kSV (Figure 7B). A decrease in quenching rate indicates that increase in NaCl concentration in the medium discourages interaction of curcumin with monomeric (preaggregated) COL, thus confirming an electrostatic interaction between curcumin and preaggregated form of COL. This is further supported by FT-IR spectra measured as depicted in Figure 7C. The phenolic O−H vibration of curcumin was found at ∼3510 cm−1, whereas vibration of the free hydroxyl groups of COL was observed at ∼3650 cm−1. None of these two prominent peaks were obtained in the mixture of COL and curcumin; instead, a band was found at ∼3315 cm−1. Similarly, the peak at ∼2973 cm−1 because of enolic O−H vibration (note that sp3 C−H stretching also has the vibration in the same region, but often in curcumin case 11218


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Figure 8. (A) Plot of 1/F vs 1/[COL] for 0.25 mM NaCl. (B) Variation of partition coefficient (PCOL/water) with NaCl concentration. (C) SEM image of COL nanoaggregates in the presence of NaCl; scale bar = 200 nm.

Table 4. Partition Coefficient of Curcumin in COL Nanoaggregates in the Presence of NaCl and Bile Salt partition coefficient (PCOL/water)

a

[salt]

0 mM

0.25 mM

0.5 mM

1 mM

5.0 mM

10.0 mM

NaCla NaCb NaDCb

1.23 × 10−3 1.23 × 10−3 1.23 × 10−3

5.46 × 105 8.34 × 106 2.97 × 106

1.29 × 106 1.79 × 107

1.53 × 106 9.89 × 106

2.18 × 106

2.62 × 106

Measured in postaggregated form of COL. bMeasured in preaggregated form of COL.

band in this region has been shown to be for O−H vibration) in curcumin could be identified in the mixture of COL and curcumin. The bands due to amide groups at ∼1560 and 1404 cm−1 were identified in COL; similarly, bands at ∼1627 and ∼1602 cm−1 for α−β unsaturated ketone and enol form of curcumin were clearly established. In the mixture, the amide groups of COL and ketone and enol form of curcumin were found to be in the same region. This suggests strong hydrogen bond types of interaction between COL and curcumin. 3.7. Effect of Ionic Strength on Nanoaggregated COL. To understand partition of curcumin in aggregated form of COL (above cmc) compared to aqueous medium, the curcumin partition coefficient in COL was determined based on a procedure reported earlier.42 Briefly, curcumin fluorescence intensity was monitored at 540 nm after aggregation (above 5 μM of COL) with increasing COL concentration.74 Curcumin is expected to partition between aggregated COL phase and aqueous medium. The partition coefficient PCOL/water is defined as follows:

PCOL/water =

[curcumin]nanoaggregate /[COL] [curcumin]water /[water]

where [curcumin]nanoaggreagte, [curcumin]water, [COL], and [water] represent curcumin concentration in COL nanoaggregated phase, in the aqueous medium, COL, and water concentration, respectively. Since fluorescence intensity F is proportional to curcumin concentration bound to aggregated COL F = α[curcumin]nanoaggregate

and the total curcumin concentration is given by [curcumin]water = [curcumin]nanoaggregate + [curcumin]water

Rearranging these equations for application like solubilization, it can be written as 1 55.6 1 1 = + F PCOL/waterF0 [COL] F0 11219


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Figure 9. (A) Fluorescence spectra of curcumin in different concentration of COL in the presence of 0.25 mM sodium cholate. Change in fluorescence intensity of curcumin in different concentration of COL in the presence of 0.25 mM (B), 0.5 mM (C), and 1 mM (D) of sodium cholate.

where F0 is the fluorescence intensity when curcumin is in the COL aggregates, and 55.6 mol/L represents water concentration in a dilute medium. A linear plot of 1/F versus 1/[COL] led to estimate PCOL/water from the intercept and slope (Figure 8A). As it can be inferred from Figure 8B and Table 4, the partition of curcumin into nanoaggregated COL was substantially low, about 1 in 1000 molecules partitioned into COL nanoaggregated phase. Earlier fluorescence quenching by KI also confirmed that most of the curcumin/pyrene are accessible to I−. Thus, the poor partitioning of curcumin into the nanoaggregated phase is not surprising; possibly curcumin is anchored on the surface of nanoaggregates by exposing itself to I− and aqueous phase. However, increase in NaCl concentration in the medium very remarkably enhanced the partition of curcumin into COL aggregated form, which is consistent with earlier observation at higher concentration of KI. This confirms that in nanoaggregated form the interaction between curcumin and COL is driven by hydrophobicity of COL domains and curcumin. However, the presence of salt in aqueous medium could also help in pushing curcumin into COL domains. In the presence of NaCl the shape of the COL nanoaggregates remained spherical, few elongated nanoaggregates were also observed, and the size of the nanoaggregates increased to 30−60 nm as shown in Figure 8C. Both experiment75 and molecular dynamics simulations76 have confirmed that shape of micelle changes and size of micelle increases in the presence of NaCl due to formation of salt bridges. In the present case we suspect similar formation of salt bridges, which increase the size of nanoaggregates in the presence of NaCl making few of them elongated and favors partitioning of curcumin. 3.8. Effect of Hydrophobic Bile Salt. Bile salt has unique properties, as its anion is hydrophobic in nature. Its interaction

with heterogeneous environment or aggregation is different from that of ionic salt.43 It also partitions into micelle/ membrane and alters its properties.43 In this case when bile salt containing hydrophobic anion such as sodium cholate was used, the fluorescence intensity of curcumin was not quenched in the preaggregated form of COL; rather, an enhancement in curcumin fluorescence intensity (Figure 9A) was observed. The initial increase in fluorescence intensity in preaggregated form in the presence of sodium cholate is similar to one observed in the presence of CPB earlier and suggests a cooperative interaction between cholate (hydrophobic) and curcumin along with COL that helps in stabilizing the excited state of curcumin. However, the enhancement was much higher in the lower than higher concentration of COL (Figure 9B), and a blue-shift in the emission maximum of curcumin (Figure 9A) was obtained in higher concentration of COL. The blueshift suggests a different microenvironment in higher concentration signifying aggregation of COL. Based on two different trends in fluorescence intensity, the cac was estimated to be 1.7-fold higher than without sodium cholate (see Table 1). This result is in contrast with that of presence of NaCl where a marginal decrease in cac was found. The increase cac value in the presence of sodium cholate could be hypothesized based on the fact that bulky and hydrophobic cholate group becomes part of aggregation process along with COL to form large aggregates/micelle; therefore, the nanoaggregates formed in the presence of sodium cholate are different from COL alone and need higher concentration of COL to accommodate cholate ion in micellization process, whereas in the presence of NaCl the ions remain in aqueous phase and help the hydrophobic domain of COL to come close toward each other to form nanoaggregates in lower concentration of COL. Similarly, at 0.5 mM (Figure 9C) and 1 mM (Figure 9D) of 11220


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Figure 10. (A) Plot of cac (critical aggregation concentration) vs sodium cholate concentration. (B) Plot of partition coefficient of curcumin vs sodium cholate concentration. (C) Change in fluorescence intensity of curcumin in different concentration of COL in the presence of 0.25 mM sodium deoxycholate. (D) SEM image of COL nanoaggregates in the presence of sodium deoxycholate; scale bar = 200 nm.

sodium cholate the fluorescence intensity of curcumin increased in preaggregated form but almost remained constant in post-cac. The cac value increased linearly with sodium cholate concentration (Table 1 and Figure 10A), suggesting presence of cholate in the solution does not help COL to selfassemble to form nanoaggregates. However, the presence of cholate increased partition coefficient of curcumin into preaggregated form of COL (Figure 10B), indicating direct interaction of cholate and curcumin. When 0.25 mM sodium deoxycholate was used (Figure 10C), the fluorescence intensity of curcumin increased in preaggregated form, but in post-cac the fluorescence intensity of curcumin decreased. Based on the fluorescence intensity alteration, the cac was estimated in the presence of sodium deoxycholate as summarized in Table 4. It was found that the cac value increased with sodium cholate concentration. Even while changing from NaCl to sodium cholate to sodium deoxycholate, the cac value increased. Since deoxycholate is more hydrophobic compared to cholate, as discussed earlier, cac is expected to increase. Formation of larger nanoaggregates, ∼100 nm, in the presence of deoxycholate was further confirmed by SEM images shown in Figure 10D, which establishes the present hypothesis. The partition coefficient of curcumin in preaggregated form was found to be higher in the presence of sodium cholate and sodium deoxycholate. However, the values were slightly lower in higher concentration of sodium cholate and in the presence of deoxycholate. This lower value could be due to possibility of some kind of direct interaction between oxy/deoxy-cholate with curcumin beside COL−curcumin interaction. 3.9. Water Permeability Rate of COL−Curcumin Film. Since chitosan-based films can be used for food packaging

applications,77 we tested the effect of curcumin of COL film for water vapor permeability. Water vapor can transfer from the internal or external environment through the polymer package walls. Therefore, it is generally investigated as one of the important permeants in food packaging applications.78 COL solution was prepared by dissolving 0.5% w/w COL in 1% w/w aqueous acetic acid solution and stirring until a homogeneous and clear solution was obtained. A curcumin:methanol solution was also prepared, 1:20 w/w, and added to obtain the desired curcumin concentrations of 0, 2.7 × 10−6, 1.4 × 10−5, and 2.7 × 10−5 M. Consequently, the solution was cast onto polystyrene plates and left to dry overnight at room temperature. The COL film and curcumin−COL film were examined for the water vapor permeability (see Table 5). We found a 34% decrease in Table 5. Water Vapor Permeability Rate of COL Films in the Presence of Curcumin curcumin concentration (mol/L) 0.000 2.7 × 10−6 1.4 × 10−5 2.7 × 10−5

water vapor permeability rate (g mm/(h m2 Pa)) 8.22 5.45 5.25 4.79

× × × ×

10−4 10−4 10−4 10−4

± ± ± ±

4.14 2.73 1.34 3.87

× × × ×

10−5 10−5 10−5 10−5

water vapor permeability rate despite the concentration of curcumin was as low as 2.7 μM, and the decrease in water vapor permeability rate with curcumin concentration was linear. Similar results have been obtained for other curcumin−chitosan systems.79 Water vapor permeability is influenced by several factors which include the chemical structure, morphology, the nature of the permeant, and the surrounding environment.80 11221


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The Journal of Physical Chemistry C There is possibility of swelling of COL based film due to many hydrophilic groups such as hydroxyl groups, carbonyl groups, etc., of COL molecules that could interact with water;81 therefore, this marginal decrease in the presence of curcumin could be rationalized based on the interaction of curcumin and COL as observed and discussed earlier in FTIR (Figure 7C). This is consistent with hydrophobic nature of curcumin that will not allow water to penetrate the film.

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4. CONCLUSIONS The present study brings new insight into understanding drug molecules with polymer that is crucial to drug delivery application. COL formed nanoaggregation above critical aggregation concentration of ∼5 μM by the self-assembly process. These nanoaggregates were found to have diameters in the range 10−30 nm. During folding one COL chain could make around five independent hydrophobic microdomains, and such pockets would play important role during contact between COL and drug molecule. The hydrophobic drug curcumin molecule interacts with COL in both preaggregated and nanoaggregated form, but the mechanism of contact between curcumin and COL was different in two forms of COL. A strong ground state interaction was found between curcumin and nanoaggregated COL. The association constant was remarkably 3.91 × 104 L/mol at 298 K with negative ΔH° and ΔS°, which suggests introduction of van der Waals interactions as consequences of hydrophobic effects. Partition of curcumin exponentially increased with increase in NaCl (ionic strength) concentration. The presence of NaCl in the solution increased the size of the nanoaggregates to ∼30−60 nm and marginally lowered the cac. In contrast, the study found an electrostatic interaction between curcumin and preaggregated form of COL, which was dampened in the presence of NaCl in the medium. Interestingly, hydrophobic bile salt surged the cac as well as increased the size of nanoaggregates to ∼100 nm; these results suggest a cooperative binding between curcumin, bulky and hydrophobic cholate group and COL to form large nanoaggregates. It was shown that curcumin blocks the hydrophilic part of COL in COL film, and because of the hydrophobic nature of curcumin, the water vapor permeability of COL film decreased linearly with curcumin concentration.

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AUTHOR INFORMATION

Corresponding Author

*Tel +9611350 000 ext 3985; Fax +9611365217; e-mail dp03@ aub.edu.lb (D.P.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support provided by American University of Beirut, Lebanon, through URB, Kamal A. Shair Research Fund, and Kamal A. Shair Central Research Science Laboratory (KAS CRSL) facilities to carry out this work is greatly acknowledged.

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REFERENCES

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