Interactions of phenothiazine drugs with bile salts: Micellization and binding studies

Journal of Colloid and Interface Science 387 (2012) 194–204

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Interactions of phenothiazine drugs with bile salts: Micellization and binding studies Suruchi Mahajan, Rakesh Kumar Mahajan ⇑ Department of Chemistry, UGC-Centre for Advanced Studies, Guru Nanak Dev University, Amritsar 143 005, India

a r t i c l e

i n f o

Article history: Received 15 June 2012 Accepted 25 July 2012 Available online 3 August 2012 Keywords: Phenothiazines Bile salts Micellization Benesi–Hildebrand plots Binding constant

a b s t r a c t An evaluation of the interactions of phenothiazine tranquilizer drugs (promazine hydrochloride; PMZ and promethazine hydrochloride; PMT) with bile salts viz., sodium cholate (NaC) and sodium deoxycholate (NaDC) in aqueous medium, investigated through different physicochemical measurements is presented in this work. The mixed micellization behavior and surface properties of the phenothiazine–bile salt systems have been analyzed by conductivity and surface tension measurements. Application of different theoretical approaches to all the phenothiazine–bile salt mixtures shows a non-ideal behavior. Further, the spectroscopic techniques such as UV–visible and steady state fluorescence have been employed to study the binding of phenothiazines with bile salts. The stoichiometric ratios, binding constants (K), and free energy change (DG) for the phenothiazine–bile salt complexes were estimated from the Benesi–Hildebrand (B–H) double reciprocal plots obtained by using the changes in spectral intensities of phenothiazines on addition of bile salts. The results are discussed in the light of use of bile salts as promising drug delivery agents for phenothiazines and hence improve their bioavailabilty. Ó 2012 Elsevier Inc. All rights reserved.

1. Introduction Oral route is the most convenient, economic, and frequently used route of drug administration, but it suffers from a major drawback of poor gastrointestinal membrane permeability. Penetration enhancers may be incorporated into various formulations in order to overcome the problem of low permeability and bioavailability of drugs across the biological membranes. They include surfactants, fatty acids, bile salts, medium chain glycerides, calcium chelators such as ethylenediaminetetraacetic acid (EDTA), acyl carnitine and alkanoylcholines, N-acetylated a-amino acids and N-acetylated non-a-amino acids, chitosans, and other mucoadhesive polymers [1,2]. Bile salts have been extensively used as penetration enhancers and are also the most important biosurfactants of anionic type that are biosynthesized from cholesterol in the liver, stored in the gall bladder, and then secreted through the bile duct into the small intestine [3]. The micellization behavior of bile salts is very controversial. The most accepted primarysecondary micelle model known as Small’s model [4] proposed that micellization in bile salts is a stepwise aggregation process. At low concentrations, the bile salt monomers aggregate to form primary aggregates containing 2–10 monomers through hydrophobic interactions between the nonpolar side of the monomers. As the concentration of bile salt monomer is raised, these ⇑ Corresponding author. Fax: +91 183 2258820. E-mail address: [email protected] (R.K. Mahajan). 0021-9797/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jcis.2012.07.085

aggregates further interact to form larger secondary aggregates via hydrogen bonding among the hydroxyl groups located on the surface of primary micelles. Due to the unusual self-assembly behavior of bile salts, their critical micelle concentration (cmc) is considerably lower, and the micelles formed just above the cmc are characterized by much smaller aggregation numbers than in the case of simple surfactants [5]. Bile salts are involved in a variety of important physiological functions like solubilization and transport of fats and lipids, assistance to hydrolysis of triglycerides by pancreatic enzymes along their transport, cholesterol homeostasis and in the formulation of food, cosmetics, and several other chemicals [4,6,7]. Owing to these widespread applications and unique facial amphiphilic structure, the physicochemical properties of bile salts have been extensively investigated [3,8,9]. In addition to these applications, bile salts have also been used as drug delivery media for the transport of some drugs through the intestine mucous membrane due to their biocompatibility and solubilizing property [10]. The surfactants are useful in drug delivery as they minimize drug degradation and loss, increase the bioavailability, and protect the body from any unwanted side effects of the drug and at the same time, achieving the desired concentration of drug at the target site [11,12]. For instance, colloidal suspensions of the bile salt, sodium deoxycholate have been commercially used as a medium for Amphotericin B delivery [13]. Phenothiazines represent a group of biologically important heterocyclic compounds endowed with dopamine receptor antagonistic activities in the central nervous system (CNS), which are commonly employed as

S. Mahajan, R.K. Mahajan / Journal of Colloid and Interface Science 387 (2012) 194–204

Cl

H

N

H

Cl

S N

S N

N

(a) Promazine hydrochloride (PMZ)

(b) Promethazine hydrochloride (PMT) + O- Na

+ O- Na

OH

HO

H

O

OH

(c) Sodium cholate (NaC)

195

OH

HO

O

H

(d) Sodium deoxycholate (NaDC)

Fig. 1. Chemical structures of phenothiazines (a and b) and bile salts (c and d).

antipsychotics, neuroleptics, and antihistamines [14]. Promazine hydrochloride (PMZ) and promethazine hydrochloride (PMT) are important phenothiazine derivatives with the sole difference being the additional secondary methyl group on the side chain of PMT (Fig. 1a and b). For the antipsychotic activity of the phenothiazines, there should be three carbon atoms between both the nitrogen atoms of the alkylamino chain (in the case of PMZ), whereas the phenothiazine derivatives with chains consisting of only two carbon atoms, as in PMT are very powerful antihistaminic, antiallergic and sedative drugs, but have no antipsychotic activity. Because of their potentially useful pharmacological and biological applications, it is very important to investigate the physicochemical aspects of the interactions of phenothiazine derivatives with model membranes such as micelles of surfactants and phospholipid bilayers, and accordingly, several literature studies have been reported in this field [15–18]. Caetano and Tabak [18] reported the interactions of two phenothiazine derivatives, chlorpromazine (CPZ), and trifluoperazine (TFZ) with anionic surfactant, sodium dodecyl sulfate (SDS), by means of electronic absorption and fluorescence spectroscopy and evaluated the binding constants of these drugs in the micelles by using the red1 shifts of the maximum absorption band and absorbance changes upon alkalization or in the presence of SDS. Recently, our research group reported the detailed physicochemical investigation of interactions between pyridinium gemini surfactants, that is, [12-(S-2-S)-12], [14-(S-2-S)-14], [16-(S-2-S)16], and PMT by employing conductivity, surface tension, steady state fluorescence, UV–visible, and NMR measurements [17]. In continuation of our interest in phenothiazines, we have now studied the interactions of bile salts with phenothiazines (PMZ and PMT). Since bile salts are very often used as penetration enhancers, so their binding with phenothiazines will improve their bioavailability due to which they can reach the systemic circulation and hence become available for distribution to the intended site of action. Bile salts possess a rigid steroid backbone with polar hydroxyl groups on the concave a-face and hydrophobic methyl groups on the convex b-face. The bile salts chosen for the present study are sodium cholate (NaC) and sodium deoxycholate (NaDC), which differ only in the number of hydroxyl groups (Fig. 1c and d). The hydrophobic bile salts such as NaDC promote absorption more 1 For interpretation of color in Figs. 2, 3, 5 and 6, the reader is referred to the web version of this article.

effectively than NaC, which has more polar surface area [19]. Further, the trihydroxy bile salt, that is, NaC has higher cmc value as compared to the dihydroxy bile salt, NaDC. Various bulk, interfacial, and thermodynamic parameters for the mixtures of phenothiazines (PMZ and PMT) with bile salts (NaC and NaDC) have been evaluated from conductivity and surface tension techniques. Moreover, the photophysical properties of phenothiazines have been utilized to study their binding behavior with bile salts by using the UV–visible and steady state fluorescence spectroscopy. The Benesi–Hildebrand (B–H) equation has been used to calculate the binding constants (K), stoichiometric ratios, and free energy change (DG) for the drug–bile salt complexes. These parameters predict the extent of binding between the phenothiazines and the bile salts.

2. Experimental section 2.1. Materials The phenothiazines, promazine hydrochloride (PMZ, purity P 98%) and promethazine hydrochloride (PMT, purity P 98%), and bile salt, sodium deoxycholate (NaDC, purity P 97%), were purchased from Sigma–Aldrich. The bile salt, sodium cholate (NaC, purity P 99%), was purchased from Alfa Aesar, UK. All the chemicals were used as received. Sartorius analytical balance with a precision of ±0.0001 g was used for weighing the amount of different substances. The deionized double distilled water having conductivity in the range of 1–2 lS cm1 was used for preparing all the solutions. Different mole fractions of drug–bile salt mixed systems were prepared from the stock solutions of different concentrations of drugs and bile salts. 2.2. Methods 2.2.1. Conductivity measurements Conductometric measurements were taken on an EQUIP-TRONICS auto temperature conductivity meter model EQ.661 equipped with a dip-type conductivity cell having a cell constant of 1.01 cm1. Temperature was maintained constant at 25.0 ± 0.1 °C using a constant temperature bath. The conductivity values were recorded when their fluctuation was less than 1% within 2 min. The reproducibility of these measurements was within ±0.2%.

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2.2.4. 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 spectra of PMZ and PMT were recorded in the range of 390– 550 nm wavelengths by exciting at 300 nm using an excitation and emission slit width of 10 nm. The fluorescence titrations were carried out in a manner similar to that of UV–visible measurements.

2.2.2. Surface tension measurements The surface tension values (c) were measured with a KRUSS Easy Dyne Tensiometer from Kruss Gmbh (Hamburg, Germany) using the Wilhelmy plate method at 25.0 ± 0.1 °C. The surface tension of pure water (i.e., 72.8 mN m1) was measured to calibrate the tensiometer and also to check the cleanliness of the glassware. For cmc determination, the concentrated stock solution was added progressively to a known volume of water and the c values were then measured after thorough mixing and temperature equilibration. Harkins and Jordan [20] correction in-built in the instrument software was taken into account to measure the surface tension values. The accuracy of c measurements was within ±0.15 mN m1. The regular solution theory (RST) proposed by Rubingh [21], widely used for the treatment of a non-ideal mixing owes much of its success to its simplicity. The most essential assumption in the regular solution model is that the adsorbed layer can be treated as a pseudo-phase, that is, the composition of the layer is fixed [22] and the model is accordingly often known as the pseudo-phase model. This approach will be applied here for the phenothiazine– bile salt mixed systems, thus allowing the evaluation of the micelm lar mole fraction (X m 1 ) and micellar interaction parameter (b ). This is exactly the sort of information that industrial researchers in particular are looking for.

3. Results and discussion 3.1. Drug–bile salt interactions in mixed micelles and mixed monolayer The bile salts, NaC and NaDC, form mixed micelles with PMT and PMZ. The cmc values of pure bile salts (NaC and NaDC), drugs (PMZ and PMT), and their mixtures have been measured by means of conductivity and surface tension techniques (Table 1). The cmc determined from conductivity and surface tension techniques show some differences in their values. This is due to the fact that cmc determination by surface tension reveals the onset of micelle formation in the surfactant solution, whereas the conductivity measurements indicate mainly the end of the micellization process [23]. For the determination of cmc by conductivity measurements, the micellar aggregate interfaces must contain a structure and composition suitable to give an aggregate with a degree of dissociation different from unity [24], which is responsible for a clear break in the plot of specific conductivity (j) versus [surfactant]. As shown in Fig. 2a and b, the plots of specific conductivity versus concentration of surfactant show two linear segments with the intersection at the cmc of surfactants. The smaller slope of the

2.2.3. UV–visible measurements The absorption spectra were recorded on Shimadzu UV-1700 UV–visible spectrophotometer with a quartz cuvette (path length, 1 cm). The titrations were performed by successive additions of 1 M stock solutions of bile salts (NaC and NaDC) directly into the quartz cuvette containing 3 mL of 25 lM drug solution, so as to give a final bile salt concentration in the range of 8.264– 62.500 mM for NaC and 3.322–16.393 mM for NaDC.

Table 1 Physicochemical parameters in mixed micelle and monolayer formation of mixtures of phenothiazines (PMZ and PMT) with bile salts (NaC and NaDC): Experimental cmc values (cmcexp) obtained from conductivity and surface tension techniques, ideal cmc (cmc), counterion binding (g), interaction parameters (bm and br), and activity coefficients (f1m ; f2m ; f1r and f2r ).

adrug

cmc (mM)

g

bm

f1m

f2m

br

f1r

f2r

7.98 3.26 4.53 7.31 4.34 26.98

7.98 9.29 11.11 13.83 18.29 26.98

– 0.202 0.183 0.224 0.208 0.272

– 6.33 4.51 2.82 6.06 –

– 0.069 0.177 0.387 0.248 –

– 0.461 0.521 0.608 0.194 –

– 8.95 7.53 4.60 7.55 –

– 0.036 0.079 0.249 0.176 –

– 0.256 0.265 0.394 0.130 –

2.90 0.66 1.80 3.53 1.15

2.90 3.53 4.51 6.24 10.14

– 0.282 0.218 0.185 0.170

– 10.51 5.60 3.20 9.33

– 0.014 0.081 0.238 0.073

– 0.256 0.543 0.706 0.127

– 8.34 2.87 – 8.06

– 0.012 0.120 – 0.055

– 0.544 0.945 – 0.275

1.68 4.12 6.88 3.98 45.71

9.56 11.91 15.82 23.50 45.71

0.417 0.366 0.196 0.375 0.318

9.87 5.62 3.93 7.50 –

0.020 0.100 0.232 0.142 –

0.259 0.483 0.550 0.165 –

12.04 5.70 – 10.74 –

0.008 0.072 – 0.044 –

0.192 0.558 – 0.103 –

0.99 1.64 2.77 1.56

3.56 4.63 6.62 11.56

0.744 0.827 0.796 0.738

9.30 6.98 5.13 8.86

0.014 0.044 0.107 0.062

0.386 0.468 0.553 0.180

10.64 9.14 – 17.10

0.005 0.015 – 0.005

0.384 0.392 – 0.037

cmcexp (mM) (Cond.)a (S.T.)b

PMZ + NaC 0.0 0.2 0.4 0.6 0.8 1.0

– 3.39 4.64 7.18 4.20 30.33

PMZ + NaDC 0.0 0.2 0.4 0.6 0.8

– 0.25 0.24 0.25 0.24

PMT + NaC 0.2 0.4 0.6 0.8 1.0

3.07 4.27 8.58 3.64 41.39

PMT + NaDC 0.2 0.4 0.6 0.8

0.32 0.52 2.26 0.42

(–) Not detected. a Conductivity. b Surface tension.

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(a)

(b)

600

1000

500

800

400 -1

PMZ

300

0.2 0.4 0.6 0.8

200

600

κ/μScm

κ/μScm

-1

α

α

PMT

400

0.2 0.4 0.6 0.8

200 100

0

0 0

2

4

6

8

0

10

2

4

6

8

C /mM T

(c)

10

12

14

16

C /mM T

(d)

65

70 α

α

PMT

PMZ

65

60

0.2 0.4 0.6 0.8

0.4 0.8 -1

50

0.6

60

γ /mNm

γ /mNm-1

55

0.2

55

50

45 45 40

40

35

35 -4

-3.6

-3.2

-2.8

-2.4

-2

log C

T

-4

-3.6

-3.2

-2.8

-2.4

-2

log C

T

Fig. 2. Specific conductivity (j) versus total concentration (CT) plot for drug–bile salt mixed systems (a) PMZ + NaC and (b) PMT + NaC and surface tension (c) versus log CT plot for (c) PMZ + NaC and (d) PMT + NaC. In (b), the scale shown is for aPMT = 0.2 and other curves have been shifted upwards by 0.5, 1, and 1.5 scale units, respectively.

linear segment above cmc is due to the confinement of a fraction of the counter ions to the micellar surface resulting in an effective loss of ionic charges, and furthermore, the micelles can contribute to the charge transport to a lesser extent than the free ions owing to their lower mobility [25]. However, in the present work, for the cmc determination of pure NaC and NaDC, there was no clear change in slope over a wide range of concentrations, thus rendering the conductance method not suitable for pure bile salts [4,9]. This means that the micelles of NaC and NaDC are completely ionized and donot have the ability to bind the Na+ counterion, that is, the micellar interface would not exist in the micellar aggregates of bile salts. But the specific conductivity plots of phenothiazine–bile salt mixed systems give much clear breaks than the bile salts individually. This is due to the amount of counterion bound to the micellar surface in the mixed systems, which contributes to the appearance of break. Fig. 2a and b shows the plots for cmc determination of drug–bile salt systems (PMZ + NaC and PMT + NaC) at

different mole fractions of drug by conductivity measurements. However, the corresponding plots for PMZ + NaDC and PMT + NaDC systems are not shown here. The change in conductivity values on addition of drugs indicates the interaction emanating from the drug–bile salt mixtures. Further, the values of degree of counterion binding (g) have been evaluated from the degree of dissociation of drug–bile salt mixtures, which is obtained from the ratio of post- to pre-micellar slopes using the specific conductivity (j) isotherms [26] (Fig. 2a and b). It can be observed that the values of ‘‘g’’ for the mixed systems of drugs with bile salts are almost similar and besides, the values are low that may be attributed to complex formation by the drug and bile salt molecules, which in turn reduces the micellar surface charge density. The mixed micellization studies of bile salts with drugs were further carried out by employing surface tension technique. The surface tension values of pure bile salts, drugs, and their mixed systems are found to decrease linearly with an increase in

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concentration, which ultimately attains a minimum value beyond which no considerable change in surface tension values was observed. This breakpoint is taken as cmc of pure as well as mixed drug–bile salt systems. The break in the surface tension-log[surfactant] curves is clear and without any minimum close to cmc indicating their purity. Hence, the surface tension plots (Figs. 2c and d, S1 and S2) have been employed to evaluate various micellization, interfacial, and thermodynamic parameters of drug–bile salt systems. The cmc values of pure PMZ and PMT (Fig. S2a) are found to be in close agreement with their literature values (i.e., 36 mM and 44 mM, respectively) [27]. Both of these drugs have no substitution on phenothiazine ring, but differ only in the branching of the alkyl side chain. Branching of an aliphatic chain is equivalent to shortening the chain length and lowering surface activity; therefore, PMT has a higher cmc value than PMZ. Florence and Parfitt [28] reported that on micelle formation, the hydrophilic N-methyl groups of phenothiazine derivatives are concentrated at the surface of the micelle and the bulky phenothiazine rings are arrayed in some manner to form the hydrophobic micellar core. The cmc values of pure bile salts, NaC (trihydroxy bile salt), and NaDC (dihydroxy bile salt) (Fig. S2b) were also found to be close to their literature values (i.e., 4–20 mM and 2–5 mM, respectively) [3,8]. The decrease in the number of hydroxyl groups renders the molecules of NaDC more hydrophobic as compared to NaC, leading to lower solubility and hence lower cmc values for NaDC than NaC. The cmc values of pure phenothiazines are higher than that of bile salts due to the differences in their micellization behavior. From Table 1, it can be observed that the experimental cmc values (cmcexp) of mixed micelles of bile salts with drugs are lower than those of bile salts and drugs individually. The electrostatic repulsive interactions between cholate ions in case of NaC (and deoxycholate ions in case of NaDC) are reduced by the added drug molecules through the electrostatic attractive interactions between the carboxylate group of bile salts and +NH(CH3)2 groups of drug molecules (Fig. 3a). This results in the decrease in cmc values of mixed micelles of bile salts with drugs, and hence, they aggregate more easily in the aqueous solution. It is also apparent from Table 1 that as the mole fraction of drug (adrug) increases, the cmc values of mixed systems initially increases, which may be attributed to increased repulsive interactions between the same charged head groups (+NH(CH3)2) of drug molecules that overcome the attractive interactions between the carboxylate group of bile salts and +NH(CH3)2 groups of drug molecules. But at higher mole fraction of drug, the cmc of mixed

S

S

Electrostatic Interactions

N

N

X

X NH

NH O O

O

Hydrophobic Interactions

O R3

R3

H

H H R1

H

(a)

H R2

H R1

H R2

H

(b)

Fig. 3. Schematic diagram showing interactions between phenothiazines (PMZ; X = –(CH2)2– and PMT; X = > CH–CH3 and bile salts (NaC; R1 = R2 = R3 = OH and NaDC; R1 = R3 = OH and R2 = H) at (a) low mole fraction of drug and (b) high mole fraction of drug.

micelles decreases, owing to the hydrophobic interactions between the phenothiazine rings of drugs and the steroid rings of bile salts that play their role at high mole fraction of drug (Fig. 3b). The behavior of mixed micelles of bile salts with drugs has been studied by considering Clint’s phase separation model [29] from which the ideal cmc (cmc) of the drug–bile salt mixed systems has been calculated by using the following equation:

1 a1 ð1  a1 Þ ¼ þ cmc cmc1 cmc2

ð1Þ

Here, the cmc1 and cmc2 refers to the cmc’s of pure drugs (PMT and PMZ) and pure bile salts (NaC and NaDC), respectively, and a1 is the mole fraction of drug (PMT and PMZ). This equation is the difference between ideal and nonideal mixtures. From Table 1, it can be observed that the cmcexp values of different combinations of phenothiazine–bile salt systems (PMZ + NaC, PMZ + NaDC, PMT + NaC, and PMT + NaDC) determined from the surface tension (c) – log[surfactant] plots (Figs. 2c and d, and S1) are lower than the ideal one, indicating that synergistic interactions are stabilizing the mixed micelles of bile salts with drugs. The nature and strength of interactions between bile salts and drugs can be analyzed by using RST [21] that allows us to evaluate the micellar mole fraction (X m 1 ) and micellar interaction parameter (bm) between the two components in the mixed micelles from the following equations: 2 m m m ½ðX m 1 Þ  lnða1  C 12 =X 1  C 1 Þ

½ð1 

bm ¼

2 Xm 1Þ

ln



m m  ln½ð1  a1 Þ  C m 12 =ð1  X 1 Þ  C 2 

Cm 12 a1 m Cm 1 X 1

¼1

ð2Þ



2 ð1  X m 1Þ

ð3Þ

m m where C m 1 ; C 2 and C 12 denote the experimental cmc values of the pure drugs (PMZ and PMT), pure bile salts (NaC and NaDC), and their mixed systems, respectively, and a1 is the mole fraction of drugs (PMZ and PMT). Eq. (2) was solved iteratively for X m 1 , which is then substituted into Eq. (3) to calculate bm values. Analogous to Rubingh’s Eqs. (2) and (3) for mixed micelles, Rosen et al. [30,31] proposed a model to evaluate the micellar mole fraction of first component (X r1 ) at the mixed adsorbed film and the interfacial interaction parameter (br) using above equations but m m m with X m (in Eqs. (2) and (3)) replaced by 1 ; C 1 ; C 2 and C 12 X r1 ; C S1 ; C S2 and C S12 respectively. The symbols C S1 ; C S2 and C S12 represent the molar concentrations of pure drugs (PMZ and PMT), pure bile salts (NaC and NaDC), and their mixtures at different mole fractions of drugs (a1), required to produce a given surface tension reduction at air–water interface. r As seen from Figs. 4 and S3, the values of both X m 1 and X 1 increase with an increase in the mole fraction of drug, which suggests that as adrug increases, more number of drug molecules contribute to the mixed micelle and mixed monolayer formation. The b values (bm and br) reflect the extent of interactions between bile salts and drugs and hence their deviation from ideality on mixing. A negative b value indicates attractive interactions, a positive value to incompatible surfactant species and repulsion while zero value means ideal mixing. The bm values, given in Table 1, are negative with the respective average values (bm av ) being 4.93, 7.16, 6.73 and 7.57 for PMZ + NaC, PMZ + NaDC, PMT + NaC and PMT + NaDC, respectively, signifying attractive interactions in the mixed micelle formation. It can be observed that the PMZ/ PMT + NaDC systems have higher values of bm av , which may be attributed to the more hydrophobic nature of NaDC than NaC. The magnitude of bm values decreases on increasing the mole fraction of drug except at higher mole fraction of drug. Moreover, the br values (Table 1) for all the phenothiazine–bile salt systems

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(a)

(b)

1

0.8

1

,X

0.6

1

ideal

ideal m

0.4

1

X ,X

1

X ,X

m

0.6

1

,X

1

σ

σ

0.8

1

0.4

m 1

X

m 1

X

ideal X 1

0.2

0.2

ideal 1

X

σ 1

X

σ 1

X

0

0 0

0.2

0.4

0.6

α

0.8

1

0

0.2

0.4

0.6

0.8

1

α

PMT

PMZ

r ideal Fig. 4. Plots of micellar mole fraction (X m ) versus mole fraction (a) of drug for (a) PMZ + NaC and (b) 1 ), interfacial mole fraction (X 1 ), and mole fraction in ideal state (X 1 PMT + NaC mixed systems.

throughout the mole fraction of drug are also negative having average values (brav ) of 7.16, 6.42, 9.49 and 12.29 for PMZ + NaC, PMZ + NaDC, PMT + NaC and PMT + NaDC, respectively, suggesting attractive interactions in mixed monolayer formation. The values of b (bm and br) are related to activity coefficients [21] in the micelles (f1m and f2m ) and monolayers (f1r and f2r ) of drugs and bile salts respectively by the following equations:

f1 ¼ exp½b  ð1  X 1 Þ2 

ð4Þ

f2 ¼ exp½b  ðX 1 Þ2 

ð5Þ m

Xm 1

In Eqs. (4) and (5), the b and values are used for evaluating f1m and f2m , whereas for f1r and f2r , the br, and X r1 values are utilized. The values of activity coefficients for all the drug–bile salt systems are less than unity (Table 1) indicating non-ideal behavior for mixed micelles as well as mixed monolayers. Motomura and Aratono [32] developed a model that is an attempt to overcome the limitations of Rubingh’s regular solution theory [33,34] and improve the predictions of the phase separation model. Basically, it is a thermodynamic method that considers mixed micelles as a macroscopic bulk phase where the energetic parameters of such systems can be evaluated in terms of excess thermodynamic quantities. In the present study, only the micellar mole fraction of the mixed sytems in the ideal state (X ideal ) has been 1 computed using Motomura’s approximation as follows:

X ideal ¼ 1

a1 

Cm 2

a1  C m2

þ ð1  a1 Þ  C m 1

3.2. Drug–bile salt interactions at the air–water interface When surfactants concentrate in an adsorbed monolayer at an interface, the physical properties of the interface can be very important in all types of natural phenomena and industrial processing operations. For example, many industrial processes involve colloidal dispersions, such as foams, emulsions and suspensions, all of which contain large interfacial areas and hence the properties of these interfaces may also play a large role in determining the properties of the dispersions themselves [35]. When surfactant molecules adsorb at an interface, they provide an expanding force and cause the interfacial tension to decrease (at least up to the cmc). This is illustrated by the general Gibbs adsorption equation from which the maximum surface excess concentration [36] (concentration of surfactant at the surface in excess of the bulk concentration) at the air/water interface, Cmax (mol m2) can be calculated as:

Cmax ¼ 

ð7Þ

where dc/dlog C is the maximum slope, c is the surface tension of the solution, R is the universal gas constant (8.314 JK1 mol1), 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 Umax values (Table 2) were further used to evaluate the minimum area per surfactant molecule, Amin (Å2) at the air/water interface [36] using the following equation:

ð6Þ

r Figs. 4 and S3 illustrates the comparison of the X m 1 and X 1 (evalideal uated using Rubingh’s and Rosen’s equations) with X 1 as a function of adrug for all the phenothiazine–bile salt systems. It becomes very clear that all the mixed systems are far from an ideal behavior. r It is evident from the figure that both X m 1 and X 1 values are greater than X ideal for all the mole fractions of drug–bile salt mixed sys1 tems, indicating that the mixed micelles formed by these systems contain more contribution of drug molecules than in its ideal mixing state and less transfer of bile salts from the solution to the micellar phase.

  1 @c 2:303nRT @ log C

Amin ¼

1020 NA Cmax

ð8Þ

where NA is Avogadro’s number. These values suggest whether the surfactant molecule is closely or loosely packed at air/water interface. The Umax and Amin values for all drug–bile salt systems, as shown in Table 2, do not follow any regular trend with an increase in the mole fraction of drug. The lower value of Umax of pure NaC (0.89) than that of NaDC (1.04) may be ascribed to more polar nature of NaC due to which it has a tendency to stay preferably more in the bulk solution as compared to NaDC. On the other hand, the lower value of Umax of pure PMT (1.22) than PMZ (1.73) may be

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Table 2  Interfacial and thermodynamic parameters: surface tension at cmc (ccmc), surface excess (Cmax), minimum area per molecule (Amin), pC20, free energy of micellization (DGm ), free  s energy of adsorption (DGad ), surface free energy (Gmin ), excess free energy (DGex) of mixtures of phenothiazines (PMZ and PMT) with bile salts (NaC and NaDC).

adrug

ccmc (mN m1)

106 Cmax (mol m2)

Amin (Å2)

pC20

DGm (kJ mol1)

DGad (kJ mol1)

Gsmin (kJ mol1)

DGex (kJ mol1)

PMZ + NaC 0.0 0.2 0.4 0.6 0.8 1.0

42.9 40.3 38.6 40.2 40.3 41.7

0.89 1.21 1.40 1.38 0.95 1.73

186.55 137.21 118.59 120.31 174.77 95.97

2.95 3.36 3.23 2.82 3.19 2.02

21.93 24.15 23.33 22.14 23.43 18.91

54.51 49.52 46.33 54.25 55.54 35.61

48.20 33.30 27.57 29.13 42.42 24.10

– 3.57 2.63 1.70 3.75 –

PMZ + NaDC 0.0 38.0 0.2 40.9 0.4 40.2 0.6 42.9 0.8 41.2

1.04 1.08 1.00 1.33 0.92

159.64 153.73 166.03 124.83 180.47

3.52 3.94 3.84 2.90 3.90

24.44 28.12 25.62 23.95 26.72

56.07 55.16 58.12 45.07 58.90

36.54 37.87 40.20 32.25 44.78

– 6.00 3.07 1.75 5.76

PMT + NaC 0.2 0.4 0.6 0.8 1.0

41.0 41.2 51.6 41.5 44.1

1.01 1.04 0.92 0.79 1.22

164.39 159.64 180.47 210.16 136.09

3.66 3.22 2.20 3.56 2.09

25.79 23.56 22.29 23.65 17.60

55.59 53.56 44.68 62.51 41.88

40.60 39.61 56.09 52.53 36.15

5.70 3.21 2.32 4.64 –

PMT + NaDC 0.2 41.6 0.4 38.2 0.6 50.2 0.8 39.1

0.86 1.00 0.80 0.61

193.06 166.03 207.54 272.18

3.92 4.08 2.70 4.15

27.10 25.84 24.55 25.97

61.28 59.74 52.17 77.28

48.37 38.20 62.75 64.10

5.01 3.82 2.85 5.41





(–) Not detected.

attributed to more steric hindrance in PMT due to branching close to the hydrophilic group. A magnitude frequently used to measure the efficiency of adsorption (pC20) is the negative logarithm of the concentration of surfactant in the bulk phase required to reduce the surface tension of the solvent by 20 mN m1. The molar concentration of the surfactant at this point is called C20. The larger the pC20 value, better is the adsorption and more efficiently it reduces the surface tension. Most of the phenothiazine–bile salt systems have higher pC20 values than the pure bile salts (NaC and NaDC) and drugs (PMZ and PMT) (Table 2). However, the PMT + NaDC system shows better adsorption efficiency at air/water interface than the other drug–bile salt mixed systems. 3.3. Synergism in drug–bile salt interactions Phenothiazine–bile salt mixed systems have much higher surface activities and lower cmc values as compared to their individual components. The existence of synergism in mixtures containing two surfactants has been shown to depend not only on the strength of the interaction between them (measured by the values of the b parameter) but also on the relevant properties of the individual surfactant components of the mixture. The synergism in mixed micelle formation [37] exists when the cmc of mixture is less than that of the individual surfactants of the mixture and its conditions are the following: (a) bm must be m s s r m negative, (b) jbm j > j lnðC m 1 =C 2 Þj and ðcÞ jb  b j > ½j lnðC 1 =C 2 Þj m m m j lnðC m =C Þj where C and C represent the cmc values of pure 1 2 1 2 drugs (PMZ and PMT) and pure bile salts (NaC and NaDC), respectively. From Table S1, it can be observed that all drug–bile salt systems satisfy these conditions and hence exhibit synergism in their mixed micelles. On the other hand, the conditions for synergism in surface tension reduction efficiency [37] are same as (a and b) but m S S r with |bm|, C m 1 and C 2 replaced by |b |, C 1 and C 2 respectively, S S where C 1 and C 2 represent the molar concentrations of pure drugs (PMZ and PMT) and pure bile salts (NaC and NaDC) respectively, required to produce a given surface tension reduction at air–water

interface. It is apparent that all phenothiazine–bile salt mixtures exhibit synergism in surface tension reduction efficiency, since both these conditions are satisfied by them (Table S1). 3.4. Thermodynamics of micellization and interfacial adsorption phenomenon By applying the phase separation model of micellization, the  standard free energy of micellization [38] (DGm ) is calculated from the following equation 

DGm ¼ RT ln X cmc

ð9Þ

where Xcmc is the cmc in mole fraction units. As shown in Table 2,  the negative DGm values of drug–bile salt systems indicate sponta neity of micellization. It can also be observed that DGm values for most of the mole fractions of drug–bile salt mixtures are more negative than their individual components, signifying more spontaneity of the mixed micelle formation. Alternatively, the standard  free energy of adsorption [39] (DGad ) at the air/water interface has been evaluated using the following equation 



DGad ¼ ðDGm Þ 



pcmc

Cmax

 ð10Þ

where pcmc (mN m1) is the surface pressure at cmc and can be obtained as per the relation:

pcmc ¼ co  ccmc

ð11Þ

where co is the surface tension of pure solvent and ccmc is the sur face tension of surfactant solution at cmc. The DGad values are also negative suggesting the spontaneity of adsorption (Table 2). The   less negative values of DGm as compared to DGad show that the micelle formation is a secondary and less spontaneous phenomenon as compared to adsorption. Another thermodynamic quantity known as the free energy of a surface at equilibrium (Gsmin ) has been used to evaluate the synergism in the mixed adsorbed monolayer formation [40]:

S. Mahajan, R.K. Mahajan / Journal of Colloid and Interface Science 387 (2012) 194–204

Gsmin ¼ Amin ccmc NA

ð12Þ

The low magnitude of Gsmin values (Table 2) of pure bile salts (NaC and NaDC), pure drugs (PMZ and PMT), and their mixtures indicate that thermodynamically stable surfaces are formed by these systems and hence the drug–bile salt interactions are favorable. The excess free energy of micellization, DGex can be evaluated by utilizing the activity coefficients in micelles (f1m and f2m ) through the following equation

DGex ¼ ½X 1 ln f1 þ ð1  X 1 Þ ln f2 RT

ð13Þ

As shown in Table 2, all the calculated DGex values are negative, indicating the energetic stabilization accompanied by the mixed micelle formation. 3.5. Spectroscopic studies The interactions of phenothiazine derivatives (PMZ and PMT) with bile salts (NaC and NaDC) have further been investigated by employing the spectroscopic techniques such as UV–visible and steady state fluorescence. The aromatic ring of phenothiazine moiety in PMZ and PMT is responsible for their significant absorption and fluorescence properties, which vary with its local environment and thus can be used to probe drug–bile salt interactions. It is to be noted here that the bile salts showed no absorption and emission background. 3.5.1. UV–visible studies The absorption spectra of phenothiazines, PMZ and PMT, in aqueous solutions with varying concentrations of bile salts, that is, NaC and NaDC are shown in Figs. 5 and S4 respectively. The spectra of PMZ presented two maximum absorption wavelengths (kmax) at 252 and 300 nm regions. The shorter wavelength band (at 252 nm) was attributed to p–p transition, whereas the longer

Fig. 5. Absorption spectra of PMZ (25 lM) with increasing concentration of (a) NaC and (b) NaDC.

201

wavelength band (at 300 nm) was mainly due to n–p transition. Aaron et al. [41] pointed out that the interpretation of the latter band was supported by the presence of the lone pairs of electrons on sulfur atom in the phenothiazine ring. From the Figs. 5 and S4, it can be observed that gradual addition of bile salts to both of the drugs is associated with red shift of the kmax at 252 nm for PMZ (and at 249 nm for PMT) with a simultaneous increase in absorbance. On the other hand, the kmax at 300 nm shows no spectral shift (just 1–2 nm) and is only accompanied by hyperchromic effect. The red shift for PMZ is about 4 nm in both 62.5 mM of NaC and 16.393 mM of NaDC solutions compared to its position in aqueous solution (Fig. 5). Similarly, for PMT, the shift is 5 nm in 62.5 mM of NaC as well as 16.393 mM of NaDC solutions (Fig. S4). These spectral shifts may be the first indication of the interaction between drugs and bile salts, although nothing concrete can be derived from such a response due to their low values. Only the quantitative estimate of the extent of binding of drugs to bile salt micelles has been made by using the Benesi–Hildebrand equation [42] (discussed later on). 3.5.2. Steady state fluorescence studies Phenothiazines, in general, have charge-transfer transitions and the emission from these fluorophores occur at wavelengths longer than those at which absorption occurs. The fluorophore is usually excited from the ground state (S0) to the first singlet state (S1) in which the electron density is transferred from the sulfur atom of the middle ring of phenothiazine moiety to the benzene rings, possessing greater p character. This charge transfer is related to a decrease in torsion angle, leading to an increase in the dipole moment of the excited state relative to that of the ground state. Following excitation, the dipoles of the surrounding molecules can reorient or relax around the excited state that lowers its energy and hence increase the wavelength in fluorescence measurements of phenothiazines [43,44]. It has also been observed that the emission maxima are more dependent on polarity of the surrounding

Fig. 6. Fluorescence spectra of PMZ (25 lM) with increasing concentration of (a) NaC and (b) NaDC.

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environment of probe than the corresponding absorption maxima due to the fact that the molecule stays in the excited state for more time in fluorescence than in the absorbance measurements. Moreover in absorption spectra, the molecule is exposed to the same local environment in the ground and excited states, whereas in emission spectra, the emitting fluorophore is exposed to the relaxed environment, which contains solvent molecules oriented around the dipole moment of the excited state [43]. In the present study, PMZ shows emission maxima at 446 nm with a shoulder at 419 nm and PMT fluorescence at 440 nm in aqueous solution (Figs. 6 and S5). It is to be noted here that the emission maxima for both the drugs are very close to each other which shows that there is no contribution of the N-alkyl chain to the emission properties of the phenothiazine derivatives, which is in agreement with the fact that the emission maxima of the parent phenothiazines are close to those of the corresponding 10-alkylated derivatives [45]. Contrarily to the effects of bile salts observed for the absorption spectra, the emission maxima were shifted toward the blue with decreasing polarity of the surrounding environment. It can be observed from Figs. 6 and S5 that the gradual addition of both NaC and NaDC micelles to the aqueous solutions of phenothiazines (PMZ and PMT) is associated with a blue shift in the emission maximum along with an increase in fluorescence intensity, suggesting that the environment around the drug gets perturbed in presence of bile salts. The blue shift for PMZ is about 9 nm in 62.5 mM of NaC compared to its position in aqueous solution, whereas the shift is approximately 8 nm in only 16.393 mM of NaDC (Fig. 6). Similarly, for PMT, the shift is 11 nm in 62.5 mM of NaC and 13 nm in only 16.393 mM of NaDC (Fig. S5). The blue shift of the emission maxima may be due to the lowering of polarity of immediate surroundings of the probe that arises from its movement, in course of binding with bile salts, from highly polar (aqueous) environment to the less polar (hydrophobic) environment of bile salts. As a result of the movement of probe to a more hydrophobic environment, the possibility of non-radiative decay reduces to a greater extent. This is then marked by the simultaneous increase in intensity of emission maximum band due to the enhancement of the energy gap between the excited and ground states.

(a)

3.5.3. Determination of stoichiometric ratio and binding constant of drug–bile salt complex The stoichiometric ratio and the binding constant of all the phenothiazine–bile salt complexes were estimated from the Benesi– Hildebrand (B–H) equation [42,46]. The change in the absorbance is related to the concentration of bile salts, that is, [BS] according to the following equation

1 1 1 ¼ þ A  A0 KðA1  A0 Þ½BSn A1  A0

where K is the binding constant for the formation of drug–bile salt complexes and n is the stoichiometry of the equilibrium reaction in relation to bile salts, A and A0 are the values of absorbance of drugs in the presence and absence of bile salts respectively, and A1 is the absorbance due to formation of drug–bile salt complex. The fluorescence enhancement of phenothiazines (PMZ and PMT) in the presence of both of the bile salts was also analyzed by B–H equation to reflect the changes in fluorescence intensity as follows:

1 1 1 ¼ þ I  I0 KðI1  I0 Þ½BSn I1  I0

ð15Þ

where I0 and I are the fluorescence intensities of drugs in the absence and presence of bile salts, respectively, and I1 is the fluorescence intensity of the drug–bile salt complex formed. The double reciprocal B–H plots of (A  A0)1 or (I  I0)1 versus 1/[BS]n for all the phenothiazine–bile salt complexes (Figs. 7 and S6) should show a linear relationship for the correct stoichiometry (n). The values of K for these complexes were calculated from the ratio of intercept and slope of B–H plots, obtained from both UV– visible and steady state fluorescence experiments, and are given in Table 3 along with the values of correlation coefficient (R) that justifies the excellence of the linear fit. As shown in Figs. 7 and S6, a linear B–H plot was obtained for all the complexes when n = 2 indicating 1:2 stoichiometry for these complexes, that is, Drug : (BS)2 by both of the spectroscopic techniques. However, the B–H plots assuming a 1:1 stoichiometry results in a distinctly curvilinear fit, suggesting that this is an incorrect assumption and hence the actual stoichiometric ratio for the drug–bile salt

(b)

18

ð14Þ

0.25 NaC

NaC

16

NaDC

NaDC

0.2

14 0

1/I-I

1/A-A

0

0.15 12

0.1 10

0.05

8

6

0 0

5000

10000

15000 -2

[BS] /M

-2

20000

25000

0

20000

40000

60000 -2

[BS] /M

80000

100000

-2

Fig. 7. Benesi–Hildebrand plot using (a) changes in absorption spectra of PMZ (measured at 256 nm for both NaC and NaDC) and (b) changes in fluorescence spectra of PMZ (measured at 437 nm for NaC and 438 nm for NaDC).

S. Mahajan, R.K. Mahajan / Journal of Colloid and Interface Science 387 (2012) 194–204 Table 3 Estimated binding constants (K), free energy change for the drug–bile salt complexation (DG), and the correlation coefficients (R) for various phenothiazine–bile salt complexes. Drug–bile salt complex

K (M2)

DG (kJ mol1)

R

Absorbance data PMZ + NaC PMZ + NaDC PMT + NaC PMT + NaDC

1244.396 10414.239 416.823 2117.360

17.665 22.931 14.954 18.982

0.9817 0.9999 0.9921 0.9988

Fluorescence data PMZ + NaC PMZ + NaDC PMT + NaC PMT + NaDC

1504.620 13140.196 628.292 2568.659

18.135 23.508 15.971 19.462

0.9921 0.9953 0.9999 0.9980

complexes is primarily 1:2. The equilibrium process may then be expressed as follows:

203

of bile salts to the Benesi–Hildebrand (B–H) equation. The values of K and DG for these complexes, calculated from both the spectroscopic measurements, are comparable to each other. It was also observed that the stoichiometric ratio of these complexes obtained from B–H plots of both of the spectroscopic measurements is dominated by 1:2. The highest value of K for PMZ + NaDC system signifies strong association between them, suggesting that NaDC binds strongly with phenothiazines than NaC and can improve their bioavailability to a better extent. These results have been explained on the basis of more hydrophobic nature of NaDC and PMZ than NaC and PMT respectively. Further, the negative values of DG indicate the spontaneity of drug–bile salt complexation. Acknowledgment Suruchi Mahajan thanks the UGC-SAP, New Delhi, India, for the award of Junior Research Fellowship. Appendix A. Supplementary material

K

Drug þ 2BS $ Drug : ðBSÞ2 K¼

½Drug : ðBSÞ2  ½Drug½BS2

From the values of K, the free energy change for this process of complexation can be obtained by employing the relation DG = RT ln K. Table 3 shows that the values of K and DG obtained from both of the spectroscopic measurements are comparable to each other. The negative values of DG show that the drug–bile salt complexation is energetically favorable. As can be observed from the values of K, the binding of drugs is stronger for NaDC than NaC due to the slight difference in their hydrophobicity leading to their different binding abilities. Further, it was also observed that among PMZ + NaC and PMT + NaC systems, the binding is stronger for the former one which may also be attributed to the more hydrophobic nature of PMZ than PMT. Similarly, PMZ + NaDC system has higher value of K than PMT + NaDC. The coulombic attraction forces between the positively charged drug and negatively charged bile salts play only a minor role in bringing them close to each other at the start of the interaction process. 4. Conclusions Bile salts are biocompatible surfactants and a very promising class of penetration enhancers, so the present study is valuable in understanding the drug–bile salt interactions. As far as we are aware, there is no report in the literature that deals with a detailed insight on the micellization as well as binding studies of phenothiazine tranquilizers (PMZ and PMT) with bile salts (NaC and NaDC), hence we pursued the study presented here. From the micellization studies, it can be concluded that cmc values of phenothiazine–bile salt mixed micelles is much lower than their individual components due to which the toxicity of drugs is reduced and their permeability and bioavailability is enhanced. Some bulk and surface properties of the phenothiazine–bile salt systems were investigated by conductivity and surface tension techniques. Synergism was observed in micelle as well as at the interface for all the phe nothiazine–bile salt mixed systems. The negative values of DGm  and DGad signifies the spontaneity of micellization and adsorption phenomenon, whereas a negative value of DGex ensures the stability of drug–bile salt mixed micelles. The UV–visible and steady state fluorescence measurements have also been employed to determine the stoichiometric ratios, binding constants (K), and free energy change for the drug–bile salt complexation (DG) by fitting the changes in spectral intensities of phenothiazines on addition

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