Optical Spectroscopic and TEM Studies of Catanionic Micelles of

Mar 17, 2004 - The ground-state electronic transition of the anionic form of piroxicam is n, π*.15 For a molecule showing n, π* transition, a 10 nm ...
0 downloads 8 Views 226KB Size
Download PDF
Langmuir 2004, 20, 3551-3558

3551

Optical Spectroscopic and TEM Studies of Catanionic Micelles of CTAB/SDS and Their Interaction with a NSAID Hirak Chakraborty and Munna Sarkar* Chemical Sciences Division, Saha Institute of Nuclear Physics, 1/AF, Bidhannagar, Calcutta-700 064, India Received November 14, 2003. In Final Form: February 11, 2004 If a vesicle is a better model of a membrane in the context of the hydrophobic effect, then from the charge distribution point of view, a catanionic micelle is a closer model to a biomembrane. We have prepared and characterized two different types of catanionic micelles of sodium dodecyl sulfate (SDS) and cetyl N,N,Ntrimethylammonium bromide (CTAB) having different surface charge ratios using optical spectroscopy and transmission electron microscopy. The average size of both types of mixed micelles was found to be much larger than that of micelles containing uniformly charged headgroups. Catanionic micelles containing higher concentrations of positively charged headgroups (CTAB) are larger in size, less compact, and more polar compared to the micelles containing higher concentrations of negatively charged headgroups (SDS). We have used these catanionic micelles as membrane mimetic systems to understand the interaction of piroxicam, a nonsteroidal anti-inflammatory drug (NSAID) of the oxicam group, with biomembranes. In continuation of our work on membrane mimetic systems, we have used spectral properties of the drug itself to understand the effect of the presence of mixed charges on the micellar surface in guiding the interaction of catanionic micelles with piroxicam. Our earlier studies of the interaction of piroxicam with micelles having uniform surface charges have shown that the charge on the micellar surface not only dictates which prototropic form of the drug will be incorporated in the micelles but also induces a switch-over between different prototropic forms of piroxicam. The equilibrium of this switch-over is extremely sensitive to the environment. In this study, we demonstrate how even small changes in the electrostatic forces obtained by doping the uniformly charged surface of the micelles with oppositely charged headgroups (as in catanionic micelles) are capable of fine-tuning this equilibrium. This implies that the surface charge of biomembranes, which are quite diverse in vivo, might play a significant role in selecting a particular form of the drug to be presented to its targets.

Introduction Micelles, dynamic nanostructures of surfactant molecules, have the capability to solubilize a wide variety of organic molecules with different polarities and hydrophobicities. Several reactions, such as polymerization and catalysis, are carried out in micelles to get a better yield. In these applications, the size and the stability of the micelle play an important role. The tailoring of micellar properties may be achieved by adding salts, organic solvents, or a second surfactant forming the so-called mixed micellar system. The larger size and better thermodynamic stability of the mixed micelles would enhance the incorporation capability of solutes in the micellar phase. In this work, we have prepared and characterized mixed micelles of cationic [cetyl N,N,N-trimethylammonium bromide (CTAB)] and anionic [sodium dodecyl sulfate (SDS)] surfactants (called “catanionic micelles”), in which two oppositely charged headgroups are distributed. We have taken two sets of catanionic micelles, one in which positive charge is more and another with more negative charge compared to the other. Competition between various molecular interactions (van der Waals, hydrophobic, electrostatic, hydration forces, etc.) may result in a variety of microstructures, viz., catanionic salts, mixed micelles, and catanionic vesicles.1-3 So the phase behavior of cationic/anionic surfactant mixtures strongly depends * Corresponding author. Fax: 91-33-23374637. E-mail: munna@ nuc.saha.ernet.in or [email protected]. (1) Kaler, E. W.; Murthy, A. K.; Rodriguez, B. E.; Zasadzinski, J. A. N. Science 1989, 245, 1371-1374. (2) Kaler, E. W.; Herrington, K. L.; Murthy, A. K.; Zasadzinski, J. A. N. J. Phys. Chem. 1992, 96, 6698-6707. (3) Brasher, L. L.; Herrington, K. L.; Kaler, E. W. Langmuir 1995, 11, 4267-4277.

on the molar ratio, actual concentration of individual surfactant relative alkyl chain lengths, number of alkyl chains per surfactant, and temperature, resulting in a rich array of aggregates.2-9 In a particular region of composition, two oppositely charged surfactants could form catanionic micelles.4,6,10 So to prepare catanionic micelles one has to be very cautious about the composition of two oppositely charged surfactants. Generally, mixtures with an excess of either cationic or anionic surfactants have been shown to form catanionic micelles. As a result, the ratio of two oppositely charged headgroups in these micelles is far from 1:1 and polydispersity in the micellar size and charge distribution occurs. We have characterized the catanionic micelles by using optical spectroscopic techniques. Both steady-state and time-resolved fluorescence studies were done with pyrene as the reporter chromophore. Transmission electron microscopy (TEM) has been used to confirm the formation of catanionic micelles, and the average diameter of these micelles was calculated from TEM photographs. We have also studied the interactions of these mixed micelles with a nonsteroidal anti-inflammatory drug (NSAID), piroxicam [4-hydroxy-2-methyl-N-(pyridin-2-yl)(4) Yatcilla, M. T.; Herrington, K. L.; Brasher, L. L.; Kaler, E. W. J. Phys. Chem. 1996, 100, 5874-5879. (5) Soderman, O.; Herrington, K. L.; Kaler, E. W.; Meller, D. D. Langmuir 1997, 13, 5531-5538. (6) Tomasic, V.; Stefanic, I.; Filipovic, N. Colloid Polym. Sci. 1999, 277, 153-163. (7) Marques, E. F. Langmuir 2000, 16, 4798-4807. (8) Jung, H. T.; Coldren, B.; Zasadzinski, J. A.; Iampietro, D. J.; Kaler, E. W. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 1353-1357. (9) Edlund, H.; Sadaghiani, A.; Khan, A. Langmuir 1997, 13, 49534963. (10) Karukstis, K. K.; McCormack, T. M.; McQueen, T. M.; Goto, K. F. Langmuir 2004, 20, 64-72.

10.1021/la0361417 CCC: $27.50 © 2004 American Chemical Society Published on Web 03/17/2004

3552

Langmuir, Vol. 20, No. 9, 2004

Figure 1. Structure of different prototropic forms of piroxicam.

2H-1,2-benzothiazine-3-carboxamide1,1-dioxide] (Figure 1). The physiological target of this drug is cyclooxygenase, which is a membrane active enzyme.11 Catanionic micelles serve as a better model of biomembranes in the context of charge distribution, and hence the study of the interaction of this drug with catanionic micelles would be of biological relevance. In our earlier work, we have studied the interactions of this drug with simple micelles having uniform headgroup charges.12 A switch-over or change from one prototropic form of piroxicam to another in the presence of differently charged micelles was observed, which was correlated to the change in the pKa value in the presence of charged surfactants. This change in pKa values may be due to either electrostatic interaction and/or hydrophobic interaction, which in turn depends on the location and orientation of the drug in the micellar phase. In this present study, we have made an attempt to see the effect of changing electrostatic interaction on the switchover between different prototropic forms. That is why we have taken two sets of catanionic micelles, one in which a predominantly positively charged micellar surface is doped with a small amount of negative charges and the other in which the negatively charged micellar surface is doped with a small amount of positive charges. The interaction of piroxicam with the catanionic mixed micelles was studied using the intrinsic absorption and fluorescence properties of the drug. Piroxicam can exist in three different prototropic forms, viz., anionic, neutral, and zwitterionic forms.13 The neutral and zwitterionic forms are spectroscopically indistinguishable and are thereby termed together as the “global neutral” form. The incorporation of different prototropic forms in different catanionic micelles has been shown by optical spectroscopic techniques. We have also measured the change in free energy (∆G) value of the switch-over in the presence of different catanionic micelles to probe how the change in electrostatic interaction modulates this equilibrium. Experimental Section CTAB and SDS were purchased from Merck and USB, respectively. Piroxicam was purchased from Sigma Chemicals (U.S.) and was used without further purification. Water was distilled thrice before use. Stock solutions of piroxicam of concentration 0.5 mM were prepared in ethanol (Merck, Germany), and the exact concentration was adjusted by tripledistilled water. Each sample contains a maximum of 6% (v/v) of ethanol. The pH of the working solutions was adjusted by adding dilute HCl to them. The volume of acid (HCl) added to the working (11) Hawkey, C. J. Lancet 1999, 353, 307-314. (12) Chakraborty, H.; Banerjee, R.; Sarkar, M. Biophys. Chem. 2003, 104, 315-325. (13) Tsai, R. S.; Carrupt, P. A.; Tayar, N. E.; Giroud, Y.; Testa, A. B. Helv. Chim. Acta 1993, 76, 842-854.

Chakraborty and Sarkar solutions is exactly equal to the volume of acid that is needed to acidify a volume of water equal to the working solution to attain that particular pH. Solution at pH 5.5 indicates that no acid or alkali was added to the aqueous solutions. Samples were deoxygenated by passing argon gas for about 20 min before scanning to avoid photochemical changes. The temperature was kept constant at 298 K throughout all experiments. We have prepared the catanionic micelles of two different compositions by mixing aliquots of concentrated micellar solutions of SDS and CTAB followed by sonication for 15 min. The solutions were then left for 24 h before making any measurements. In one, the concentration of CTAB is higher than the SDS concentration (the concentration of SDS was kept constant at 0.03 mM, and the concentration of CTAB was varied from 0.4 to 12 mM), and the opposite is true for the other (the concentration of CTAB was kept constant at 0.1 mM, and the concentration of SDS was varied from 2 to 30 mM). We have measured the critical micellar concentration (cmc) of the catanionic micelles of different concentrations of SDS and CTAB at different pHs using pyrene as the chromophore following the environmental effect on the vibronic band intensities (3/1) of pyrene.14 Below the cmc, there are no micelles present and the pyrene fluorescence spectrum has a 3/1 band ratio that is the same as in water. However, as the detergent concentration increases above the cmc, pyrene is solubilized in the hydrophobic interior as illustrated by the increased 3/1 ratios. The micelle formation was followed by the sharp increase in the 3/1 vibronic band ratio of the fluorescence spectrum of pyrene, which corresponds to the cmc value of the mixed surfactants. Pyrene solution was prepared in dimethyl formamide (DMF), and a very low concentration of pyrene (5 × 10-6 M) was used to avoid excimer formation. The maximum concentration of DMF in the working solution was 0.1% (v/v). The fluorescence lifetime of pyrene was also used to monitor the formation of mixed micelles. Fluorescence lifetimes were determined from total emission intensity decay measurements, using a time-resolved fluorimeter assembled in our laboratory with components from Edinburgh Analytical Instruments (EIA, U.K.) and EG&G ORTEC (U.S.) and operated in the timecorrelated single-photon-counting mode. A pulsed high-pressure (1.5 atm) N2 lamp operating at 25 kHz repetition rate was used as a source. The pulse profile had a full width at half-maximum (fwhm) of 1.2 ns. Pyrene was excited by the 337 nm N2 line, and its emission was monitored at 373 nm. Slits with 32 nm bandpass were used in both excitation and emission channels. Intensity decay curves could be fitted to a biexponential series:

I(t) ) A0 + A1 exp(-t/τ1) + A2 exp(-t/τ2) where A1 and A2 are pre-exponential factors representing the fractional contribution of the time-resolved decay of the component with a lifetime τ1 and τ2, respectively. A0 is a constant. The decay parameters were recovered using a software package supplied by EIA, which used the Marquardt iterative nonlinear least-squares fitting procedure. Statistically, the goodness of the fit was evaluated by the reduced χ2 value. Absorption spectra were recorded with a Shimadzu UV-visible spectrophotometer model UV2101PC. Baseline correction was done with water before recording each set of data. Fluorescence measurements were performed using a Hitachi spectrofluorimeter model F 4010. All emission spectra were corrected for instrument response at each wavelength. The concentration of the drug was measured from Lambert-Beer’s law, as the extinction coefficients are known at the characteristic wavelength of the global neutral and anionic forms.15 A 2 × 10 mm2 path length quartz cell was used for all fluorescence measurements to avoid any blue edge distortion of the spectrum due to inner filter effect.16 TEM was done with a Hitachi electron microscope model 600 operating at 75 kV with a resolution of 5 Å. The samples were spread over a copper grid coated with carbon. Phospho tungstic (14) Kalyansundaram, K.; Thomas, J. K. J. Am. Chem. Soc. 1997, 999, 2039-2044. (15) Banerjee, R.; Chakraborty, H.; Sarkar, M. Spectrochim. Acta, Part A 2003, 59, 1213-1222. (16) Lackowicz, J. R. Principles of Fluorescence Spectroscopy; Kluwer Academic/Plenum Publishers: New York, 1999.

Catanionic Micelles of CTAB/SDS

Langmuir, Vol. 20, No. 9, 2004 3553

acid (PTA) was used as the stain for catanionic micelles containing a higher concentration of CTAB, and uranyl acetate was used for the catanionic micelles containing a higher concentration of SDS. Samples were negatively stained with those heavy metal compounds. All measurements were done with solutions incubated at 298 K.

Results and Discussion (A) Characterization of Catanionic Micelles. Characterization by Pyrene Fluorescence 3/1 Band Ratio and Lifetime. Interplay between various molecular interactions may result in a variety of microstructures, catanionic surfactant salts, mixed micelles, catanionic vesicles, and so forth.6 Spontaneous vesicle formation has rarely been observed in single tailed chain systems without adequate chemical and mechanical treatment, but in cationic/ anionic surfactant mixtures, spontaneous formation of vesicles is frequently observed. The major reason behind this phase transition is the electrostatic force. It was found that the phase transition proceeds in several stages with increasing mole fraction of one surfactant. Figure 2a,b shows the plot of pyrene 3/1 band ratio versus concentration of SDS at pH 5.5 and pH 3.8, respectively, with constant CTAB concentration of 0.1 mM. Figure 3a,b demonstrates the change of pyrene 3/1 band ratio versus concentration of CTAB at pH 5.5 and pH 3.8, respectively, with constant SDS concentration of 0.03 mM. The measurement at pH 3.8 has been done for the interest of the second part of this work. From the above plots, the saturation values of the peak ratio in the mixed micelles of higher concentration of SDS and CTAB at pH 5.5 are 0.96 and 0.78, respectively. Below the cmc, there are no micelles present and the pyrene fluorescence spectrum corresponds to that in water with a 3/1 ratio of ∼0.64.14 The major contribution to the change in pyrene vibronic band intensities is from specific solute-solvent dipoledipole coupling, although other effects due to π-orbital interactions between solute and solvent and the bulk dielectric constant of the solvent cannot be neglected.14 Despite the different contributions, qualitatively the 3/1 band ratio serves as a measure of solvent polarity and increases with decreasing dipole moment of the solvent. So from the peak ratio, we can compare different environments in terms of dipole moment; at least we can say which one has the higher dipole moment and which one has the lower dipole moment. Again, if two solvents have the same dipole moment, then the peak ratio increases with decreasing dielectric constant.14 The higher 3/1 ratio (0.96) for catanionic micelles containing a higher SDS concentration (Figure 2a,b) therefore reflects a less polar micellar core compared to the catanionic micelles having a larger concentration of CTAB. Generally the change of the pyrene 3/1 band ratio with surfactant concentration is very sharp and then saturates as we have seen in Figure 3a,b. But in Figure 2a,b between 4 and 7 mM concentration of SDS, the value of the 3/1 band ratio overshoots and is higher than the saturation value. This is not seen in singlesurfactant micellar systems. Measurement of the fluorescence lifetime of pyrene is also a very good tool to study the micellization process.14,17 Generally the lifetime of pyrene is increased in both restricted and less polar environments provided there is no excimer formation. As mentioned before, the concentration of pyrene was kept low to avoid excimer formation. Figure 4a,b shows the plot of lifetime with increasing (17) Siemiarczuk, A.; Ware, W. R. Chem. Phys. Lett. 1990, 167, 263268.

Figure 2. Plot of IIII/II of pyrene vs concentration of SDS at (a) pH 5.5 and (b) pH 3.8 at constant CTAB concentration of 0.1 mM.

concentration of SDS (at constant CTAB concentration) and CTAB (at constant SDS concentration) at pH 5.5, respectively. The lifetime of pyrene in the catanionic micelles containing a higher concentration of SDS is 175 ns (Figure 4a), and that in the other mixed micelles containing higher CTAB concentrations is 156 ns (Figure 4b). From the lifetime data of pyrene in two types of catanionic micelles, we can say that the catanionic micelle containing a higher concentration of SDS is much more compact and less polar compared to the one containing a higher CTAB concentration. To summarize, the micellar cores of catanionic micelles, having a higher concentration of negatively charged headgroups, are both more compact and hydrophobic than the ones containing a higher concentration of positively charged headgroups. Characterization by TEM. We have measured the size of the catanionic micelles of two different types from the TEM photographs. Figure 5a,b shows representative TEM photographs of mixed micelles at a higher concentration

3554

Langmuir, Vol. 20, No. 9, 2004

Chakraborty and Sarkar

Figure 4. Trace of the fluorescence lifetime of pyrene with increasing concentration of (a) SDS at constant CTAB concentration of 0.1 mM and (b) CTAB at pH 5.5 at constant SDS concentration of 0.03 mM.

Figure 3. Plot of IIII/II of pyrene vs concentration of CTAB at (a) pH 5.5 and (b) pH 3.8 at constant CTAB concentration of 0.03 mM.

of SDS (the concentration of CTAB is 0.1 mM, and the concentration of SDS is 10.0 mM) and a higher concentration of CTAB (the concentration of SDS is 0.03 mM, and the concentration of CTAB is 6.0 mM), respectively, at pH 5.5 (without adding acid or alkali). Figure 5c is the TEM photograph of the overshooting region obtained in Figure 2a,b. The photograph has been taken at 0.1 mM CTAB and 5 mM SDS concentration. Figure 6a-c demonstrates the bar diagrams of frequency versus diameter corresponding to Figure 5a-c, respectively. Several TEM photographs were taken, and in all cases all the systems show a high level of polydispersity. This is because of the nonuniform charge ratio of catanionic and anionic surfactants in the catanionic micelles. The average diameter of the catanionic micelles with a higher concentration of SDS is around 50 nm (the diameter of a pure SDS micelle is 3.68 nm18), and that with a higher concentration of CTAB is around 80-90 nm (the diameter of a pure CTAB micelle is 4 nm19). There have been suggestions that water can

enter the micelles and can extend up to four carbons from the headgroup.20 In micelles with compact headgroups such as SDS, water penetration is smaller than in micelles with larger headgroups such as CTAB, making their cores more hydrophobic. The larger size of catanionic micelles containing a higher CTAB (having a less compact headgroup) concentration could allow more water penetration, making their cores more polar and less compact than those of the catanionic micelles containing a higher SDS (having a more compact headgroup) concentration. The larger size of the catanionic micelles compared to single-surfactant micelles can be explained as follows. Micelle formation is a compromise between the extremes of a complete phase transition and a molecular disperse solution. The sequestering of the nonpolar tails of the surfactant in the micellar interior is driven by the solvophobic effect which is balanced by the solubilization of polar headgroups in polar solvent. Micellar aggregation is characterized both by the (18) Duplatre, G.; Marques, M. F. F.; Miguel, M. D. G. J. Phys. Chem. 1996, 100, 16608-16612. (19) Singh, M.; Trivedi, M. K.; Bellare, J. J. Mater. Sci. 1999, 34, 5315-5323. (20) Fendler, J. H.; Fendler, E. J. Catalysis in Micellar and Macromolecular Systems; Academic Press: New York, 1975.

Catanionic Micelles of CTAB/SDS

Langmuir, Vol. 20, No. 9, 2004 3555

Figure 5. TEM photographs of mixed micelles (a) at a higher concentration of SDS (concentration of CTAB ) 0.1 mM and concentration of SDS ) 10 mM), (b) at a higher concentration of CTAB (concentration of SDS ) 0.03 mM and concentration of CTAB ) 10 mM), and (c) at the overshooting region obtained in Figure 2a,b (concentration of CTAB ) 0.1 mM and concentration of SDS ) 5 mM) at pH 5.5.

cooperativity of the formation and also by the mechanism that stops aggregate growth to a macroscopic size and finally to phase separation. For typical surfactants, it is the repulsive interaction between the headgroups that emanates the stop process. The magnitude of the electrostatic repulsion guides the predominant size of the aggregates in solution.21 A standard way of achieving aggregate growth is to reduce the headgroup-headgroup repulsion. The two types of catanionic micelles studied here can be viewed as having either a positively charged micellar surface doped with negative charges or vice versa. In both cases, this doping with surfactants having oppositely charged headgroups reduces headgroup-headgroup repulsion and thereby promotes aggregate growth. This results in a much larger average diameter of the catanionic micelles compared to the micelles with uniformly charged headgroups. The average diameter of the spherical structures in the overshooting region in Figure 5c is much larger (diameter, 90-100 nm) than the average diameter of the mixed micelles with a higher SDS concentration (diameter, 50 nm). This could indicate either an actual increase in the micellar size or more likely the presence of mixed components, which could include vesicles. As has been mentioned earlier, the phase transition between monomer and mixed micelles is not as clear as in the case of pure micelle formation. The border of the phase transition in the case of mixed micelles is (21) Evans, D. F.; Wennerstrom, H. The Colloidal Domain: Where Physics, Chemistry and Biology Meet; Wiley-VCH: New York, 1999.

characterized by the presence of other microstructures such as vesicles. For constant CTAB and varying SDS concentration, the overshooting region (4-7 mM SDS concentration in Figure 2a,b) corresponds to a region close to the phase boundary6 where other microstructures are present. This is reflected in our pyrene 3/1 band ratio and TEM photograph. As we move away from the phase boundary, the predominant structures are mixed micelles. Figure 7 represents the TEM photograph of a mixture of 0.03 mM SDS and 6 mM CTAB at pH 5.5, 2 h after the preparation of the solution. Here we have found that distinct micelles have not formed; rather they are fused together. From this, it is evident that catanionic micelle formation takes a certain hydration time. Accordingly, all experiments have been done with solutions incubated at 298 K for at least 24 h. This is consistent with the observations that the nature and size of the microstructures depend on formation path, sonication, and aging.7 (B) Interaction of Catanionic Micelles with a NSAID. NSAIDs of the oxicam group can exist in different prototropic forms, that is, cationic, global neutral (neutral and/or zwitterionic), and anionic forms, in different physiological conditions.13 These forms are extremely sensitive to their microenvironment22 because of their dynamic structural features.13,23-25 The absorption maxima (22) Banerjee, R.; Sarkar, M. J. Lumin. 2002, 99, 255-263. (23) Yoon, M.; Chol, H. N.; Kwon, H. W.; Park, K. H. Bull. Korean Chem. Soc. 1998, 9, 171-175. (24) Bordner, J.; Hammen, P. D.; Whipple, E. B. J. Am. Chem. Soc. 1989, 111, 6572-6578.

3556

Langmuir, Vol. 20, No. 9, 2004

Chakraborty and Sarkar

Figure 6. (a-c) Size distribution plots of mixed micelles corresponding to Figure 5a-c.

Figure 7. TEM photograph of a mixture of 0.03 mM SDS and 6 mM CTAB at pH 5.5, 2 h after the preparation of the solution.

in water of the neutral and anionic forms are 330 and 353 nm, respectively.22 With increasing concentration of SDS at a particular concentration of CTAB, the absorption maximum does not show any shift at pH 5.5. This is because the predominant population at pH 5.5 is the anionic form, which is expected to be repelled by the negatively charged headgroup of the anionic surfactant. So the experiments with a higher concentration of SDS have been done at pH 3.8 to get an adequate amount of the global neutral form even though the major population is the anionic one as indicated by the 353 nm absorption maximum. In the presence of a higher concentration of SDS at pH 3.8, the absorption maximum shifts from 353 to 343 nm (Figure 8a). On the other hand, increasing the concentration of CTAB while keeping SDS concentration constant shifts the absorption maximum from 353 to 363 nm (Figure 8b). The ground-state electronic transition of the anionic form of piroxicam is n, π*.15 For a molecule showing n, π* transition, a 10 nm red shift of the absorption maximum indicates that it is being incorporated in the (25) Geckle, J. M.; Rescek, D.; Whipple, E. B. Magn. Reson. Chem. 1989, 27, 150-154.

Figure 8. Change in the absorption maximum with change in the concentration of (a) SDS at pH 3.8 at constant CTAB concentration of 0.1 mM and (b) CTAB at pH 5.5 at constant SDS concentration of 0.03 mM.

less polar environment within the micelles from the bulk aqueous phase. Again, with increasing concentration of SDS, keeping CTAB concentration constant, the absorption maximum shifts from 353 to 343 nm, indicating that the population of the neutral form has increased in solution. The absorption maximum has not shifted to 335 nm, which was observed in the case of pure SDS micelles.12 This will be explained in a later part of this paper. Another way of monitoring the incorporation of the drug is by following the increment of fluorescence intensity with increasing concentration of surfactant. Figure 9a represents the plot of fluorescence intensity versus concentration of SDS at pH 3.8 at constant CTAB

Catanionic Micelles of CTAB/SDS

Langmuir, Vol. 20, No. 9, 2004 3557

Figure 10. Plot of O.D363nm/O.D330nm with the change in concentration of (a) SDS at pH 3.8 at constant CTAB concentration of 0.1 mM and (b) CTAB at pH 5.5 at constant SDS concentration of 0.03 mM. Figure 9. Plot of the relative fluorescence intensity with increase in the concentration of (a) SDS at pH 3.8 at constant CTAB concentration of 0.1 mM (λexc ) 330 nm) and (b) CTAB at pH 5.5 at constant SDS concentration of 0.03 mM (λexc ) 363 nm).

concentration monitored at the excitation wavelength of the neutral form, that is, at 330 nm. Figure 9b shows the change of fluorescence intensity with concentration of CTAB at a fixed concentration of SDS (pH 5.5) when the excitation wavelength was kept at that of the anionic form at 363 nm. These two plots make it clear that the neutral form is incorporated in catanionic micelles with a higher concentration of anionic surfactant SDS and the anionic form is incorporated in the catanionic micelles with a higher concentration of cationic surfactant CTAB. Figure 10a,b shows the plot of the optical density ratio at 363 and 330 nm (the absorption maxima of the anionic and global neutral forms) with increasing SDS concentration (at constant CTAB concentration of 0.1 mM at pH 3.8) and with increasing CTAB concentration (at constant SDS concentration of 0.03 mM at pH 5.5), respectively. Interestingly, it is found that the ratio decreases with the concentration of SDS at pH 3.8 and increases with the CTAB concentration at pH 5.5 though the extinction

coefficient of the global neutral form is higher than that of the anionic form.15 This indicates that in the presence of different catanionic micelles there is a switch-over or change between two prototropic forms of piroxicam, viz., the anion and the global neutral form. The amount of neutral form is increased in the presence of negatively charged SDS, and the anionic form is increased in the presence of positively charged CTAB. In our earlier work, we have shown that this switch-over between two prototropic forms, that is, change of population between global neutral and anionic forms, also occurred in singlesurfactant systems.12 Consider the following equilibrium in the presence of catanionic micelles:

NaA

(1)

where N represents the global neutral and A the anionic form of piroxicam. Since the micellar pseudophase is spectroscopically silent, the effect of micellar equilibrium is indirectly reflected in the changes in spectral properties of the drug molecule. The equilibrium constant is given by

K ) [A]/[N]

3558

Langmuir, Vol. 20, No. 9, 2004

Chakraborty and Sarkar

presence of a higher concentration of SDS is 1.43 kJ mol-1. The change in ∆G in pure CTAB micelles was -0.3 kJ mol-1, and that in the presence of pure SDS was 1.7 kJ mol-1.12 In the presence of both types of catanionic micelles, the changes in ∆G value are less compared to those of their pure micellar counterpart. This may be due to the charge screening effect. In pure SDS micelles, drug molecules face a uniform negatively charged micellar surface, but in the catanionic micelles with a higher concentration of SDS they face a micellar surface predominantly negatively charged with a small amount of positive charge doped in it. This is reflected in a smaller change in ∆G value. The same argument is also true for the other catanionic micelles where CTAB concentration is higher. In the presence of pure SDS micelles, the maximum value of ∆G was positive, which indicated that the forward reaction (eq 1) was thermodynamically forbidden thereby allowing the formation of the neutral form only, resulting in a shift at the absorption maximum to 335 nm. In the presence of catanionic micelles with a higher concentration of SDS, the ∆G value becomes less negative but never reaches a positive value. So in the presence of this type of catanionic micelles, the reaction predominantly proceeds to the lefthand side, but the forward reaction is not thermodynamically forbidden, which allows the coexistence of both neutral and anionic forms of piroxicam. This results in a shift of the absorption maximum to 343 nm, characteristic of the mixed population, instead of 335 nm for the neutral form only. Such a strong effect of the micellar surface charge on drug incorporation and modulation of prototropic forms could indicate that the probable locus of solubilization of the drug is nearer to the interface rather than deep inside the micellar core. However, at this stage we can only speculate on the exact location of the drug in the micelles. Conclusion -1

Figure 11. Dependence of ∆G (J mol ) with concentration of (a) SDS at pH 3.8 at constant CTAB concentration of 0.1 mM and (b) CTAB at pH 5.5 at constant SDS concentration of 0.03 mM.

where [A] is the concentration of the anionic form and [N] is the concentration of the neutral form of piroxicam. The change in the free energy (∆G) can be determined from the equation

∆G ) -RT ln K The ∆G value becomes more negative in the presence of catanionic micelles with a higher concentration of positively charged CTAB (Figure 11b) and becomes less negative in the presence of a higher concentration of negatively charged SDS (Figure 11a). Negative ∆G indicates the spontaneity of the equilibrium to the righthand side, that is, formation of more anionic species, whereas positive or less negative ∆G indicates the formation of the more neutral form of the drug. The difference between the ∆G values in the presence of maximum concentration of surfactant and in water is termed as change in ∆G. The maximum change in ∆G value in the presence of catanionic micelles with a higher concentration of CTAB is -0.12 kJ mol-1, and that in the

Our study demonstrates that catanionic micelles containing a higher concentration of CTAB are larger in size, less compact, and more polar compared to the ones containing a higher concentration of SDS. The larger size and less compact nature of the catanionic micellar core containing a higher concentration of CTAB might allow some water penetration which could be the reason for it being more polar than the corresponding micelles containing a higher SDS concentration. The doping of some positively charged headgroups in a predominantly negatively charged micellar surface and vice versa modulate the equilibrium of the switch-over between prototropic forms of piroxicam in a different way compared to micelles having uniformly charged headgroups. This fine-tuning of the equilibrium even by such small changes in the electrostatic properties of the environment is extremely important in the context of biomembranes where charges vary depending on the nature of the membrane. Acknowledgment. We are extremely thankful to Ms. Rona Banerjee for her help and cooperation. We also acknowledge Mr. Pulak Ray, Mr. Tapan Kumar Ray, and Mr. Ajay Chakrabarti of the Biophysics Division of the Saha Institute of Nuclear Physics for their help in transmission electron microscopy. LA0361417