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Spectroscopic studies on the interaction between methylene blue and bile salts
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Spectroscopic studies on the interaction between methylene blue and bile saltsVijay BenadictrajKanagaraj and SusithraSelvam*
Department of Chemistry, Vel Tech Dr. RR and Dr. SR Technical University,Avadi, Chennai 600 062, Tamilnadu, India
Email: [email protected], [email protected]
Abstract
Methylene blue (MB) has a long history of diverse applications both as a staining reagent and
pharmaceutical agent. MB in aqueous environment forms dimer and larger aggregates. MB is a
well-known staining dye, but the adoption of MB as a fluorescent probe is not studied
thoroughly. Here, we attempt to probe the micellization behavior of bile salt media by MB
absorption and fluorescence. Bile salts are well known disaggregation agents and widely used as
drug delivery systems. The absorption and fluorescence data indicate increase in absorbance and
fluorescence intensity along with increase in fluorescence anisotropy. The temperature study on
the MB-bile salts system indicates that there is disaggregation of bile salts aggregates with
increasing temperature.
Keywords: Methylene blue, Sodium deoxycholate, Sodium cholate, Bile salts, Absorption,
Fluorescence spectroscopy, Aggregates
Introduction
Methylene blue (MB) has a long history of diverse applications both as a staining reagent and
pharmaceutical agent.1 MB enjoys manifold, wides spread uses such as a dye for hair, redox
indicator, antioxidant & antiseptic, stain for fixed and living tissues, antidote for cyanide and as a
treatment for malaria. MB exhibits two major absorption bands at 293 nm (π-π* transition) and
665 nm (n-π* transition) in dilute aqueous solutions, with a shoulder at 610 nm correspondingly
to the 0-1 vibronic transition.3 MB undergoes spontaneous aggregation in aqueous media. In this
present work the usability of photophysical properties of MB will be studied. Spectral properties
of MB has been studying in both homogenous and organized media like proteins, surfactants
etc.4-6 The most widely used solvent polarity functions are those obtained by Lippert-Mataga and
Kamlet-Taft ,which use both dipole moment of the medium empirical parameters. The
absorption studies are widely studied, whereas the literature on fluorescence studies are
minimal.7 Here we attempt to use MB as a fluorescence probe to study the micellization behavior
of bile salts.
Bile salts are biological compounds that are synthesized from cholesterol in the liver. The bile
salt molecules exhibit facial polarity, with their hydrophilic groups being situated on the concave
face of the molecule and most of the steroidal skeleton when its protruding methyl groups lying
on the convex face.8,9 Bile salts are widely used as solubilization and disaggregating agents in the
pharmaceutical industry.10,11 In aqueous media, bile salts undergo spontaneous aggregation
leading to primary and secondary aggregates involving a step – wise aggregation model.12 The
primary aggregates are formed due to hydrophobic interaction between the steroidal back-bone.
Secondary micelles are held together by hydrogen bond. . Among the various bile salts, sodium
cholate (NaC) and sodium deoxycholate (NaDC), primary unconjugated bile salts was chosen for
the studies. Sodium cholate is dihydroxy bile salt and sodium deoxycholate is trihydroxy bile
salts. With less number of hydroxyl groups, the dihydroxy bile salts are more hydrophobic and
attain micellization with lesser concentrations than that of the tri hydroxyl bile salts.
Materials and Methods
Methylene blue, sodium cholate and Sodium deoxycholate were purchased from Sisco Research
Laboratories Private Limited (India) and were used without any further purification. A sodium
phosphate buffer of pH 7.4 with 50 mM concentration was used for all experiments. The solvent
ethanol used was of spectroscopic grade. The stock solutions of MB are prepared by dissolving it
in ethanol and the required concentration of MB solution was prepared by further dilution with
pH buffer. All the organic solvents with high purity (spectroscopic grade) were purchased from
Research Laboratories Private Limited India. UV-visible spectrophotometric experiments were
recorded on a Shimadzu UV 1800 double beam spectrophotometer, using 10 mm quartz cuvettes.
A Horiba Jobin-Yvon Fluoromax-4 spectrofluorometer was used to record the fluorescence
spectra. Excitation and emission spectra were measured with 5 nm band width. Temperature
related experiments were done in a double walled cuvette holder carrying the fluorimetric cell.
The temperature was controlled by circulating water through jacketed cuvette holder from a
refrigerated bath (JULABO, Germany). The steady state fluorescence anisotropy is defined as, 13
Where IVV and IVH are the fluorescence intensities and the subscript indicates the vertical (V) and
horizontal (H) orientations of the excitation and emission polarizer. G is the instrumental
correction factor:
.
Preparation of MB in bile salt solutions
The stock solutions of bile salt were prepared in phosphate buffer pH 7.4 at concentration
higher, than the critical micellar concentration (cmc), NaC = 6-16 mM and NaDC = 4-8 mM. A
range of different concentrations of bile salt solutions are prepared by diluting these stock
solutions using buffer solution. MB concentration was maintained at 5 µM for all the
experiments with less than 2% ethanol contamination. The range of concentration of bile salt
varied is NaC = 0 – 43.2 mM and NaDC = 0 – 18 mM. The solutions were incubated for 2 hrs to
achieve equilibrium.
Result and Discussion
Absorption and Fluorescence Spectra of MB
The absorption and fluorescence spectra of MB in various organic solvents with various
polarities were investigated. Typically, Fig 1 A and B represents absorption and fluorescence
spectra of MB in selected organic solvents, respectively. MB concentration was maintained at 10
µM. MB shows single fluorescence band in polar solvents. As can be seen the shape and
maximum wavelength of the absorption and emission of the MB are solvent dependent.
However, the solvent has no large and regular effect on the spectral behavior can be observed
from their absorption spectra. The fluorescence maxima of the MB in a high polarizable solvent
such as DMSO located at 672 nm. The study indicates solvent polarity has no large regular
effect on the spectral behavior of the dyes, and thus they might be considered as poor
solvatochromic dyes.
Figure 1. Absorption (A) and fluorescence normalized (B) spectra of MB at 250C .
Empirical scales of solvatochromism (solvent parameters)
A B
Lippert-Mataga equation
If solvent relaxation is complete, equations for the dipole-dipole interaction solvatochromic
shifts can be derived within the simple model of spherical – centered dipoles on isotropically
polarizable spheres and within the assumption of equal dipole moments in Franck-Condon and
relaxed states. Talking in to consideration the solvatochromic shifts for the absorption and
emission, the Lippert-Mataga equation is written as
Where are the absorption and emission maxima, h is Planck’s constant, c is the
velocity of light, a is the radius of the cavity in which the solute residues and ∆f is the orientation
polarizability defined as,
This expressions of the stokes shift depends only on the absolute magnitude of the charge
transfer dipole moment and not on the angles between dipoles. The validity of
the equation 1 can be checked using various solvents and by plotting as a function of
∆f. A linear variation is not always observed because only the dipole-dipole interactions are
taken into account and the solute polarizability in neglected. Using Lippert-Mataga equation, it is
also possible to determine excited dipole moments, but under certain assumptions.
Kamlet-Taft Solvatochromic parameter
A multi – parameteric approach using π* scale of kamlet and Taft solvatochromism has been
successfully applied to the positions or intensities of maximal absorption in IR, NMR, ESR and
1
UV- visible absorption and fluorescence spectra. In case of absorption and fluorescence spectra,
the variation wave number of an absorption or emission band, as a function of solvent polarity
is given by,
Where are the wave numbers of the peak maxima in the considered solvent and in
the reference solvent (generally cyclohexane), respectively. The π* is a measure of the polarity
of the solvent; the α scale is an index of solvent HBD acidity and the β scale is an index of the
solvent HBA basicity. The coefficients s, a and b describe the sensitivity of a process to each of
the individual contributions. The advantage of Kamleft-Taft treatment is to sort out the
quantitative role of properties such as hydrogen bonding.
Absorption Spectra of MB in presence of bile salts
The absorption spectrum of MB is given in (Fig.2). It exhibits a long-wavelength maximum at
665 nm corresponding to the MB monomer peak, a shoulder at 610 nm corresponding to dimer
MB and higher aggregates at around 292 nm.14 The MB absorption increases on addition of bile
salts. The monomer absorbance at 665 nm is prominent. The absorption study reveals a possible
solubilization of MB by bile salt media.
Figure 2. Absorption spectra of MB – bile salt system at 25 °C
Fluorescence spectra of MB in presence of bile salts
The fluorescence spectra of MB when excited at 665 nm shows emission peak at 686 nm (Fig 3)
corresponding to the monomer fluorescence. To identify the dimer fluorescence of MB, a 5 µM
MB solution was prepared in 0.1 M nitric acid, according to a procedure given in literature.15 In
the absorption spectrum MB showing three peaks. Dimer peak increases correspondingly
monomer peak is decreases and new peak was observed in 744 nm. This type is called J
aggregation. In dimer peak at 610 nm the MB concentration increases and monomer fluorescence
intensity is decreases. So the dimmers and high aggregates are non-fluorescent. The results
indicate that there is an increase in the fluorescence intensity of MB monomer. There is a
minimal shift in the emission wavelength to the red region. The fluorescence anisotropy was
measured at 686 nm emission peak. The increase in emission intensity along with increase in
absorbance of MB, shift in emission wavelength and increase in fluorescence anisotropy indicate
that MB associate with the hydrophobic region of the NaC & NaDC aggregates. The data here
reveals the cmc range of NaC = 6- 16mM and NaDC to be 4-8 mM which is in agreement with
NaDCNaC
the literature data (Fig 4). The fluorescence intensity at λem 686 nm corresponding to the
monomeric MB form increase with increasing concentration of the bile salts and show saturation
behavior at higher concentrations. Thus it can be proposed that MB fluorescence can sense the
micellization behavior of bile salts aggregates.
Figure 3. Fluorescence emission spectra of MB – bile salt system at 25 °C
Figure 4. Variation of (A) with in fluorescence intensity data and (B) for fluorescence anisotropy data of MB – bile salt system at 25 °C.
A B
NaC NaDC
Temperature study of MB –bile salts system
The study was further extended to understand the temperature dependence of MB-bile salt
system (Fig 5 & 6). The photophysical parameters, fluorescence intensity and fluorescence
anisotropy were used and the temperature was varied from 150C to 45 0C. The results show that
there is a decrease in both fluorescence intensity and fluorescence anisotropy of MB – bile salt
system with increasing temperature. The temperature depends of the MB – NaC micelles is
found to be more than that of the MB – NaDC micellar system. This may be due to the higher
facial hydrophobicity of NaDC than NaC. The study indicates that MB incorporates into lesser
hydrophobic region of NaC and NaDC aggregates. Conversely the MB monomer fluorescence
senses the disaggregation of bile salts aggregates with increasing temperature.
Figure 5. Variation of (A) with in fluorescence intensity data and (B) for fluorescence anisotropy data of MB – NaC system at different temperatures.
A B
Figure 6. Variation of (A) with in fluorescence intensity data and (B) for fluorescence anisotropy data of MB – NaDC system at different temperatures.
Conclusion
The absorption and fluorescence studies of MB molecules exist as monomer, dimer and higher
aggregates. Monomer absorption is at 665 nm and emission at 686 nm. MB dimer and higher
aggregates are non-fluorescent. The solvent study indicates solvent polarity has no large regular
effect on the spectral behavior of the MB, and thus they might be considered as poor
solvatochromic dye. The increase in absorbance and fluorescence intensity of the monomer MB
with increasing bile salt concentrations indicate the monomerization of MB aggregates induced
by bile salt media. The dihydroxy bile salts (NaDC) are found to be more effective than the
trihydroxy bile salts (NaC). The solubilization effect of sodium deoxycholate on various solutes
will be achieved more easily than that of the sodium cholate bile salts. This indicates the
hydrophobic interaction between MB and bile salts. MB monomer fluorescence is useful in
probing the micellization behavior of NaC and NaDC. The temperature study on the MB – bile
salt system indicates that disaggregation of NaC and NaDC aggregates with increasing
temperature.
References
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