Surfactants are known to play a vital role in many processes of interest in
both fundamental and applied science. One important property of surfactants
is the formation of colloidal-sized clusters in solutions, known as micelles
which have particular significance in pharmacy because of their ability to increase
the solubility of poorly soluble drugs in water (Rangel-Yagui
et al., 2005a, Rangel-Yagui et al., 2005b). These
aggregates exhibit an interfacial region separating the polar bulk aqueous phase
from the hydrocarbon-like interior (Corrigan and Healy, 2002).
Hydrophobic groups of surfactants form a micelle core which is liquid hydrocarbon
like in character, while their hydrated polar groups constitute a micelle outer
shell in contact with water (Florence and Attwood, 2006).
While solubilization of very no polar organic substance takes place in the micellar
core, relatively high polar molecules tend to be located on outer region that
is solubilized in the interfacial region of the micelle. This model implies
that an organic substance which is almost completely insoluble in water will
be solubilized by micelles while a substance which is more soluble in aqueous
media would be expected to partition between aqueous and micellar phase.
|| The molecular structure of PHT
As a consequence, micellar solutions consist of a special medium in which hydrophobic,
amphiphilic or ionic compounds may be solubilized and reagents may be concentrated
or separated in aqueous solution. Therefore, the utilization of aqueous micellar
solutions for drug solubilization can be advantageous for drug delivery purposes
(Rangel-Yagui et al., 2005a).
The physical chemical interactions of drugs with surfactant micelles can be
visualized as an approximation for their interactions with biological surfaces
because surfactant micelles and lamellar phases also have been used as mimetics
for biomembranes. This provides an insight into more complex biological processes,
such as the passage of drugs through cell membranes. An important and fundamental
event in the interaction of drugs with biological tissues at the molecular level
is their binding to membranes. This is an important issue because it relates
to the mechanism of drug action (Fresta et al., 2002;
Corrigan and Healy, 2002). An important step to understand
such interactions is to characterize in detail the drug interaction sites, as
well as the effect of the microenvironment on the drug interaction sites. Physicochemical
aspects of the binding of nitrogen-containing heterocyclic drugs to model and
natural membranes have been subjects of extensive studies involving a great
variety of drugs from different therapeutic categories (Enache
and Volanschi, 2010). In recent years some studies have been published on
the characterization of phenothiazine derivatives such as thioridazin HCl, promethazine
HCl, chlorpromazine HCl in the presence of surfactants (Caetano
and Tabak, 1999, Caetano and Tabak, 2000; Erdinc
et al., 2010; Caetano et al., 2002). There is not
much work concern about distribution and binding properties of phenothiazine
in the presence of surfactants and effect of cosolvent on these interactions.
In this work interaction of sparingly water-soluble drug phenothiazine (PHT)
with Sodium Dodecyl Sulfate (SDS) and TritonX-100 (TX100) micelles was studied
in the presence of various amount of ethanol (v/v). To the best of our knowledge,
the effect of ethanol on the interaction of PHT with various surfactants has
not yet been studied. Previously, solubilization of PHT in aqueous surfactant
micelles has been studied from a thermodynamic point of view with the theoretical
background to a mechanism of solubilization (Moroi et al.,
1982). Recently, Liu and Guo reported microenvironment effect on the location
distribution of PHT in CTAB/n-pentanol/H2O and bi-continuous microemulsions
using cyclic voltammetry methods (Liu and Guo, 2007).
Phenothiazines are a group of basic drugs including a phenothiazine ring with
different substituents attached at the 2-and 10-position, which are used as
antipsychotics, neuroleptics and antihistamines. The parent of the phenothiazines,
PHT is a typical and useful organic compound with an N atom that easily looses
one electron to form cationic radical (Kamat and Seetharamappa,
2004). The molecular structure of PHT is illustrated in Fig.
MATERIALS AND METHODS
The research was carried out between 2007/2008 and 2009/2010 academic sessions in Department of Basic Pharmaceutical Sciences, Faculty of Pharmacy, Marmara University, Istanbul. All the chemicals were of analytical reagent grade. SDS, TritonX-100 and PHT were obtained from Sigma. As solvent, doubly distilled water, water-ethanol mixtures containing 5, 10, 20, 30, 40, 50% (v/v) of ethanol (absolute ethanol Ridel de Haen) were used. Spectroscopic measurements were carried out using a UV-Visible Spectrophotometer (Shimadzu 2100S) with a matched pair of cuvettes of 1 cm path length in a thermostated cell holder. The reproducibility for λmax of spectra was ±0.1 nm. All measurements were done at least in triplicate at 298 K during the study. A PHT solution with a given concentration was prepared in ethanol-water mixture. Absorption spectra of PHT in aqueous solution with a varying wide concentration range of SDS and TX100 in the presence of various concentrations of ethanol (v/v) have been recorded at 298 K (±0.1), keeping the concentration of PHT (1.0x10-5 mol dm-3) fixed in each one. Drug/micelle binding constant, Kb, was determined from the absorbances at 250 nm of a series of solutions containing a fixed concentration of PHT in the presence of various amount of ethanol and increasing concentrations of SDS and TX100. Micelle/water partition coefficients, Kx, were determined from the absorbances at the same wavelength (λ = 250 nm) of a series of solutions containing a fixed concentration of PHT in the presence of various amount of ethanol and increasing concentrations of SDS and TX100. In this study the CMC determination is based on the surface tension measurements. The surface tension of surfactant solutions was determined using an automatic tensiometer, KSV Instruments, model Sigma 701 (Helsinki, Finland) employing the Wilhelmy plate method to determine the CMC and observe the variation surface tension of surfactants in the presence of 1.0x10-5 mol dm-3 PHT at various ethanol-water mixtures.
RESULTS AND DISCUSSION
The hydrophobic cationic drug PHT exhibit two maximum absorption bands at 250
and 315 (±0.1) nm in the presence of 5, 10, 20, 30, 40 and 50% (v/v)
ethanol concentrations. The change in absorbance value at 250 nm has been used
to study the interaction between PHT and surfactants. The molar absorption coefficients
(ε0) of PHT at 250 nm in the concentration range of 1.0x10-5
to 8.0x10-5 mol dm-3 were calculated as 35607, 35656,
35669, 35834 and 35909 mol-1 dm3 cm-1 at 298
K (±0.1), respectively. The linear relation between absorbance and PHT
concentration (r: 0.9999) indicates that the validity of Lambert-Beer Law. The
absorption spectra of 1.0x10-5 mol dm-3 PHT at various
selected concentrations of SDS and TX-100 in the presence of 5% (v/v) ethanol
concentrations are shown in Fig. 2a and b,
respectively, as an example.
A similar behavior i.e., progressive enhancement in absorbance at the surfactant
concentration above the CMC was observed for SDS and TX100 in the presence of
5, 10, 20 and 30% concentrations of ethanol. Addition of surfactants did not
influence the spectral characteristics of PHT in the presence of various ethanol
concentrations. The value of λmax was constant regardless of
the surfactant concentration. In the case of SDS a decrease in the absorbance
was observed at the concentration below the CMC which indicates the molecular
complex formation between cationic PHT and anionic surfactant molecules due
to the electrostatic interaction. At the concentrations below the CMC the absorbance
of PHT remained almost constant for nonionic surfactant TX100. As seen in Fig.
2a and b, the increase in surfactant concentration above
CMC is regarded to be caused by the incorporation of PHT molecules to micelles.
PHT may be adsorbed on the surface or may be trapped in the hydrocarbon core
of the type of the surfactant. As more drug molecules are incorporated to micelles
the absorbance values of λmax reaches a limiting value and becomes
almost constant. The increase in ethanol concentration the micelle formation
of both surfactants diminished and micellization totally inhibited when ethanol
concentration reached a certain value.
||(a) The absorption spectra of 1.0x10-5 mol dm-3
PHT at various concentrations of SDS in the presence of 5% (v/v) ethanol
at 298 K. Concentrations of SDS; 1: no SDS, 2: 5.10-5, 3: 3.10-4,
4: 8.10-4, 5: 1.10-3 mol dm-3 (b). The
absorption spectra of 1.0x10-5 mol dm-3 PHT at various
concentrations of TX100 in the presence of 5% (v/v) ethanol at 298 K. Concentrations
of TX100; 1: no TX100, (2): 1.10-5, (3): 8.10-5, (4)
1.10-4 (5) 5.10-4 mol dm-3
||(a) The absorption spectra of 1.0x10-5 mol dm-3PHT
at various concentrations of SDS in the presence of 40% (v/v) ethanol at
298 K. Concentrations of SDS; 1: no SDS, 2: 1.10-4, 3: 5.10-3,
4: 8.10-3, 5: 1.10-2 mol dm-3 (b). The
absorption spectra of 1.0x10-5 mol dm-3 PHT at various
concentrations of TX100 in the presence of 40% (v/v) ethanol at 298 K. Concentrations
of TX100; 1: no TX100, (2): 8.10-5, (3): 1.10-4, (4)
4.10-4 (5) 1.10-3 mol dm-3
The absorption spectra of PHT at various concentrations of SDS and TX100 in
the presence of 40% (v/v) ethanol were shown in Fig. 3a and
b, respectively. As seen in these figures even at high surfactant
concentrations there was no interactions observed.
||(a) The absorbance change of 1.0x10-5 mol dm-3
PHT with concentration of SDS in the presence of 5, 10, 20 and 40% (v/v)
ethanol. (b) The absorbance change of 1.0x10-5 mol dm-3
PHT with concentration of TX100 in the presence of 5, 10, 20 and 40% (v/v)
The absorbance change of 1.0x10-5 mol dm-3 PHT with concentration
of SDS and TX100 in the presence of 5, 10, 20 and 40% concentrations of Ethanol
was shown in Fig. 4a and b, respectively.
Binding constant of PHT in the presence of micelles: The binding constant is quantitatively determined in terms of the pseudo-phase model (phase separation approximation) in which micelles and water are considered as separate pseudo-phases. Equilibrium scheme of PHT and micelle can be assumed as:
Benesi-Hildebrand (B-H) method was initially put forward by Benesi
and Hildebrand (1949) and has become popular method for determining the
binding constant using various spectroscopic techniques (UV-vis, fluorescence,
infrared, etc.). The extended B-H equation (Gokturk and
Tuncay, 2003; Benesi and Hildebrand (1949); Erdinc
et al., 2004) is shown below using UV-vis data:
where, [PHT] and [Sm] (Sm = total surfactant concentration-CMC)
are the initial molar concentrations of PHT and the micellized surfactant concentration,
respectively, l is the optical path length of the solution.
||The physical parameters for the interaction of 1.0x10-5
mol dm-3 PHT with SDS and TX100 micelles at various ethanol concentrations
|*Limit values of f at the 5.0x10-4 mol
dm-3 surfactant concentration. Error limit in K (dm3
mol-1) values is ±5%. The correlation coefficients are
higher than 0.9980
A and A0 are the absorbances of PHT in the presence and absence
of surfactant, respectively. εm, is the molar absorption coefficient
of the drug fully bound to micelles determined in large excess of the micelles.
The plot of l [PHT]/(A-A0) against 1/[Sm] were used to
calculate Kb values at different ethanol concentrations (v/v) from
the slope and intercept and were given in Table 1. From Table
1, it can be seen that the binding constant of PHT with micelles decreases
with the increase in ethanol concentration. The results mean that the rise of
ethanol concentration is unfavorable to the interaction between PHT and micelles.
A more precise comparison can be provided from amount of solubilized PHT by
micelles. The concentration of the solubilized PHT was also calculated from
the relationship (Bielska et al., 2009).
where, Dm is the concentration of PHT solubilized in the micelle.
Figure 5a and b represent the variation
of amount of solubilized PHT with SDS and TX100 micelles in the presence of
various ethanol concentrations, respectively. As seen in Fig.
5a and b the amount of solubilzed PHT decreased with increase
in ethanol concentration.
Partition coefficient of PHT between micelles and aqueous phase: Absorbance
values obtained at λmax, can be also used for the calculation
of partition coefficient, Kx, defined according to the pseudo-phase
model as (Sepulveda et al., 1986; Sepulveda,
where, XmPHT and XwPHT are the mole fractions of cationic dye PHT in micellar and aqueous phase, respectively. They are related with concentrations of species in the solubilization system:
where Cmsurfac tan t and Cwsurfac tan
t represent concentrations of surfactant in micellar and monomeric states
and nw = 55.5 mol dm-3 is the molarity of water.
||(a) Solubility curve of 1.0x10-5 mol dm-3
PHT as a function of SDS concentration in the presence of various amount
of ethanol (v/v), (b) Solubility curve of 1.0x10-5 mol dm-3
PHT as a function of TX100 concentration in the presence of various amount
of ethanol (v/v)
Under the present experimental conditions CmPUT +Cwsurfac
tan t<< nw, if we express Ks = Kx/nw,
we get the equation:
the fraction of the associated PHT ( f) may be defined as:
At a certain CPHT, f is equal to zero in the non-micellar region up to CMC and increases with increasing the concentration of surfactant above CMC. As Csurfactant increases up to infinity, f approaches unity since all added drug should be solubilized in micelles. f can be directly calculated from the experimental data:
||Variation of binding constants value of PHT to SDS and TX100
micelles in the presence of ethanol concentrations (v/v)
where, ΔA = A-Awater-ethanol and ΔA∞
= A∞-Awater-ethanol, A∞ being the
absorbance of PHT completely bound micelles. The change in f with ethanol percentages
(v/v), at the concentrations of 5.0x10-4 mol dm-3 for
TX100 and SDS micelles were given in Table 1. It can be seen
in Table 1, the increase in ethanol concentration caused to
decrease the fraction of PHT with two types of micelles. By using Eq.
5-8 can be written in linear form:
where, KS and Kx (Ks = Kx/nw) is obtained from the slope of the plot of 1/ΔA versus 1/(CPHT+Csurfac tan t-CMC). The results at the different ethanol concentrations are summarized in Table 1. In the presence of 1.0x10-5 mol dm-3 PHT, as seen in Table 1 CMC values are always significantly lower than the corresponding CMC values of SDS at all the studied ethanol concentrations. This confirms that PHT is capable of interacting with SDS, inducing the formation SDS aggregates. As seen in Table 1 PHT interacts with SDS more hardly and weakly with increasing in ethanol concentrations. It is also clearly seen in Fig. 6, as the ethanol concentration increases the tendency of drug-micelle association decreases. More pronounced inhibitory effect of ethanol on binding of PHT to SDS micelles than TX100 micelles can be roughly explained by decreasing electrostatic interaction as well as decreasing hydrophobic attraction as a result of incorporation of ethanol molecules in the palisade layer of micelles.
It is well known that the properties of surfactant solutions such as micellization,
micellar size and properties are affected markedly by the presence of organic
or inorganic additives (Florence and Attwood, 2006). Organic
additives are known to affect the micellization characteristics of both ionic
and nonionic surfactants (Zana, 1995). Mixed alcohol-water
systems have particularly been investigated because of their importance in the
preparation of microemulsions (Liu and Guo, 2007). The
studies have shown that the incorporation of alcohols into the micelles produces
noticeable changes in the micellar shape and their transport properties.
The inhibitory effect of cosolvent on binding can be explained in a very qualitative
manner in terms of decreasing hydrophobic attraction if the effect of the cosolvent
on the micelles is considered. It is well known the presence of cosolvents has
a negative influence on hydrophobic interaction due to its destructive action
on structured water molecules around the hydrophobic parts of the surfactant
(Zana, 1995). In our system there are many factors responsible
for the influence of ethanol on interaction such as changing water structure,
preferential solvation of PHT, changing dielectric constant of the media, changing
cohesive energy, changing micellar size and properties due to the incorporation
of ethanol molecules to micelles and etc. The distribution (or interaction)
of PHT between aqueous ethanol mixture and micellar phase is a complex phenomenon
since the quaternary systems (e.g., water-surfactant-cosolvent-substrate) are
too complicated to study quantitatively. The quaternary system contains water,
ethanol, SDS (or TX100) and PHT in this study. The latter may interact with
surfactant monomers, micelles and ethanol and also ethanol may interact with
micelles. All of these shift the CMC and influence the size and shape of the
micelles and of course changes the physicochemical properties of the bulk. Very
low CMC values obtained in case of SDS supports this explanation.
The higher Kb and Kx in the case of TX100 than in the case of SDS can be attributed to higher hydrophobicity of TX100 than that of SDS. The dependence of K values with amount of ethanol can be related to partition of the alcohol between micellar pseudophase and bulk aqueous phase. The decrease in binding and partition coefficients with increase in ethanol concentrations can be explained by incorporation of the alcohol to the micellar surface. Lastly, the partition of alcohol between the micellar pseudophase and the intermicellar solution, which decreases with increase in ethanol concentration, may affect K values.
In order to interpret the inhibiting efficiency of ethanol the interactions
between surfactants and ethanol have been taken into consideration separately.
Surface tension measurements of SDS and TX100 were performed in the presence
of varying concentrations of ethanol in the absence and presence of 1.0x10-5
mol dm-3 PHT. The surface tension (γ) versus logarithm of concentrations
curves of SDS and TX100 in ethanol-water mixtures are shown in Fig.
7. The CMC values of SDS and TX100 were obtained from the intersection point
between the straight lines in water and ethanol-water mixtures. The results
are summarized in Table 2. The CMC values obtained in water
for both surfactants are in agreement with previous values reported in literature
i.e., CMCSDS = 8.2x10-3 mol dm-3 and CMCTX100
= 0.3x10-3 mol dm-3 (Florence
and Attwood, 2006).
As seen in Fig. 7 and Table 2, CMC values
of SDS and TX100 obtained from the surface tension results clearly indicate
that the effect of ethanol on micelle formation. The difference in ethanol effects
on CMC can be ascribed to the difference of surfactant types. The CMC of SDS
was depressed in ethanol-water mixtures. The depression of CMC of SDS is due
manly to the decrease in the thickness of the electric double layer surrounding
the ionic head-groups and consequent decreased electrical repulsion between
them in the micelle.
|| Variation of surface tension of SDS and TX100 in the absence
and presence of 5 and 30 % (v/v) ethanol concentrations
||Effect of ethanol concentrations (v/v) on the CMC values of
SDS and TX100 determined from surface tension measurements in the absence
and presence of 1.0x10-5 mol dm-3 PHT
|aThe CMC values in the absence of PHT.
bThe CMC values in the presence of 1.0x10-5 mol dm-3PHT
On the other hand the CMC of TX100 did not show significant change in ethanol-water
mixtures. This nonionic surfactant has a polyoxyethylene hydrophilic head group
that interacts with water through hydrogen bonds. Therefore, the CMC does not
change in the presence of ethanol. The effect of alcohols on critical micelle
concentration is well documented in the literature (Zana
et al., 1981; Ionescu et al., 1984). Ethanol is a
border case between short-chain and long-chain alcohols (with more than four
carbons in the alcohol molecule), showing an increase of the CMC with ethanol
concentration or presenting a minimum, depending on the surfactant (Zana,
1995). The common effect of ethanol on these systems is totally inhibiting
micellization of both surfactants at a certain concentration of ethanol 30%(v/v).
Several authors have shown that the presence of cosolvents may diminish the
micelle formation and totally inhibit micellization when cosolvent concentration
reaches at a certain value (Ionescu et al., 1984; Zana,
1995; Gokturk and Tuncay, 2003; Gokturk
et al., 2006). This can be explained by the greater the similarity between
surfactant and solvent the harder the micelle formation. In the presence of
1.0x10-5 mol dm-3 PHT the CMC of SDS were lowered, whereas
no significant change but slightly increased was observed for the CMC of TX100.
In this respect it has been described that molecules solubilized in the outer
portion of the micelle core are most effective in reducing the CMC than ones
solubilized in the inner core. It can be said that PHT should be located near
the inner core of TX100 micelles, as a result of hydrophobic interactions with
the surfactant tail while in the presence of SDS PHT should be located outer
portion of micelle. The surface tension (γ) versus logarithm of concentrations
curves of SDS and TX100 containing 1.0x10-5 mol dm-3 PHT
in ethanol-water mixtures are shown in Fig. 8a and b,
respectively. As seen in Fig. 8a and b the
increase in ethanol concentration diminished the micelle formation of both surfactants
and inhibited at the ethanol concentration of 30% (v/v). As a conclusion there
is a reasonable agreement with the value obtained by spectrophotometric measurements
in terms of inhibitory effect of ethanol on interaction between PHT and both
surfactants studied (Table 1).
In order to gain further insight about the solubilization of 1.0x10-5 mol dm-3 PHT it has been monitored the dependence of surface tension of PHT in the presence of different ethanol-water mixtures. The variation of surface tension of 1.0x10-5 mol dm-3 PHT as a function of ethanol concentration has been shown in Fig. 9. It was observed that with the increase in ethanol concentration the surface tension of PHT significantly decreased. This is also supported that there is an interaction between PHT and ethanol apart from surfactant-ethanol interaction. In other words there is preferentially solvation of PHT by ethanol i.e., co-solubilization effect of ethanol. The parallelism between molar extinction coefficients and surface tension values of PHT in the presence of various amount of ethanol demonstrate that significant influence of polarity change on micellar binding and partition.
||(a) Surface tension of SDS in the presence of 1.0x10-5
mol dm-3 PHT at various ethanol concentrations % (v/v). (b) Surface
tension of TX100 in the presence of 1.0x10-5 mol dm-3PHT
at various ethanol concentrations % (v/v)
||Surface tension of 1.0x10-5 mol dm-3
PHT in the presence of various concentrations of ethanol % (v/v)
It might be said that the solubility of PHT increased as a function of EOH
concentration. Most cosolvents have hydrogen bond donor and/or acceptor groups
as well as small hydrocarbon regions. Their hydrophilic hydrogen bonding groups
ensure water miscibility, while their hydrophobic hydrocarbon regions interfere
with waters hydrogen bonding network, reducing the overall intermolecular attraction
of water. By disrupting waters self-association, cosolvents reduce waters ability
to squeeze out non-polar, hydrophobic compounds, thus increasing solubility
(Millard et al., 2002).
The importing finding here is that the addition of ethanol generally did not
contribute much to improving solubilization in the presence of two types of
micelles SDS and TX100. Moreover the addition of ethanol decreased the solubility
or drug-micelle incorpo ration as observed our system. When ethanol concentration
increased the dramatic decrease was observed for SDS-PHT system. On the other
hand, it might be said that the addition of ethanol to TX100-PHT solutions had
only minor effect on the solubilization of PHT. However addition of ethanol
with the drug to the surfactant solutions generally offered an advantage due
to decreasing CMC and caused to low toxicity of surfactants to formulation.
In conclusion much attention is required when surfactant and cosolvents are
formulated together because the cosolvents affect the micelle characteristics
This study reports the results obtained from a series measurements of the binding and partition coefficient of PHT between two types of micelles i.e., anionic (SDS) and nonionic (TX100) and aqueous phase in the presence of various amount of ethanol (v/v). The addition of ethanol reduces drug-surfactant association. Its influence can be interpreted in two ways: firstly, the increase of the ethanol content in the water-ethanol mixed solvent causes a decrease of the dielectric constant of the aqueous phase and thereby causes an increase of attractive electrical interactions; secondly ethanol is known for its negative influence on hydrophobic interactions because of its ability to break down the structured water molecules around the hydrophobic parts of solute. According to the results the first influence might be dominated by the second one as the equilibrium constants of the PHT-surfactant systems examined decrease with increasing amount of ethanol. Irrespective of surfactant type micellization decreased with the increase in ethanol concentration and totally inhibited at 30% (v/v) ethanol concentration. The results show that the binding constants and partition coefficients decreased with the increase of ethanol percentages. Thus, the rise of ethanol concentration is unfavorable to the PHT distribution in the micelles phase. Apart from decrease in hydrophobic interaction decrease in electrostatic attraction occurs in binding of PHT to SDS micelles, more pronounced effect of ethanol on the binding constants obtained for SDS supports this explanation. The greater binding constants of PHT for neutral TX100 than for ionic SDS indicate that the former micelle provides a more hydrophobic environment to PHT. Inhibitory effect of ethanol on binding to micelles can be explained by decreasing hydrophobic attraction as a result of incorporation of alcohol molecules to micelles.