Interaction of an Amphiphilic Drug Trifluoperazine ... - ACS Publications

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Interaction of an Amphiphilic Drug Trifluoperazine Dihydrochloride with Pluronic Triblock Copolymers: A Physicochemical Study Rakesh Kumar Mahajan,* Shruti Chabba, and Rabia Sharma Department of Chemistry, UGCCentre for Advanced Studies, Guru Nanak Dev University, Amritsar 143005, India S Supporting Information *

ABSTRACT: In the present study, we report the interactional behavior of the phenothiazine drug trifluoperazine dihydrochloride (TFP) with Pluronics (L64, F68, and P123) using surface tension, cloud point and fluorescence measurements. Various micellar and interfacial parameters such as critical micellization concentration (cmc), interaction parameter (β), effectiveness of surface tension reduction (πcmc), maximum surface excess concentration (Γmax), and minimum area per molecule (Amin) at the air−water interface have been evaluated using surface tension technique. The phase separation behavior of the drug is studied in the presence of Pluronics, and the corresponding thermodynamic parameters such as Gibbs free energy (ΔG°c), standard enthalpy (ΔH°c), and entropy (TΔS°c) of clouding have been calculated. Employing the steady state fluorescence measurements, the interactions between the drug and Pluronics have been determined in terms of the number of binding sites for drug molecules (n), binding constants (Ka), and Stern−Volmer quenching constants (Ksv).

1. INTRODUCTION Trifluoperazine (TFP) is an important phenothiazine compound which is commonly used as an antidepressant and antipsychotic drug and is also well-known for its biological and chemical properties.1,2 The drug molecule consists of a hydrophobic nitrogen containing heterocycle bound to a short chain containing a charged amino group which makes it amphiphilic in nature. The nature of the aromatic ring system determines the colloidal properties of amphiphilic drugs. The drug has interesting self-association characteristics under physiological conditions; it interacts with biological membranes and causes disruption and solubilization of compounds like surfactants.3 The self aggregation mechanism of amphiphilic drugs and their solubilization properties are important for understanding the relationship between the molecular architecture and physicochemical properties which helps in development of effective drug delivery systems. The interaction of drugs with surfactants is pharmaceutically important as undesirable side effects of, for example, antiarrhythmic, cardiovascular, and anticholinergic drugs may be efficiently reduced if the drugs are used as mixed micelles. Micelles have been widely used as drug delivery vehicles because they have low viscosity, small aggregate size, simple preparation, and long shelf life.4,5 Within this context, Pluronic triblock copolymers are of significant industrial interest because of their biodegradability, biocompatibility, and very low toxicity. They find extensive applications for use as formulations to obtain controlled release from gelled states at body temperatures, as components in the formulation of treatments for thermal burns, and as solubilizers of sparingly soluble drugs to increase their bioavailability, etc.6,7 Amphiphilic block copolymers are of wide use in pharmaceutical and cosmetic fields mainly because of their remarkable design flexibility for controlling nanostructure and functionality.8−12 Pluronics consist of a central poly(propylene oxide) block with terminal poly(ethylene oxide) blocks (PEO-PPO© 2014 American Chemical Society

PEO). Because of the hydrophobicity difference between the PPO and PEO blocks, they tend to micellize in the solution above a concentration named as the critical micellization concentration (cmc).13−15 These micelles are characterized by their unique core−shell architecture, which is suitable for solubilization and transportation of drugs, protecting them from chemical degradation and metabolism by biological agents and sustained release in different formulations. The dynamic PEO chains prevent particle opsonization and render them unrecognizable by the reticulo-endothelial system.16,17 These polymeric micelles, being kinetically stable, dissociate slowly extending circulation times in blood. In addition they display larger cores than surfactant micelles, leading to higher solubilization capacity than the regular micelles.18 The unique combination of advantageous features makes polymer based micelles as promising drug delivery agents.19−21 Thus it is indispensable to understand the behavior of drug−surfactant mixtures and to determine various physicochemical properties of such mixtures in order to tune their properties with respect to a desired application. Visualizing their applications and advantages in drug delivery, many studies have been reported on the binary mixtures formed by amphiphilic drugs and various surfactants. Caetano and Tabak22 investigated the bindings of chlorpromazine and trifluoperazine with anionic surfactant sodium dodecyl sulfate (SDS) by means of electronic absorption and fluorescence spectroscopy. Kabir-ud-Din et al.23 have studied the effect of poly(ethylene glycols) (PEGs) of varying molecular weights on the micellization and interfacial behavior of amitriptyline hydrochloride (AMT). In another study24 they have also reported mixed micellization of amphiphilic drug (adephenine Received: Revised: Accepted: Published: 4669

November 12, 2013 February 27, 2014 February 28, 2014 February 28, 2014 dx.doi.org/10.1021/ie403828u | Ind. Eng. Chem. Res. 2014, 53, 4669−4678

Industrial & Engineering Chemistry Research

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corresponding thermodynamic parameters (ΔGq). The aim of the study is to obtain information about the physicochemical characterization of the drug in aqueous solution and the effect of TFP on the aggregate stability of Pluronics .

hydrochloride and nortriptyline hydrochloride) with nonionic surfactants (ethoxylated sorbitan esters and triblocks) where the interactions between drug and nonionic surfactant systems were found to be attractive but quite weak for adephenine hydrochloride systems, which may be attributed to its rigid structure making micellization difficult. Alam and Mandal25 have reported the mixed micellization of a tricyclic antidepressant drug amitriptyline hydrochloride with nonionic surfactant TX-100 where the drug−surfactant systems showed an increase in synergism with the increase in surfactant concentration. Sanan and Mahajan26 developed a poly(vinyl chloride) (PVC) sensor based on neutral ion-pair complexes of dodecylmethylimidazolium bromide-sodium dodecyl sulfate for studying the micellar aggregates of imidazolium ionic liquids in the presence of drugs, viz., PMT (promethazine hydrochloride) and PMZ (promazine hydrochloride). In continuation of our work related to mixed micellization and interactional studies of drug−surfactant mixtures,27−31 the present work aims at studying the physicochemical behavior of cationic amphiphilic drug TFP (Figure 1) in the presence of

2. EXPERIMENTAL SECTION 2.1. Materials. The triblock copolymers F68, P123, and L64 and amphiphilic drug TFP (>98%) were obtained from Sigma Aldrich. All of the chemicals were used as received and were of analytical grade. An analytical balance (Sartorius Analytic) with a precision of ±0.0001 g was used for weighing the amount of different substances. The solutions were prepared by dissolving accurately weighed quantities in requisite volumes of deionized double distilled water having a specific conductivity of the range (1−2) × 10−6 S cm−1. 2.2. Methods. 2.2.1. Surface Tension Measurements. The surface tension measurements were done with a KRUSS Easy Dyne tensiometer from Kruss Gmbh (Hamburg, Germany) using the Wilhelmy plate method. The surface tension of doubly distilled pure water 72.8 mN m−1 at 25.0 ± 0.1 °C was used to calibrate the instrument. The surface tension of each solution was measured by successive additions of the stock solutions in pure double distilled water after thorough mixing and equilibrations. The series of measurements were repeated at least three times. The reproducibility of surface tension measurements is estimated to be within ±0.15 mN m−1. 2.2.2. Cloud Point Measurements. The CPs of the drug− Pluronic mixed system was determined by heating the solutions in glass tubes suspended in an oil bath, whose temperature was increased gradually with constant stirring at a rate of about 0.1 K min −1. CPs were determined visually by noting the abrupt change in appearance of solution from clear to turbid. The temperature at which turbidity first appeared was recorded. The solution was then allowed to cool slowly, and the temperature at which it became clear again was also recorded. After that the mean of both temperature was recorded as the CP. The maximum error in CP value is 0.5% calculated from at least three determinations. The results were reproducible within ±0.5 K. A 0.336 mM solution of TFP was prepared in 10 mM sodium phosphate (SP) buffer having pH 6.7. Solutions of various Pluronics were prepared by dissolving the required amount of these polymers in stock solution of TFP. 2.2.3. Fluorescence Measurements. The steady state fluorescence measurements were performed on a Varian Cary Eclipse fluorescence spectrophotometer using a 10 mm path length quartz cuvette at 25.0 ± 0.1 °C. The emission spectrum of TFP was recorded in the range of 400−600 nm wavelengths by excitation at 320 nm. The fluorescence titration experiments were performed by successive additions of 0.01 mM stock solutions of Pluronics directly into the quartz cuvette containing 3 mL of 0.166 mM drug solution so as to give a final concentration in the range of 0.625−2.857 μM.

Figure 1. Structure of trifluoperazine dihydrochloride.

nonionic polymeric surfactants, i.e., Pluronics L64, F68, and P123. The reason behind choosing these Pluronics is that they have different numbers of PEO-PPO units and hence different hydrophilic−lipophilic balance (HLB) ratios. Various techniques such as surface tension, cloud point (CP), and fluorescence measurements have been performed. Surface tension measurements have been used to evaluate various micellar and interfacial parameters such as cmc values of pure components as well as their mixtures; interaction parameter β; micellar mole fractions X1 and X1,ideal in the mixed state and the ideal state, respectively; activity coefficients (f1 and f 2); surface excess concentration (Γmax); minimum area per molecule (A min); and surface pressure at the cmc (πcmc ). The thermodynamic parameters such as Gibbs free energy of micellization (ΔG°m), Gibbs energy of adsorption (ΔG°ad), and excess free energy of micellization (ΔGex) have also been calculated. In addition to the evaluation of physicochemical parameters, cloud point and fluorescence measurements have been done to get a deep insight into the interactional behavior of Pluronics with the drug. The cloud point measurements have helped to study the thermodynamics of the clouding behavior of the drug in the presence of Pluronics. Fluorescence quenching studies have been utilized to study the binding behavior of the drug with Pluronics in terms of the number of binding sites (n) in the drug molecule, binding constants (Kq and Ka), Stern−Volmer quenching constants (Ksv), and

3. RESULTS AND DISCUSSION 3.1. Polymer−Drug Interactions in the Mixed Micelles. The cmc values of pure drug TFP as well as pure Pluronics have been evaluated tensiometrically and are found to be in good agreement with the literature values.26,31,32 The cmc of pure drug has been worked out to be 0.042 mM from the surface tension (γ) vs concentration plot shown in Figure S1 of the Supporting Information (SI). The cmc values for various Pluronics in the presence of drug have been determined from 4670

dx.doi.org/10.1021/ie403828u | Ind. Eng. Chem. Res. 2014, 53, 4669−4678


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Figure 2. Plot of surface tension (γ) vs log [Pluronic] for (a) L64, (b) F68, and (c) P123 in the presence of different concentrations of drug TFP at 25 ± 0.1 °C.

the drug (TFP) has been studied by using Clint’s equation, eq 1, based on a pseudophase thermodynamic model:34

the surface tension plots shown in Figure 2, and the cmc values are presented in Table 1. As initially drug is added to the aqueous solutions of polymers, the decrease in surface tension is caused by drug monomers adsorbing at the air−water interface. Further increase in drug concentration leads to more drug molecules binding to the surface which lowers the surface tension. When free concentration of drug becomes high enough to form micelles, it will do so and surface tension then remains constant thereafter.33 For L64/F68 + TFP systems, it can be observed from Table 1 that the cmc values obtained for the binary mixtures are lower than cmc values of pure Pluronics, which may be because of penetration of drug molecules inside the Pluronic micelles. However, there is an increase in cmc values of the mixtures with increasing concentration of drug which can be related to a steric factor that comes into play on account of a bulky headgroup of TFP at higher concentrations which leads to inefficient packing of drug molecules in the mixed state. In the case of the P123 + TFP system the cmc value of the mixtures is greater than the value for pure P123 which signifies unfavorable mixing of the components. The ideality in the interaction of Pluronics (L64/F68/P123) with

α1 (1 − α1) 1 = + cmc* cmc1 cmc 2

(1)

where cmc1 and cmc2 are the cmc’s of pure TFP and pure Pluronics, respectively, α1 is the mole fraction of drug TFP, and cmc* is the ideal state mixed cmc. From Table 1 it is clear that the cmc values lie lower than cmc* for the L64 + TFP system. This negative deviation in cmc from ideal cmc* values indicates favorable mixing of these surfactants in binary mixtures. In the case of F68 and P123 cmc remains lower than ideal cmc except for cases when higher concentration of the drug is present (3 and 5 mM) where the values obtained are higher than cmc* giving a positive deviation which indicates unfavorable mixing of triblock polymer and drug. This may be due to the reason that repulsive interactions dominate between the Pluronics and the drug at higher concentration of drug, and hence systems exhibit antagonistic interaction behavior. Clint’s model does not take into account the interaction of two components in mixed micelles. Thus to study the interactions between polymers and drug, micellar composition 4671



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Table 1. Micellar Parameters of Mixed Systems of Pluronics (L64,F68,P123) with Trifluoperazine Dihydrochloride (TFP) at 25 ± 0.1°C (Experimental cmc (cmc), Ideal cmc (cmc*), Micellar Compositions (X1 and X1ideal), Interaction Parameter (β), and Activity Coefficients (f1 and f 2))a [drug] (mM)

cmcb (×104 mol dm−3)

0.0 0.5 1.0 3.0 5.0

50.0 0.41 0.89 2.88 3.80

0.0 0.5 1.0 3.0 5.0

10.0 0.41 1.25 2.51 4.36

0.0 0.5 1.0 3.0 5.0

0.01 0.08 0.12 1.47 2.23

cmc* (×104 mol dm−3)

X1

X1ideal

β

f1

f2

0.50 0.53 0.64 0.79

0.48 0.67 0.86 0.91

− 16.78 − 10.82 − 5.13 − 2.29

0.015 0.091 0.514 0.904

0.015 0.047 0.122 0.239

0.51 0.56 0.81 0.71

0.50 0.67 0.86 0.91

− 10.58 − 4.49 10.95 17.23

0.078 0.419 − −

0.063 0.244 − −

0.51 0.56 0.42 0.56

0.50 0.67 0.83 0.91

− 4.74 − 4.21 5.89 10.92

0.320 0.153 − −

0.291 0.267

L64 + TFP 25.92 16.90 7.37 4.86 F68 + TFP 5.17 3.59 1.79 1.31 P123 + TFP

a

0.24 0.32 0.40 0.43

−, not detected. bThe error limit in cmc is ±0.003 × 10−4 mol dm−3.

(X1) and interaction parameters β in the micelle have been calculated in light of Rubingh’s regular solution approximation,35 eq 2: X12 ln(α1cmc/X1cmc1) 2

(1 − X1) ln[(1 − α1)cmc/(1 − X1)cmc 2]

interactions whether attractive or repulsive. The existence of synergism in mixtures of surfactants depends not only on the strength of interaction between different components but also on the associated properties of each surfactant in the mixture. The β values for L64/F68/P123 + TFP mixtures are listed in Table 1. The values of interaction parameter β are negative for the L64 + TFP system, which indicates attractive interaction between the components interacting in the mixture. The synergism in the L64 + TFP system can be accounted for on the basis of smaller PEO-PPO units in L64 which allow penetration of TFP in the mixed micelle quite efficiently in comparison to other Pluronics having a larger number of PEOPPO units. Also for F68/P123 + TFP mixtures, β values are negative which is an indication of synergistic interactions, but as the drug concentration increases, β is found to become less negative and attains a positive value at higher drug concentrations. The positive β values suggest a nonideal behavior due to repulsive interactions between the components. In the F68/P123 + TFP system the decrease in synergistic interactions and increase in antagonistic interactions may be associated with unfavorable mixing behavior mainly due to predominance of Pluronics in the mixed micelle and the incapability of the drug molecules to intercalate between the Pluronic micelles.37 In nonionic−cationic systems initially when the concentration of the cationic component is less, nonionic micelles will be present with the monomers of cationic micelles dissolved in them. As the concentration of the cationic component increases, it leads to demixing in the micelles and hence formation of two different kinds of micelles, one cationic rich and other nonionic rich, takes place.26 The activity coefficients ( f1 and f 2) for both of the components within the micelle are related to interaction parameter β through the following eqs 5 and 6:

=1 (2)

where X1 is the mole fraction of drug in the mixed micelle at cmc, α1 is the mole fraction of drug in the bulk, and all other terms carry their usual meanings. The preceding equation was solved iteratively to obtain the value of X1, from which the interaction parameter β was evaluated using the following eq 3:

(

ln β=

cmcα1 cmc1X1

)

(1 − X1)2

(3)

The micellar mole fraction of mixed systems in the ideal state, X1,ideal, has been computed using Motomura’s approximation,36 eq 4: α1cmc 2 X1,ideal = α1cmc 2 + (1 − α1)cmc1 (4) Table 1 shows that X1 values are lower than X1,ideal for all of the binary mixtures. Smaller values of X1 indicate that mixed micelles contain more contribution of Pluronics than in its ideal mixing state and less transfer of drug molecules from the solution to the micellar phase. The value of interaction parameter β in the mixed micelles, is a magnitude of degree of interactions operating between the unlike components in the mixed micellar state relative to their self-interaction before mixing under similar conditions. A negative β indicates an attractive interaction or synergism indicating that the interactions between the two components in the mixed micellar phase are more attractive or less repulsive than the interactions occurring between the individual components. A positive β value indicates repulsive interactions or antagonism, and zero value indicates approximately ideal mixing. A higher value of β indicates a higher magnitude of 4672

f1 = exp[β(1 − X1)2 ]

(5)

f2 = exp[β(X1)2 ]

(6)



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Table 2. Interfacial and Thermodynamic Parameters of Mixed Systems of Pluronics with Drug Trifluoperazine Dihydrochloride (TFP) at 25 ± 0.1 °C (Surface Pressure at cmc (πcmc), Surface Excess (Γmax), Minimum Area per Molecule (Amin), Free Energy of Micellization (ΔG°m), Free Energy of Adsorption (ΔG°ad), Effective Free Energy (Geff) and Excess Free Energy (ΔGex)z ). [drug] (mM)

π

cmc

(mN m−1)

Γmax × 106b (mol m−2)

0 0.5 1.0 3.0 5.0

36.8 25.0 18.5 23.5 19.3

0.60 0.26 0.24 0.28 0.36

0 0.5 1.0 3.0 5.0

31.2 17.5 13.2 11.7 15.8

0.45 0.38 0.30 0.42 0.28

0 0.5 1.0 3.0 5.0

38.4 18.3 25.4 18.1 11.5

0.79 1.96 1.88 1.12 0.60

Aminb (Å2)

ΔG°m (kJ mol−1)

L64 + TFP 276.71 −13.12 638.57 −25.03 691.79 −23.11 592.96 −20.02 461.19 −19.51 F68 + TFP 368.95 −17.11 436.92 −25.03 553.43 −22.27 395.50 −20.54 592.96 −19.17 P123 + TFP 210.16 −34.23 84.70 −34.78 88.31 −27.97 148.24 −21.86 276.71 −20.83

ΔG°ad (kJ mol−1)

Geff (kJ mol−1)

ΔGex (kJ mol−1)

− 74.45 −121.18 −100.19 −104.12 − 73.12

− − − − −

61.33 96.14 77.07 83.91 53.60

− −10.92 − 8.81 − 4.69 − 1.93

− − − − −

86.44 71.08 66.27 48.39 75.60

− − − − −

69.33 46.04 44.00 27.85 56.42

− − 6.43 − 3.46 − −

− − − − −

82.84 44.12 41.48 38.02 40.00

− 48.60 − 9.33 −13.50 −16.15 −19.16

− − 8.23 − 1.69 − −

−, not detected. bThe uncertainty limits in Γmax and Amin are ±0.01 × 10−6 mol m−2 and ±0.03 Å2, respectively.

z

area per molecule (Amin) at the air−water interface38 is calculated using eq 8:

The activity coefficients are unitless thermodynamic quantities and have a value of unity in an ideal solution. Any deviation from unity values indicates the increase of attractive or repulsive interactions. The sum of the activity coefficient values, i.e., f1 and f 2, less than unity for the L64 + TFP system supports the existence of synergistic interactions between the two components in the mixed state. For the F68/P123 + TFP system, the sum of the activity coefficients is less than unity until the addition of 1 mM drug, which indicates attractive nonideal behavior of the components. Above this drug concentration, the sum of the activity coefficients is greater than unity suggesting the repulsive nonideal behavior of the components. 3.2. Polymer−Drug Interactions at the Air−Water Interface. The surface properties of the drug−Pluronic systems have been studied using the surface tension method. The orientation of adsorbed amphiphilic molecules at the air− water interface decreases the surface tension of the aqueous phase. By increasing the concentration of adsorbed amphiphilic molecules, a further decrease in surface tension occurs. The extent of adsorption per unit of surface area can be calculated from the Gibbs adsorption equation.38 The Gibbs surface excess concentration, Γmax, was evaluated from the slope of tensiometric isotherms using the Gibbs adsorption isotherm. Γmax = −

1 (dγ /(d log C)) 2.303nRT

A min /Å2 = 1020 /(NA Γmax)

(8)

where NA is the Avogadro number. The values of Γmax and Amin are summarized in Table 2. The hydrophobic interactions among the nonpolar moieties and cross-sectional area of the polar headgroups of the surfactants determine the formation of the compact adsorption layer of the surfactant molecules at the air−water interface which in turn dictate the surface excess concentration as well as area per molecule of the surfactants at the interface. As Per Table 2, P123 exhibits highest value of Γmax in comparison to L64 and F68 indicating that P123 molecules tend to adsorb more at air−water interface. The addition of drug molecules to Pluronics brings about varied changes in the Γmax values depending upon the nature of the Pluronic and the concentration of drug added. In the case of the L64/F68 + TFP mixed system, addition of drug molecules is seen to lower Γmax values suggesting the lesser tendency of molecules to get adsorbed at the air−water interface. It has earlier been suggested by Kabir-ud-Din et al. that formation of mixed micelles of drugs and nonionic surfactant is accompanied by loosening of the monolayer.24 However the reverse has been observed for P123 + TFP mixtures. This can be due to higher hydrophobicity and lower cmc of P123 in comparison to L64 and F68.26 The increase in the concentration of drugs in Pluronic + drug systems does not produce any regular trend in Γmax values which might be due to different molecular interactions coming into play on mixing two surfactants having different structures and different properties. These can be electrostatic repulsive interactions between ionic hydrophilic headgroups, ion−dipole attractions between the ionic and nonionic hydrophilic groups, the steric repulsions between bulky hydrophilic or hydrophobic groups, and the attractive van der Waals interactions depending on the closeness of packing of the hydrophobic groups. Because of the complex chemical structures of the surfactants being used, the evaluation of these specific interactions is difficult.

(7)

In the preceding equation dγ/(d log C) is the maximum slope, γ is the surface tension of the solution, R is the universal gas constant (8.314 J K−1 mol−1), C is the total concentration of surfactant in solution, T is the temperature, and n is the number of chemical species whose concentration at the interface changes with the bulk phase concentration. The surface excess concentration (Γmax), determined at surface saturation using the Gibbs isotherm, is a measure of the effectiveness of the surfactant adsorption at the interface, since it is the maximum value of adsorption attained. The minimum 4673



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3.3. Thermodyamics of Micellization and Interfacial Adsorption. The effect of drug TFP in the mixture on the micellization and adsorption phenomenon can be quantified with various thermodynamic parameters. The standard Gibbs free energy of micellization,38 ΔG°m for pure components and their mixtures is calculated by using eq 9 ΔG°m = RT ln cmc

(9)

where cmc is the critical micelle concentration of pure components or a mixture. Table 2 shows that the ΔG°m values are negative for all of the systems because thermodynamically stable micelles form spontaneously. However the values become less negative in the presence of cationic drug indicating that the micellization process becomes less spontaneous with the addition of drug molecules. The standard Gibbs energy of adsorption ΔG°ad39 was calculated from standard Gibbs free energy of micellization ΔG°m using eq 10: ΔG°ads = ΔG°m − πcmc/Γmax

(10)

where πcmc is the surface pressure at cmc and is calculated by using relation π = γ − γcmc and Γmax is the surface excess concentration. The second term in the above equation is the work involved in going from zero surface pressure to surface pressure at cmc at constant minimum surface excess concentration. Table 2 shows that ΔG°ad values are negative for all of the systems which indicate spontaneity of the adsorption process. Also the ΔG°ad values are found to be more negative than corresponding ΔG°m which indicates that surface adsorption is more spontaneous than micellization because work has to be done to transfer surfactant molecules from the surface to the micelle. However, with an increase in the concentration of the drug, the ΔG°ad values were found to decrease which suggests that the adsorption process becomes less facile. The difference between ΔG°ad and ΔG°m is called effective Gibbs free energy (ΔG°eff). From Table 2 it is clear that the ΔG°eff values became less negative and follow the order L64 > F68 > P123 indicating the minimum energy requirement for micellization. As a result micellization is favored over adsorption. The values of excess free energy of micellization, ΔGex, were calculated by using eq 11: ΔGex = [X1 ln f1 + (1 − X1) ln f2 ]RT

Figure 3. Effect of addition of Pluronics on the cloud point of TFP solution prepared in SP buffer of pH 6.7.

of Pluronics, there is an increase in the cloud point of the drug due to formation of mixed micelles between amphiphilic drug and triblock polymers which contains relatively more hydrated water in comparison to that of the micelle of pure drug.40,41 However at low concentrations, P123 behaves differently from L64 and F68. In the cases of L64 and F68 there is an initial decrease in CP of the drug with addition of Pluronics whereas P123 raises the CP of the drug. This suggests that P123 owing to its comparatively much lower cmc than L64 and F68 tends to form mixed micelles with the drug even at very low concentrations. On the other hand, L64 and F68 prefer to get adsorbed on the micellar surface resulting in a lowering of the CP of the drug.42 Various thermodynamic parameters for Pluronic−drug systems such as standard Gibbs free energy, ΔGoc, standard enthalpy, ΔHoc, and entropy, ΔSoc, of clouding have been evaluated. The clouding species become insoluble when the temperature increases above the CP; thus CP can be considered as the limit of its solubility as it phase-separates at this temperature. We can consider that the phase equilibrium (separation) at CP is an ideal one and ΔGoc41 can be evaluated from the following relation:

(11)

From Table 2, it is clear that the values obtained are low; hence stable micelles are formed. But the values decrease with an increase in the concentration of the drug molecules; hence stability of the system also decreases which is in accordance with their interaction parameters. 3.4. Phase Separation Behavior of the Drug in the Presence of Pluronics. The phase separation behavior of amphiphilic drug TFP in the presence of various Pluronics has been studied. The temperature at which the solution separates into surfactant rich and surfactant lean phases is known as the cloud point since the process involves an increase in the turbidity of the solution. For all of the experiments, the concentration of the drug is kept constant, i.e., at 0.336 mM, and at this concentration the value of CP of TFP is 308.5 K. It is to be mentioned here that the cmc value of TFP is 0.042 mM.31 As the concentration of TFP was fixed (0.336 mM) which is above the cmc value, the drug must be present in the micellar form. The variation in the CP value of TFP on the addition of various Pluronics has been shown in Figure 3. The results depict a difference among behavior of Pluronics at low concentration and higher concentrations. At high concentration

ΔG°c = RT ln X

(12)

where X is the mole fraction concentration of the clouding species, R is the gas constant, and T is the clouding temperature in Kelvin scale. ΔHoc can be determined from the slope of ΔGoc/T vs T plot (representative Figure S2 of the SI) following eq 13. ΔSoc can be evaluated subsequently by use of eq 14: ΔH °c = −T 2

∂(ΔG°c /T ) ∂T

T ΔS°c = ΔH °c − ΔG°c

(13) (14)

The thermodynamic parameters calculated using the above equation are presented in Table S1 of the SI. ΔGoc is found to be negative for all of the systems, and its value increases with the increase in the concentration of Pluronics which indicates 4674



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Figure 4. (a) Fluorescence quenching spectra of pure TFP (1) in the presence of increasing concentration of L64 from lines 2 to 7 at 25 ± 0.1 °C. (b) Stern−Volmer plot of fluorescence quenching of TFP by L64. (c) Representative plot of log [(I0 − I)] vs log [L64].

the spontaneity of the clouding process. ΔH°c has been found to be negative for all of the systems in the presence of Pluronics. In the clouding process, dehydration of the headgroup region takes place with the absorption of heat followed by the association of dehydrated molecules involving the release of heat.43 The resultant heat is reflected on the measured ΔH°c. In addition to this, other associated factors such as orientation, change in configuration, and interfacial adsorption, etc., also contribute to the observed ΔH°c. The standard entropy (TΔS°c) of clouding has been found to be positive for all of the systems except for the TFP + P123 system where values become negative at higher concentrations of polymer. The positive TΔS°c values suggest formation of loose and disordered aggregates in the solution. Because the aggregates are loose, they contain more water in their headgroups, which is confirmed from the increase in their CP behavior. The incorporation of Pluronic micelles containing a hydrophilic corona of PEO units into the drug micelles increases the number of water molecules near the headgroup of the micelles thus affecting the water structure. This increases

the randomness of the system; hence values of TΔSoc are positive. From the positive values of TΔSoc, it can be concluded that the system is in a disordered state at the cloud point. 3.5. Fluorescence Measurements of TFP + L64/F68/ P123 Systems. In order to have a better understanding of interactional behavior of drug with Pluronics, steady state fluorescence has been used. The aromatic ring of the phenothiazine moiety in trifluoperazine is responsible for its fluorescence properties. This spectroscopic technique provides information on the drug microenvironment and enables us to determine how strongly the different polymers bind to the drug. As evident from Figure 4a (and Figure S3a,b of the SI), the addition of Pluronics decreased the fluorescence intensity of the drug when excited at 320 nm without changing the λmax. The quenching of fluorescence emission shows the occurrence of interaction between drug and polymer leading to formation of the complex between TFP and Pluronics which may be due to weak ion−dipole and hydrophobic interactions. However since the emission maximum is unchanged, the microenvironment around the drug did not undergo obvious changes during 4675



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Table 3. Stern−Volmer Quenching Constants (Ksv), Binding Constants (Ka), Quenching Rate Constant (Kq), Number of Binding Sites (n), and Corresponding Thermodynamic Parameter (ΔGq) for the Binary Mixtures of TFP + L64/F68/P123 Using Fluorescence Technique Ksv × 10−5 (mol dm−3)

Ka × 10−6 (mol dm−3)

0.799

3.06

0.601

0.08

0.701

0.27

Kq × 10−12 (mol dm−3 s−1) TFP + L64 7.99 TFP + F68 6.01 TFP + P123 7.01

the binding process. Similar decrease in intrinsic fluorescence of insulin at various triblock copolymer concentrations has been reported by Sarbolouki et al.44 The quantitative estimate of the extent of binding of drug to the triblock copolymers has been made using the Stern−Volmer45equation, which is as follows: I0/I = 1 + Kqτo[Q] = 1 + K sv[Q]

Ka =

(15)

0.99959

−27.969

1.02

0.99907

−27.264

1.11

0.99938

−27.646

[D0 ] − [drug] [surfactant]n [drug]

(19)

(20)

4. CONCLUSION The interactions of Pluronics (L64, F68, and P123) with drug trifluoperazine dihydrochloride (TFP) have been studied using surface tension, cloud point, and fluorescence quenching measurements. The critical micellization concentration (cmc) values of Pluronics (L64 and F68) are found to decrease in the presence of TFP but increase in the case of P123 on addition of TFP. The value of the interaction parameter (β) is found to be dependent on the amount of TFP present in the mixture as well as the nature of the Pluronics. A decrease in synergistic interactions and an increase in antagonistic interactions have been observed with the increase in concentration of TFP. The mixed micelles are found to be rich in Pluronics as indicated by lower experimental micellar mole fraction (X1) than the ideal micellar mole fraction values (X1,ideal). The Γmax and Amin values are found to increase and decrease, respectively, with an increase in the concentration of the drug for L64/F68 + TFP mixtures while for the P123 + TFP system an opposite behavior is observed. The thermodynamic evaluation of the mixtures indicates that the surface adsorption is more spontaneous than the micellization process. Further Pluronics have been found to increase the CP of the drug TFP. The quenching of fluorescence emission of TFP on addition of Pluronics signifies the binding of drug molecules to the Pluronics. A higher binding affinity has been observed for the TFP + L64 mixture in comparison to those of F68/P123 + TFP systems. It is hereby expected that the results of the present study will prove

Ka

(17)

where the (surfactant)ndrug is the new complex formed with binding constant Ka; when the system achieves equilibrium, the binding constant can be written as [(surfactant)n drug] [surfactant]n [drug]

1.29

where n is the number of binding sites and Ka is the binding constant. The values of Ka and n can be obtained at the same time using a linear regression of the fitting curve of log[(I0 − I)/I] versus log [surfactant]. A representative plot for L64 is shown in Figure 4c, and the plots for F68 and P123 are not shown. Table 3 lists the values of binding constants and binding sites for Pluronics associated with the drug (TFP). The nonionic Pluronics interact through weak ion−dipole and hydrophobic interactions with the drug. The exact mechanism involved in quenching is difficult to predict. The values of n approximately equal to 1 indicate the existence of a single binding site in the drug for Pluronics. From the values of binding constants, it is clear that the TFP + L64 system shows higher binding affinity in comparison to F68/P123 + TFP systems.

The ΔGq values are presented in Table 3, and the values are negative indicating that the quenching process is spontaneous and is favorable for systems under consideration. For the static quenching process, under the assumption that there are the same and independent binding sites n in the drug; that is, at each binding site, there is the same capacity for the surfactant binding with the drug. The scheme is given as

Ka =

ΔGq (kJ mol−1)

log[(I0 − I )/I ] = log K a + n log[surfactant]

(16)

drug + n(surfactant) ⇄ (surfactant)n drug

R

Using the spectral ratio between fluorescence intensity and the unbound drug molecule [drug]/ [D0] = I/I0 and transforming the equation,45 we obtain

where I0 and I are the steady state fluorescence intensities in the absence and presence of quencher respectively, Kq is the quenching rate constant, and τo is the average lifetime of TFP without quencher and its value is 10−8 s.46 Ksv and [Q] are the collisional quenching constant (Stern−Volmer constant) and concentration of the quencher (here Pluronics act as quencher), respectively. Table 3 presents the values of Ksv and Kq which are obtained from linear variation of I0/I vs [Q] shown in Figure 4b. The larger Ksv value has been obtained for L64 as compared to those of F68 and P123 indicating that former is more efficient in quenching the fluorescence of drug TFP. The quenching constants have been found to be larger than the limiting diffusion constant of the biomolecules (on the order of 2.0 × 1010 M−1 s−1),47 so dynamic quenching originating from collisions between the quencher and fluorophore molecules is not responsible for fluorescence quenching, but static quenching from complex formation is the main mechanism for fluorescence quenching. The free energy change for the quenching process can be calculated from the equation ΔGq = −RT ln K sv

n

(18)

If the total amount of drug molecules (bounded and unbounded with the surfactant) is D0, thereupon [D0] = [(surfactant) ndrug] + [drug] and [(surfactant) ndrug] = [D0] − [drug], so that eq 18becomes 4676



Article

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helpful in designing and formulating better mixed systems for use in drug delivery purposes.

■

ASSOCIATED CONTENT

S Supporting Information *

Figures showing surface tension as a function of the log of the concentration for pure drug (TFP), representative plot for calculation of binding constant and binding site, and fluorescence quenching plots of TFP by F68 and P123 and table containing thermodynamic parameters of clouding for drug TFP in the presence of Pluronics. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*Fax: +91 183 2258820. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS

■

REFERENCES

This work was financially supported by the Department of Science and Technology (DST), Government of India, New Delhi as a part of Project NO.SR/S1/PC-02/2011. S.C. is thankful to DST for the award of a Junior Research Fellow (JRF).

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