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: susithra.selvam@yahoo.com, ssusithra@veltechuniv.edu.in

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.

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