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Colloids and Surfaces A: Physicochem. Eng. Aspects 436 (2013) 704– 713

Contents lists available at ScienceDirect

Colloids and Surfaces A: Physicochemical andEngineering Aspects

jo ur nal ho me p ag e: www.elsev ier .com/ locate /co lsur fa

ixed micelles of sodium cholate and Brij30: Their rheologicalehaviour and capability towards solubilization and stabilizationf rifampicin

asrat Maswal, Aijaz Ahmad Dar ∗

epartment of Chemistry, University of Kashmir, Hazratbal, Srinagar 190 006, J&K, India

i g h l i g h t s

NaC aggregates showed micellargrowth upon addition of Brij30.The solubility of rifampicin washigher in NaC micellar systems.The solubilization capacity of NaCdecreased with increase in Brij30.The mixed micelles are more protec-tive for oxidation of drug.The formulation enhances solubility,stability and bioavailability of thedrug.

g r a p h i c a l a b s t r a c t

r t i c l e i n f o

rticle history:eceived 19 May 2013eceived in revised form 27 July 2013ccepted 30 July 2013vailable online xxx

eywords:ile saltrij30ormlike micelles

a b s t r a c t

Rifampicin is wide-spectrum antibiotic but being poorly water soluble and unstable towards oxidation, itrequires high dose in order to reach therapeutic plasma concentrations thereby making its adverse effectslike hepatotoxicity more prominent. Sodium cholate (NaC), Brij30 and their mixed micellar systemswere evaluated for their solubilization and stabilization capability towards rifampicin. NaC aggregatesshowed micellar growth upon addition of Brij30 surfactant inferred by characterization using rheologyand were found to form both wormlike micelles and vesicles. The solubility of rifampicin was found to behigher in NaC micellar systems due to an appreciable interfacial solubilization favoured by electrostaticinteractions. The solubilization capacity of mixed micellar systems decreased with increase in Brij30surfactant concentration. A comparative study of stability of rifampicin against oxidation with H2O2

esiclesolubilizationifampicin

showed that the micelles were able to stabilize the drug. The mixed micellar systems possessed muchmore solubilization efficiency than Brij30 micellar systems and at the same time proved markedly moreprotective against oxidation of rifampicin when compared to NaC besides being stable to large stressand having the desirable characteristics of their viscoelasticity, thus proving to be the ideal systems for

zationity, st

solubilization and stabilifor enhancing the solubil

. Introduction

Surfactant molecules self-assemble to form various micellartructures above their cmc, whose geometry depends on surfac-ant concentration. The increase in concentration of the surfactant

∗ Corresponding author. Tel.: +91 194 2424900; fax: +91 194 2421357.E-mail addresses: aijaz n5@yahoo.co.in,

ijazdar@kashmiruniversity.ac.in (A.A. Dar).

927-7757/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.colsurfa.2013.07.039

of rifampicin. These experimental results are expected to be importantability and hence bioavailability of rifampicin.

© 2013 Elsevier B.V. All rights reserved.

induces a transition from dilute individual spherical micellesto semi dilute and entangled wormlike micelles imparting vis-coelasticity to the system followed by formation of more orderedvesicular micelles at higher surfactant concentrations [1]. Worm-like micelles are of considerable interest both from theoretical andindustrial/technological applications point of view. From funda-

mental perspective they act as model of ‘equilibrium polymers’ alsocalled ‘living polymers’ [2–4] as these are extended linear macro-molecular structures constantly breaking and reforming withoutany mechanical degradation. The spontaneous restoring property

: Phys

taitmtemocb

smpizggmmdispbwiepmrblsmnfcmsamn

pdmeauhipmgdmeidoto[b

M. Maswal, A.A. Dar / Colloids and Surfaces A

ogether with the drag reduction capability makes them very usefuls heat-transfer fluids, hard-surface cleaners [5–8], fracturing flu-ds in oil fields [9] and in personal care products [10] for which bothheir high viscosity and elastic properties are exploited. Wormlike

icelles of biocompatible or biodegradable surfactants have poten-ial applications as drug delivery systems [11]. These have beenmployed as carriers of fatty acids and amino acid derived oils inedicines, cosmetics and foods [12], for the transdermal transport

f caffeine or theophylline [13], for transferring cytotoxic drugs toells [9] and as sieving matrixes for the separation of DNA fragmentsy capillary electrophoresis [14].

Polyoxyethylene ether (POE) surfactants like Brij30 have beentudied extensively in pharmaceutical systems [15,16] due to theirinimum toxicity. Bile salts are physiologically relevant, biocom-

atible and biodegradable molecules which undergo aggregationn aqueous solution. They have been employed for the solubili-ation of many poorly water soluble drugs like Griseofulvin [17],lutethimide [17], digoxin [18], leucotriene-D4 antagonists [19],emfibrozol [20], etc. owing to their good acceptability for phar-aceutical products. Since dilute bile salt solutions present smallicelles with low aggregation number due to the steric hin-

rance of the large steroidal skeleton, their solubilization capacitys limited. However, with the increase in concentration of bilealt like deoxycholates [21–23] they adopt a helical structure orolymer-like aggregation aided by the intermolecular hydrogenonding between the hydroxyl and the carboxyl groups togetherith partial back-to-back hydrophobic interaction resulting in

ncrease in hydrophobic domain of the micelles. This consequentlynhances its solubilization capacity towards hydrophobic com-ounds [22]. Further, the same could be achieved by employingixed micelles of bile salts and surface active agents which are

eported to form wormlike cylindrical micelles having, therefore,etter solubilization capability [24–26]. While going through the

iterature, we found most of the investigations having objective oftudying formation of mixed micelles and their consequent phar-aceutical applications involve phospholipids and bile salts which

eed the use of organic solvents like chloroform and methanol forabrication. Therefore, in this endeavour we present rheologicalharacterization, i.e. flow behaviour and viscoelastic properties ofixed micelles of sodium chloate (NaC) and conventional non-ionic

urfactant Brij30. Such mixed micelles are easy to prepare withoutid of organic solvents together with its good acceptability in phar-aceutical research. To the best of our knowledge, such a study has

ot been reported in the literature.In continuation, these mixed micelles were evaluated for their

otential to act as solubilization medium towards hydrophobicrug like rifampicin. Rifampicin is a strategic medication recom-ended by the World Health Organization for the treatment of

ndemic diseases [27] and is one of the principal chemical ther-pies in combating tuberculosis and hanseniasis [28]. It is alsosed to treat osteomyelitis and prosthetic joint infections [29] andas some effectiveness against vaccinia virus [30,31]. Rifampicin

s a hydrophobic drug with low aqueous solubility [32] and henceroblems with its bioavailability in both fixed and single dose for-ulations are reported [33]. The therapeutic activity of rifampicin

ets hampered due to its poor bioavailability attributed to itsegradation in gastro-intestinal tract, variability in absorption andetabolism, pH dependent solubility, changes in crystalline nature,

tc. [34,35]. For the effective treatment the dose requirementncreases which makes the adverse effects of rifampicin like liveramage (hepatotoxicity) more prominent. Also the overexposuref bacterial species to rifampicin makes them resistant due to

he mutations that alter residues of the rifampicin binding siten RNA polymerase resulting in decreased affinity for rifampicin36]. An increase in the solubility, effectiveness, stability and thusioavailability of rifampicin without altering the dose requirement

icochem. Eng. Aspects 436 (2013) 704– 713 705

is demanded and a number of previous studies are reported in thisregard. Oliveira et al. [37] increased aqueous solubility of rifampicinthrough formation of cyclodextrin molecular complexes. Bhasinet al. [33], however, used microemulsion composed of oleic acid andTween 80 as drug delivery system for rifampicin. On the other hand,Arguelho et al. [38] employed chitosan as carrier material for theprotection and release of rifampicin. They observed that rifampicinis absorbed by chitosan in an acid medium, and released in analkaline medium which is vital information for optimization of theabsorption of rifampicin. In view of this, mixed micelles of sodiumcholate and Brij30 were evaluated for their potential to act as suit-able solubilization media for rifampicin with the focus on the extentof solubility enhancement and determining location of solubili-zation site within these nanostructures using spectrophotometricand fluorimetric techniques. Further stability of rifampicin againstoxidation by H2O2 within these aqueous micellar systems was stud-ied to evaluate the prospective potential of such systems as validmedium for solubilization. The experimental results of this studyis important from both fundamental as well as technological pointof view for enhancing the solubility, stability and hence bioavail-ability of rifampicin which may pave way to develop its suitablepharmaceutical formulation required for various medical compli-cacies.

2. Experimental

2.1. Materials

Rifampicin was obtained from Himedia laboratories (India, 98%).Brij30 and cholic acid sodium salt hydrate (NaC) amphiphiles wereAldrich products (>98%). H2O2 used was of analytical grade. Allthe chemicals were used as received. The chemical structures ofrifampicin and surfactants are presented in Scheme 1.

2.2. Methods

2.2.1. CMC determinationThe cmc values were determined from the surface tension

(�) vs logarithm of surfactant concentration (log [surfactant])plots (Fig. 1). Surface tension measurements were made with aKruss 9 tensiometer by the platinum ring detachment method.Surfactant concentration was varied by adding concentrated sur-factant solution in small instalments and reading were taken afterthorough mixing and temperature equilibration. The tempera-ture was maintained at 25 ± 0.1 ◦C by circulating water from aHAAKE GH thermostat. The accuracy of measurements was within±0.1 dyne cm−1.

2.2.2. Rheological measurementsSteady and dynamic rheological experiments were performed

on Anton Paar MCR-102 rheometer equipped with a peltier tem-perature control system with an accuracy of ±0.01 ◦C. Cone-plategeometries (diameter of 49.985 with cone angle of 1.006◦) wereused for the measurements. In steady shear experiments the shearrate was typically increased from 0.0001 to 10,000 s−1. In oscil-latory measurements a frequency sweep at constant stress wasperformed to obtain the dynamic viscoelastic functions such as theshear storage modulus G′ and loss modulus G′′ as a function of angu-lar frequency ‘ω’. The experiments were carried out at 25 ◦C and foreach experiment a new sample was used and the measurementswere carried out in duplicate with good reproducibility.

2.2.3. Solubility and stability experimentsThe solubility of rifampicin and its stability against oxidation

with H2O2 was determined spectrophotometricaly with a Schi-mazdu Spectrophotometer (model UV-1650). The drug was added

706 M. Maswal, A.A. Dar / Colloids and Surfaces A: Physicochem. Eng. Aspects 436 (2013) 704– 713

Scheme 1. Structures of surfactants and drug used in this study.

1296-1-2-330

35

40

45

50

55

60

65

Su

rfa

ce T

en

sio

n(m

Nm

-1)

Log[Surfactant(mM)]

Brij30

Brij30+NaC (1:1)

NaC

Fig. 1. Variation of surface tension with surfactant concentration at 25 ◦C.

M. Maswal, A.A. Dar / Colloids and Surfaces A: Physicochem. Eng. Aspects 436 (2013) 704– 713 707

ate: (a

itauTdaotow

2

tMMaes

3

3

0uwcftntbaimmiets[

Fig. 2. Plot showing variation of viscosity with shear r

n excess to the vials containing surfactant solution which werehen sealed with screw caps and agitated for a period of 24 h on

magnetic stirrer maintaining the temperature at 25 ± 0.5 ◦C. Then-dissolved drug was removed by centrifugation at 13,400 rpm.he concentration of drug in different micellar systems wasetermined from their respective absorbance values at its char-cteristic wavelength of 477 nm using the extinction coefficientf 12,697.5 mM−1 cm−1 determined from the calibration curve ofhe drug in 50% water–methanol system. Kinetics of degradationf rifampicin was followed as the decrease in absorbance at �max

ith time after the addition of 50 mM H2O2.

.2.4. Fluorescence measurementsThe fluorescence spectra of rifampicin in various micellar sys-

ems were obtained on Spectrofluorometer (Schmadzu, Japan,odel RF-5301) operating in the steady-state mode at 25 ± 0.1 ◦C.easurements were made in 1 cm path length quartz cuvette using

5 mm excitation slit width and 3 mm emission slit width. Thexcitation wavelength used was 390 nm and fluorescence emissionpectra were recorded in the range of 400–700 nm.

. Results and discussion

.1. Cmc and interaction of surfactants in mixed micelles

The experimental cmc values obtained for Brij30 and NaC were.033 and 9.8 mM, respectively, tallying well with the literature val-es [39,40]. The cmc value obtained for 1:1 mixed micellar systemas 0.03 mM which is lower than the cmcideal value of 0.065 mM

alculated using Clint equation [39] indicating negative deviationrom ideal behaviour for mixed micelle formation. Rubingh’s equa-ion [39] based on regular solution theory was used to estimate theon-ideality of this mixed binary surfactant system and the interac-ion parameter ‘ˇ’ which is an indicator of the degree of interactionetween two surfactants in mixed micelles and accounts for devi-tion from ideality with a negative value implying an attractiventeraction, was found to be −8.77 indicating strong synergism for

ixed micelle formation. The reason being that for ionic–non-ionicixed surfactant systems, the electrostatic self repulsion of ion-

cs and steric self-repulsion of non-ionics are reduced by dilution

ffects after mixing [39] and the consequent ion–dipole interac-ion between the hydrophilic head groups of ionic and non-ionicurfactants within mixed micelles favours the mixed micellization41–43]. The favourable possibility of hydrogen bonding in addition

) pure micellar systems and (b) drug loaded micelles.

to the polar attractions of the hydrophilic head groups of these twosurfactant systems and the strong hydrophobic interactions of theirtail groups may account for the obtained degree of non-idealitywhich is in conformity with some earlier studies on such types ofmixed micellar systems [44,45]. The large negative value of ‘ˇ’ insuch mixed surfactant system indicates the possibility of micel-lar growth with the formation of giant micelles having expectedlyviscoelastic properties.

3.2. Rheological behaviour

In order to study rheological behaviour of the two surfactantsin single and mixed states, the total surfactant concentration wasfixed at 50 mM since it was found to be the minimum concentra-tion where viscoelastic behaviour in some mixtures was observed.Viscosity‘�’ was found to be low and close to that of pure solvent incase of Brij30 and NaC micellar solutions in their pure states pos-sessing Newtonian flow behaviour (Fig. 2). The variation of � asa function of shear rate ‘� ’ for mixed Brij30–NaC micellar systemcorresponding to different mixing mole fraction of surfactants isshown in Fig. 2(a) and clearly indicates formation of different typesof aggregates at different mixing mole fractions reflected from thechanges in rheological properties. Viscosity vs shear rate behaviourshows no noticeable change up to the mixing mole fraction ofBrij30, XBrij30, equal to 0.4 wherein low value of � as well as Newto-nian behaviour was prevalent and characteristic of single surfactantsystems. However, at XBrij30 = 0.6 viscosity of the system increasesnoticeably which remains constant up to certain shear rate calledcritical shear rate and thereafter shows shear thinning behaviourindicating the formation of wormlike micelles in the system [46].The rapid increase in viscosity reflects the exponential growth ofcylindrical micelles developing eventually into long, flexible andrandomly oriented micelles thereby imparting viscoelasticity to thesystem. These wormlike micelles become aligned and flows in thedirection of shear above critical shear rate thus exhibiting shearthinning behaviour at large � values. With increase in XBrij30 upto 0.7, viscosity increases further with the concomitant increasein critical shear rate. Increase in the XBrij30 in mixed micellar sys-tem is expected to result in decrease in the interfacial curvatureof aggregates invoking an increase in the energy requirement forthe formation of hemispherical end-caps of the cylindrical micelles.

This is therefore minimized by fusing of the ends with cylindri-cal part of its own or other micelles resulting in the formation ofmicellar joints and/or branching in the network structure regis-tering an increase in the viscosity. The wormlike micelles become

708 M. Maswal, A.A. Dar / Colloids and Surfaces A: Physicochem. Eng. Aspects 436 (2013) 704– 713

ormlik

eaipcvictptllpviXsiocaestrchcssii

ftcsoc

Fig. 3. Oscillatory rheograms of (a) w

ntangled into transient network and the slipping of these jointslong the cylindrical body attributes the system a faster and eas-er way of stress relaxation [46,47] together with the branchingoints which restricts the alignment of micelles under shear [48]ausing an increase in critical shear rate and thus enhancement ofiscoelasticity of the system. Fig. 2(a) clearly shows that increasen XBrij30 to 0.8 causes structural transition from wormlike to vesi-le shaped micelles in this mixed micellar system as reflected byhe change in the rheological behaviour. It is well known that largeacking parameter of Brij30 surfactant system favours the forma-ion of planer lamellar micelles at higher concentrations [49]. Suchamellar micellar phase contains defects in the form of dislocationoops [50] or thermodynamically stable defects in the vicinity ofhase transition boundaries [51] and tends to reorient and formesicles by the application of shear rate confirmed by the increasen viscosity [51]. Therefore in the mixed micellar systems withBrij30 = 0.8 the staked lamellar micelles get converted into vesiclehaped micelles when subjected to shear leading to shear thicken-ng behaviour at low shear rate in tune with the reported results ofther micelles showing this type of transition [52,53]. These vesi-le shaped micelles are not elastic and thus flow in the direction ofpplied shear exhibiting shear thinning behaviour at low to mod-rate � values [54]. The mixed micellar systems with XBrij30 = 0.9hows typical behaviour of vesicle shaped micelles with an addi-ional shear thickening behaviour at an intermediate shear rateange. The reason for this shear thickening at moderate shear rateould be that the higher mole fraction of Brij30 in the vesicularydrophobic bilayers or palisade layers decreases the interfacialurvature of the membranes to an extent that the vesicles becomeensitive to shear. The outer shells of the multilamellar vesicles aretripped off under shear at moderate shear rate, leading to increasen number density of aggregates resulting in an increase in viscosityn tune with the results reported for vesicle shaped micelles [55].

The presence of different types of the micelles at different moleraction of Brij30 surfactant in mixed surfactant system was fur-her assessed by the dynamic rheological studies performed at the

−1

onstant shear rate of 0.01 s . In this case the plots of elastic ortorage modulus (G′) and viscous or loss modulus (G′′) as a functionf oscillatory shear frequency (ω) for viscoelastic solutions formedorresponding to XBrij30 = 0.6 and 0.7 in mixed micellar systems is

e micelles and (b) vesicular micelles.

given in Fig. 3(a). The elastic behaviour (G′ > G′′) at higher ω and vis-cous behaviour (G′′ > G′) at low ω values indicate again formation ofwormlike micelles [55] in such solutions. The intersection point ofG′ and G′′ gives the crossover frequency ‘ωc’. The entanglement oflong and flexible wormlike micelles imparts viscoelasticity to thesolution and has been described by considering two processes ofstress relaxation, viz., reptation or reptile like motion of the micelleand reversible scission of micelles which occurs at two time scales,reptation time �rep and breaking time �b. The viscoelastic behaviourof wormlike micelles follow Maxwell [54,56,57] model at low shearfrequency described by the following equations:

G′ = G0ω2�2

R

1 + ω2�2R

(1)

G′ ′ = G0ω�R

1 + ω2�2R

(2)

where �R is single stress relaxation time given by (�rep�b)1/2 and G0is called the shear (plateau) modulus which is the constant valueattained by G′ at high ω. The relaxation time is related with ‘ωc’ as

�R = 1ωc

(3)

The zero shear viscosity �0 is obtained from G0 and �R as

�0 = G0�R (4)

The zero shear viscosity calculated for the wormlike micellescorresponding to XBrij30 = 0.7 and 0.6 comes out to be 0.195 Pa sand 0.0489 Pa s which are about 100 and 10 times respectivelymore than that of the solvent. The Maxwellian fitting to the datapoints is represented by the solid lines in Fig. 3(a). The rheologi-cal behaviour fits appreciably with the Maxwell model in low ωregion while at higher ω experimental data shows significant devi-ation. The reasons for such deviations can arise from various factors.

Micelles are in dynamic equilibrium with monomers, the timetaken for reversible breakage and recombination is shorter thanthe reptation time of the wormlike micelles at higher frequencyand as �b/�rep increases the deviation from Maxwellian behaviour

M. Maswal, A.A. Dar / Colloids and Surfaces A: Physicochem. Eng. Aspects 436 (2013) 704– 713 709

65432100.0

0.5

1.0

1.5

2.0

2.5

3.0

G" (

Pa)

XBrij30

= 0.7

XBrij30

= 0.6

btafirlfimiX

a

(

GeocsMqbcomtqiTs

3

uapMtth

6005004003000.0

0.5

1.0

1.5

2.0

2.5

3.0

Ab

so

rba

nc

e

Water

Brij30= 0.005mM

NaC =5mM

Brij30 =50mM

XBrij30

= 0.9

XBrij30

= 0.7

XBrij30

=0.1

NaC=50mM

Wavelength (nm)

solubilizate like rifampicin. Since rifampicin exists as zwiterionat physiological pH [43], it therefore is expected to show moreinclination towards solubilization within ionic surfactants due tomore favourable electrostatic interactions between the charged

Table 1Solubility of rifampicin in aqueous, premicellar, micellar and various mixed micellarsystem.

System Solubility (mg/ml)

Water 2.770.005 mM Brij30 2.915 mM NaC 3.5350 mM Brij30 27.9

G' (Pa)

Fig. 4. Cole–Cole plots of wormlike micelles.

ecomes more prominent [54,56,57]. In addition, tube-length fluc-uations (‘breathing’ modes) and polydispersity in micellar lengthlso contributes to the deviation of wormlike micelles at higherrequencies [54,56,57]. In Fig. 3(a) ‘ωc’ shifts to lower value withncrease in XBrij30 from 0.6 to 0.7 which corresponds to longer �R

eflecting the presence of longer micelles [54,56,57] indicating theength of wormlike micelles increases with increase in Brij30 moleraction within the mixed micellar systems. Since the value of G0s related to the number of entanglements between the wormlike

icelles and a higher G0 corresponds to denser wormlike micelles,t shows that more elongated and denser micelles are formed withBrij30 = 0.7 when compared to wormlike micelles with XBrij30 = 0.6.

By combining Eqs. (1) and (2) we get the equation of semi-circles:

G′ ′)2 +(

G′ − G0

2

)2

=(

G0

2

)2

(5)

The Cole–Cole plot is obtained by plotting G′ ′ as a function of′ for Maxwell fluids which describes a semicircle with radiusqual to G0. Cole–Cole plots provide more precise determinationf relaxation behaviour of samples than simple frequency sweepurves [57]. Cole–Cole plots for the two wormlike micellar compo-itions are given in Fig. 4. The experimental results closely followaxwell model at low frequencies and show deviations at high fre-

uencies. This deviation from the semi-circular behaviour occursecause relaxation process does not only involve diffusion pro-ess described by Maxwell model but also complex mechanismf dissociation-recombination of surfactant aggregations. For theixed micellar compositions with XBrij30 = 0.8 and 0.9 (Fig. 3(b)),

he samples show a little increase in G′ with frequency and fre-uency independent value of G′ ′ in low frequency range and steep

ncrease at higher frequency with absence of crossover frequency.his observation confirms [57] the presence of vesicles in the mixedurfactant systems containing XBrij30 = 0.8 and 0.9.

.3. Solubility of rifampicin

The aqueous solubility of rifampicin was found to be 2.77 mg/mlsing spectrophotometric technique tallying well with the liter-ture value of 2.5 mg/ml [38]. Such a low aqueous solubility ofharmaceuticals is the major challenge in formulation science.

icelles have particular significance in pharmacy as they increase

he solubility of sparingly soluble substance in water [58,59], dueo their capability of solubilizing non-polar molecules into theirydrophobic interiors, enabling them to get dispersed freely in

Fig. 5. Absorbance vs wavelength of rifampicin in aqueous, premicellar, micellarand various mixed micellar system.

otherwise insoluble aqueous phase. The UV spectra of rifampicinand the solubilization capacity within aqueous, pre-micellar, postmicellar single and mixed surfactant systems of Bij30 and NaC sys-tems are given in Fig. 5 and Table 1, respectively. As observedfrom the figure and data in table, the solubility of rifampicin insingle Brij30 surfactant system below its cmc is comparable tothat of its aqueous solubility, but shows slightly higher value inNaC surfactant below its cmc value. This enhancement in pre-micellar phase of NaC surfactants is attributed to weak interactionsbetween the solubilizate and the NaC monomers, as reported ear-lier [60,61] between different solubilizates and bile salt surfactantmonomers. However, the solubility of rifampicin increases appre-ciably in post-cmc region due to its encapsulation favoured byboth polar attractions with the palisade layer and hydrophobicinteractions with the hydrophobic core of the micelle. It can beattributed to the amphiphilic nature of rifampicin which has naph-thohydroquinone group acting as polar head group and an aliphaticchain with a piperazine side chain attachment behaving as thelipophillic tail. From Table 1, we find that in post-micellar region,rifampicin has lower maximum solubility in Brij30 systems thanin NaC. Brij30 provides a larger micellar core volume for solubili-zation owing to its larger aggregation number (101) [39] and higherpacking parameter [49] with relatively less possibility of palisadelayer solubilization due to lesser number of oxyethylene groups (4)present per surfactant molecule. NaC, on the other hand, at higherconcentration have oblate ellipsoidal structure with an appre-ciable core volume and very significant charged interfacial areaboth of which are potential solubilization sites for an amphiphilic

XBrij30 = 0.9 30.47XBrij30 = 0.7 36.54XBrij30 = 0.1 39.3850 mM NaC 41.21

7 : Physicochem. Eng. Aspects 436 (2013) 704– 713

gascsriifpemiru

3

flTteRaacwiiibtptc

almmptfrcitBtmwbitwoiitbseflwb

7006506005505000.0

0.2

0.4

0.6

0.8

1.0

No

rma

lize

d In

ten

sit

y

Wavelength (n m)

CCL4

Diethyl ether

Methanol

Water

(a)

700650600550500

0

10

20

30

40

50

60

Wavelength (nm)

Inte

nsit

y

water

[Brij30] (mM)

0.0099

0.0199

0.0298

0.0398

0.0497

0.0597

0.0696

0.0796

0.0895

0.0995

0.1094

0.1194

0.1293

(b)

700650600550500

0

20

40

60

80

100

120

140

Inte

nsit

y

Wavelength (nm)

Water

[NaC] (mM)

1.247

2.493

3.74

4.987

6.234

7.48

8.72

9.97

11.22

12.46

13.71

14.96

16.21

17.45

18.70

19.95

21.19

22.44

23.69

24.93

(c)

10 M. Maswal, A.A. Dar / Colloids and Surfaces A

roups as compared to that of nonionics [38,62]. Hence, NaC beingn anionic surfactant system with two favourable solubilizationites shows more solubilization efficiency towards rifampicin whenompared to Brij30 surfactant system. In the mixed surfactantystems, increase in NaC mole fraction facilitates the uptake ofifampicin with consequent increase in the solubilization capac-ty. Therefore, mixed micellar systems at all mole fractions of NaCrrespective of micellar structures present within the system wasound to be better solubilization medium of rifampicin when com-ared to pure Brij30 micellar systems though the solubilizationfficiency of mixed micelles is slightly lower than that of pure NaCicelles as shown in Table 1. Further, the encapsulation of drugs

ncreases the viscosity of all studied micellar systems although theirheological behaviour and hence structural characteristics remainsnchanged Fig. 2(b).

.4. Locus of solubilization of rifampicin

The polarity of a solvent influences the emission spectra ofuorophores by changing the quantum yields and spectral shifts.he modifications induced by a solvent in the emission spec-ra of solubilized molecules can offer information on the locallectric field acting on the spectrally active molecule [63–65].ifampicin is sparingly soluble in water and undergoes aggregationbove its solubility limit [66] resulting in excimer emission of theggregated and interacting molecular units causing week fluores-ence signal at longer wavelength when compared to chloroformhich brings about the better solvation of rifampicin. Rifampicin

s weakly fluorescent in polar solvents but the emission intensityncreases in hydrophobic environment confirming its good solubil-ty in these media due to reduction in intermolecular aggregationy the non-polar solvents. Further rifampicin shows positive salva-ochromisim, i.e. large bathochromic shift with increase in solventolarity as shown in Fig. 6(a) in different solvents indicating thathe excited state of rifampicin is more dipolar zwiterionic whenompared to its ground state.

Micelles are highly cooperative non-covalent aggregates with variety of organization and dynamics of micellar environmentike the non-polar core, the slightly polar palisade layer and the

ore polar micelle-water interface. Therefore, the microenviron-ent of solubilized molecules bound to such regions of different

olarity determines its activity and stability making it worth inves-igation. Fig. 6(b) shows the fluorescence spectra of rifampicin as aunction of Brij30 surfactant concentration. The figure shows thatifampicin is weakly fluorescent in aqueous and pre-micellar con-entrations of Brij30 but the fluorescence intensity of rifampicinncreases in post-cmc region indicating its solubilization withinhe Brij30 micelles. Moreover the hypsochromic shift observed inrij30 post-micellar concentrations (Fig. 6(b)) clearly indicates theransfer of rifampicin towards more hydrophobic region revealing

icellar core to be the desired area of solubilization of rifampicinhere it is solubilized efficiently and stabilized by the hydropho-

ic interactions. In NaC (Fig. 6(c)), however, rifampicin fluorescencentensity increases significantly in post micellar phase illustratinghe solubilization within the micellar aggregates which is coupledith the bathochromic shift in the wavelength of maximum flu-

rescence emission indicating the solubilization site of rifampicinn more polar regions of micelle like micelle-water interface. Thiss expected because of strong electrostatic interaction betweenhe highly polar excited state of rifampicin and polar groups ofile salt at interface, which might lead to stabilization of excitedtate of solubilizate with concomitant decrease in energy differ-

nce between ground and excited state and hence emission ofuorescence intensity at higher wavelength. For mixed micellesith XBrij30 = 0.1 (Fig. 7(a)) the fluorescence spectra almost resem-

les with that of pure Bile salt micelles with slight hypsochromic

Fig. 6. Florescence spectra of rifampicin: (a) various solvents, (b) Brij30 and (c) NaCsurfactant systems.

shift which occurs due to increase in hydrophobicity of the mediumindicating that rifampicin mostly occupies the polar regions of

micelle like the interfacial layer for its solubilization. Similar rea-son explains the slight bathochromic shift of mixed micelles withXBrij30 = 0.9 (Fig. 7(d)), where the fluorescence spectra is almostidentical to that of pure Brij30 micelles indicating an appreciable

M. Maswal, A.A. Dar / Colloids and Surfaces A: Physicochem. Eng. Aspects 436 (2013) 704– 713 711

700650600550500

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2.493mM

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(a)Brij30(1):NaC(9) mixed system

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Brij30(6):NaC(4) mixed system

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water

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0.0597mM

0.0696mM

0.0796mM

0.0895mM

0.0995mM

(c)

erent

aooBmfltRtlccuIwhos

Fig. 7. Florescence spectra of rifampicin in mixed surfactant systems of diff

mount of rifampicin is solubilized within the hydrophobic cavityf the vesicles. For wormlike micelles with XBrij30 = 0.7/0.6, the flu-rescence spectra shows bathochromic shift with respect to purerij30 micelles and hypsochromic shift with respect to bile salticelles with the magnitude depending upon the relative mole

raction of two surfactants (Fig. 7(b) and (c)). For these worm-ike micelles two well defined solubilization sites are presented:he inner hydrophobic canal and outer polar periphery of tube.ifampicin owing to its structure is expected to occupy both ofhe sites so the fluorescence peak in these types of mixed micel-ar systems is the superposition of two unresolved peaks, one peakorresponding to that fraction of rifampicin solubilized within theanal of the tube and other one for the fraction of rifampicin sol-bilized in the polar peripheral region of the worm-like micelle.

ncreasing the Bile salt mole fraction for the worm-like micelles

ith XBrij30 = 0.6, results in more denser peripheral surface andence increase in degree of solubilization within the polar regionf the worm-like micelles explaining the obtained experimentalhift.

mole fractions: (a) XBrij30 = 0.1, (b) XBrij30 = 0.6, (c) XBrij30 = 0.7 (d) XBrij30 = 0.9.

3.5. Kinetics of rifampicin oxidation

Rifampicin is unstable in aqueous solution and is readily oxi-dized to rifampicin quinone. Therefore, efforts have been made todevise methods for enhancing its stability in aqueous environment[38,39]. In this endeavour the stability of rifampicin in all the stud-ied systems was determined by following the decrease in intensityat the �max of rifampicin with time after the addition of 50 mMH2O2. The kinetics of oxidation of rifampicin was studied belowthe saturation level at a constant concentration of 2 mg/ml in allthe systems. The initial rate of oxidation of rifampicin was mod-elled by assuming a first-order reaction in accordance with earlierstudies [39] so that

C(t) = C0 exp(−kt) (6)

where C0 is the initial rifampicin concentration, C(t) is therifampicin concentration remaining at time t and k is the degra-dation rate constant. The variation of ln(C(t)/C0) with time in allthe systems is shown in Fig. 8. The values of k were determined by

712 M. Maswal, A.A. Dar / Colloids and Surfaces A: Phys

Fv

crtltmtwdcltmtsdforwlhlilpb

wwrscpmmadwbamm

[

[

[

ig. 8. Variation of ln(C� /C0) with time (t) for aqueous, premicellar, micellar andarious mixed micellar system.

arrying out a linear regression on plots of ln(C(t)/C0) versus t. Theate of oxidation of rifampicin in aqueous and pre-micellar concen-rations of both the surfactants is very high although it is slightlyess in pre-micellar concentration of NaC which may be attributedo the weak interactions between the surfactant monomer and drug

olecule that makes the drug molecule less potent towards oxida-ion. The appreciable decrease in the rate of oxidation of rifampicinithin the micellar systems indicates that the encapsulation ofrug within the micellar systems causes its isolation from the radi-als and other pro-oxidant species present in aqueous environmenteading to its stabilization. Brij30 micelles proved to be much bet-er stabilization medium of rifampicin when compared to bile salt

icelles which could be safely attributed to the different loca-ion of solubilized rifampicin within these micellar systems. NaCurfactants form micelles with greater inter head group spacingue to electrostatic repulsions between the head groups whichavours penetration of water containing free radicals and other pro-xidant species in the space. Further, appreciable solubilization ofifampicin at the micelle-water interface exposes it to pro-oxidantshich get concentrated at the interface due to polar interactions

eading to acceleration of degradation of rifampicin. The inter-ead group spacing is relatively small in Brij30 micelles leading to

ess amount of water trapped between the head groups and moremportantly the main solubilization site is the micellar core iso-ating rifampicin more adequately from the pro-oxidant speciesrevailing in the system thus enabling Brij30 micellar systems toe more potent stabilization medium for rifampicin.

The stabilization capability of mixed micellar systems increasesith increase in Brij30 mole fraction in NaC micelles makingormlike and vesicular micelles efficient stabilization media of

ifampicin. Since in wormlike micelles slightly more appreciableolubilization occurs at the micelle-water interface so the rateonstant for oxidation of rifampicin is a little higher than that ofure Brij30 micelles though very less than that of pure bile salticelles. Therefore, in mixed micellar systems, increase in bile saltole fraction increases solubilization capacity for rifampicin but

t the same time decreases its stabilization capability towards oxi-ation. Brij30 micelles are efficient only in stabilizing rifampicinhile NaC micelles are only potent solubilization media rendering

oth of the pure micellar systems inefficient for the generation of viable pharmaceutical formulation of rifampicin. The worm-likeicelles having only slightly lesser solubilization capacity than NaCicelles and relatively smaller stabilization capability than Brij30

[

[

icochem. Eng. Aspects 436 (2013) 704– 713

micellar system have additional desirable viscoelastic propertiesmaking them ideal system for solubilization and stabilization forrifampicin.

4. Conclusions

The study focuses on the possibility of formation of variousmicellar structures as a function of mole fraction of two differ-ent surfactants viz. Brij30 and NaC. These systems were subjectedto the solubilization and stabilization studies with respect to avery important antimycobaterial drug rifampicin used for thechemotherapy of tuberculosis. Rifampicin is slightly soluble andunstable in water which necessitated micellar formulation forreducing the dosing frequency and hence better patient compli-ance. Pure NaC micellar system proves to be better solubilizationmedium of rifampicin but a poor stabilization medium against itsoxidation while the pure Brij30 micellar system has weak solu-bilization capacity but a far better stabilization capability thoughboth the systems being Newtonian are stable to external stress.Wormlike micelles have much more solubilization efficiency thanBrij30 micellar systems and at the same time prove markedly moreprotective against oxidation of rifampicin when compared to NaCbesides being stable to large stress and having the desirable charac-teristics of their viscoelasticity, thus proving to be the ideal systemsfor solubilization and stabilization of rifampicin.

Acknowledgments

We are thankful to the Head, Department of Chemistry, Univer-sity of Kashmir, for his constant encouragement and inspiration.MM thanks University of Kashmir for providing the financial sup-port in the form of fellowship to meritorious students.

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