P

DMa

b

a

ARRAA

KXMPBP

1

oafcmc

alosgtpf(

1h

Journal of Photochemistry and Photobiology A: Chemistry 247 (2012) 8– 15

Contents lists available at SciVerse ScienceDirect

Journal of Photochemistry and Photobiology A:Chemistry

journa l h o me pag e: www.elsev ier .com/ locate / jphotochem

hotophysical properties and interactions of xanthene dyes in aqueous micelles

iogo Silva Pellosi a, Bianca Martins Estevãoa, Juliana Semensatoa, Divinomar Severinob,auricio S. Baptistab, Mario J. Politi b, Noboru Hiokaa, Wilker Caetanoa,∗

Departamento de Química, Universidade Estadual de Maringá, Av. Colombo, 5.790, CEP 87.020-900, Maringá, Paraná, BrazilDepartamento de Bioquímica, Universidade de São Paulo, São Paulo, Brazil

r t i c l e i n f o

rticle history:eceived 16 May 2012eceived in revised form 22 July 2012ccepted 24 July 2012vailable online xxx

eywords:anthenesicelles

olymeric micelles

a b s t r a c t

Photosensitizers (PS) photodynamic activities are regulated by their location in the biological target,which modulates their photophysical and photochemical features. In this work the PS partition for theXanthene Dyes Fluorescein (FSC), Eosin Y (EOS), Erythrosin B (ERY) and Rose Bengal B (RBB) in biomimeticmodels (SDS, CTAB and Pluronic P-123 micelles) and the effects on their photophysical characteristics areevaluated. The hydrophobic and electrostatic forces that govern the PS–micelle interaction are analyzed.At physiological pH (7.25), the ability of the dianionic protolytic form of the dyes to be positioned intothe micelle palisade and its micelle interaction depends not only on the hydrophobicity of the dye butalso on the micellar surface charge. The Binding Constants obey exactly the same order of the PartitionCoefficients for the dyes in P-123 and CTAB micelles. The Stern–Volmer treatment pointed out that dyes

inding constantshotodynamic therapy

are located inside the micelle, especially ERY and RBB. The magnitude of the dye–micelle interactionincreased from SDS, P-123 and finally CTAB micelles due to the charges between dye and micelle, andamong the xanthenes, their hydrophobic characteristics. Within the micelle pseudo phase, ERY and RBBare still very efficient photosensitizers exhibiting high quantum yield of singlet oxygen, which turns themvery attractive especially with P-123 polymeric system as drug delivery systems in photodynamic therapy.

. Introduction

Photodynamic therapy (PDT) and photodynamic inactivationf microorganism (PDIMO) are therapeutic modalities that actgainst abnormal biological tissues and localized infections. Theundament of the photodynamic activity is a photosensitizer (PS)ompound that can be activated by visible light in presence ofolecular oxygen producing highly reactive species, which are

ytotoxic [1–4].The photosensitizers are promoted to the excited singlet state

nd can cross to the triplet state. At this state, the compounds showong enough lifetimes to react directly with the biological substrater with oxygen. Reactive species are the excited reactive oxygenpecies (EROS) (type I photochemical reaction), and/or singlet oxy-en 1O2 (type II photochemical reaction). The 1O2 is pointed out ashe main PDT and PDIMO agent that can lead to necrosis and apo-tosis of the tissues [5–7]. In consequence usual photosensitizers

or PDT applications exhibit high quantum yields of singlet oxygen˚�

1O2).

∗ Corresponding author. Tel.: +55 44 3011 5153; fax: +55 44 32614125.E-mail address: wcaetano@uem.br (W. Caetano).

010-6030/$ – see front matter © 2012 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.jphotochem.2012.07.009

© 2012 Elsevier B.V. All rights reserved.

Selectivity and extent of partition of PS to target tissue is animportant pre-requisite to photodynamic clinical efficiency. Usu-ally in cells the targets are lysosomes, mitochondria and plasmaticmembranes [8]. In microorganism the damage is pointed out tooccur mainly in the cell membrane [9,10]. The previous knowledgeof the incorporation and of the cyto-localization is fundamen-tal for successful in PDT treatments, which makes necessary theevaluation of the interaction parameters of PS toward cell mem-brane [11–13]. The biological cell complexity makes this interactionstudies difficult, but it is possible to study these properties indepen-dently. Cell affinity depends on the hydrophobic and electrostaticcharacteristics of the PS and can be estimated using biomimeticsystems [14–16].

The hydrophilic–lipophilic balance (HLB) of the PS is usuallyestimated in water/1-octanol mixture, which is considered the sim-plest membrane model [17]. However, more interesting studies ofthe penetration and specific interaction of drugs and biomimeticsystems can be realized with aqueous micelles that reasonablymimetize bio-membranes and furnish useful information of orga-nized systems [18–21]. In addition to biomimetic studies, micelles

can also be used as drug delivery systems for medications [14,22–24].However, ionic micelles as SDS and CTAB are not used in medi-cal formulations due to their low drug protection and toxicity inbiological fluids [25]. Very interesting formulations are obtained

y and Photobiology A: Chemistry 247 (2012) 8– 15 9

wPtlpfntt

(dae1

ebd

stbAobvpdpC

2

2

wY(CsrwretaNcsa

2

2

asbt

K

wo

Table 1˚F values of xanthene dyes in water used as standard, pH 9.2 [38].

Dye ˚F �exc (nm)

FSC 0.920 470EOS 0.200 493

D.S. Pellosi et al. / Journal of Photochemistr

ith micelles constituted by polymeric surfactants, in particularluronics® (or Poloxamers®). These surfactants are constituted byri-block monomers of polyethylene oxide (PEO) and polypropy-ene oxide (PPO) forming (PEO)(PPO)(PEO) molecules where PPOrovides the hydrophobic core while PEO portion is the water-riend counterpart. These set of polymers are non-ionic, stable,on-toxic, and biocompatible, providing large and adequate siteshat solubilize several hydrophobic drugs and are less affected byhe environmental changes in biological fluids [14,25–27].

The PS agents in this study are xanthene derivatives. FluoresceinFSC) is an important fluorescent compound used in eyes diseasesetection due to its high selectivity for ocular neo-vasculaturend high fluorescent quantum yield (˚F) [28,29]; FSC does notxhibit photodynamic activity once it has low quantum yield ofO2 (˚�

1O2). Despite it, some halogenated derivatives of FSCxhibit high production of 1O2 [30] and can be evaluated as possi-le compounds for eye diseases treatment as age-related macularegeneration and pathologic myopia.

The chemical structures of the xanthene compounds (Fig. 1)how that the molecule presents two distinct moieties, the xan-hene and benzoate rings, where the orthogonality (and rotation)etween these planes depends on their substituents [31–33].ccording to Nagano et al. [34] the photophysical characteristicsf these dyes are determined by the xanthene part, whereas theenzoate moiety controls the excited state decay processes (byibration and rotation energies dissipation). In the present workroperties of Eosin (EOS), Erythrosin (ERY) and Rose Bengal (RBB)yes (Fig. 1) that show high ˚�

1O2 in water at neutral and alkalineHs [30,35,36] are investigated in aqueous micellar systems (SDS,TAB and P-123).

. Materials and methods

.1. Materials

All solvents employed were analytical grade and were usedithout further purification. Fluorescein (FSC, Carlo Erba), Eosin

(EOS, Reagen), Erythrosin B (ERY, Vetec) and Rose Bengal BRBB, Nuclear) were analyzed and identified by 1H NMR. SDS,TAB and P-123 were purchased from Sigma–Aldrich and theolutions were prepared by weight of the previously dried mate-ials in desiccators under vacuum for 24 h. The experimentsere conducted in a UV–Vis spectrophotometer Cary-50 appa-

atus or in a fluorescence spectrofluorometer Cary-Eclipse. Allxperiments were conducted at 30.0 ◦C in aqueous solutions withhe pH 7.25 controlled by buffer (McIlvaine, [Na2HPO4] = [citriccid] = 7.5 × 10−3 mol L−1) and the ionic strength controlled byaCl addition (0.10 mol L−1). For absorbance experiments the dyesoncentrations were 5.00 × 10−6 mol L−1 and for fluorescence mea-urements were 5.0 × 10−7 mol L−1 (absorbance lower than 0.05 tovoid internal filter problems).

.2. Methods

.2.1. Partition coefficient (Kp)To a biphasic mixture containing octanol/water at 50% (v/v) was

dded the xanthenes dyes (3.0 × 10−6 mol L−1), and after intensetirring and 48 h rest in the dark, the xanthene concentrations inoth phases was evaluated by UV–Vis. The partition coefficient ofhe octanol phase (Kp) was calculated by

P = [PS]oct

[PS]water(1)

here [PS]oct and [PS]water are the molar concentrations of dyes inctanol and water, respectively.

ERY 0.020 508RBB 0.018 515

2.2.2. Binding constant (Kb)The binding constants (Kb) of xanthenes with micelles were

evaluated by the fluorescence emission spectra of the dyes(5.0 × 10−7 mol L−1). Aliquots of surfactant were added using con-centrated stock solutions of surfactants directly to a cuvettecontaining xanthenes in water. The experimental data were the-oretically fitted using Eq. (2) [37].

F = Ff + (F0 − Ff )

(1/Kb([S] − CMC)N) + 1(2)

where F, fluorescence emission intensity of xanthenes; Ff, fluores-cence of the bounded PS to the surfactant; F0, fluorescence in theabsence of surfactant; [S], surfactant concentration; and N, numberof surfactant by PS molecules.

2.2.3. Stern–Volmer suppression constant (KSV)Fluorescence quenching experiments were conducted with

titration of xanthenes solutions (5.0 × 10−7 mol L−1) in water and inthe presence of micellar systems using iodide ion as a water-solublequencher. The NaI stock solutions were 1.00 mol L−1 for SDS and P-123, and 0.10 mol L−1 for CTAB systems. The KSV were calculatedfrom Eq. (3).

F0

F= 1 + KSV[I−] (3)

where F0 and F, fluorescence intensities in the absence and presenceof the suppressor, respectively, and [I−], iodide concentration.

2.2.4. Fluorescence quantum yield (˚F)˚F values were obtained in water and in aqueous micellar SDS,

CTAB and P-123 solutions by use of Eq. (4). The standard compoundsused were the xanthenes in pure water (Table 1).

�F = AbsStdFDn2Std

AbsDFStdn2D

· �Std (4)

where Abs, absorbance intensity; F, fluorescence spectrum area;and n, refraction index. The subscripts in the symbols refer to thestandard (Std) and to the dye (D). The concentration of the surfac-tants was kept fixed and above their CMC.

2.2.5. Quantum yield of the singlet oxygen (˚�1O2) of the dyes

˚�1O2 were determined from the phosphorescence inten-

sity decays at 1270 nm. Data were recorded with a time-resolvedNIR fluorometer (Edinburgh Analytical Instruments) equippedwith Nd:YAG LASER (Continuum Surelite III) �exc = 532 nm(pulse ∼ 30 ns). The emitted light passed through a silicon and aninterference filter and a monochromator before detection with aNIR Photomultiplier (Hamamatsu Co. R5509). The singlet oxygenlifetime was determined by applying first-order exponential fittingto the curve of the phosphorescence decay. In all the experimentsthe absorbance at 532 nm was kept around 0.3 (determined in aShimadzu spectrophotometer UV-240 PC).

2.2.6. Resonance light scattering (RLS)Spectrofluorometer operating in synchronous mode

(�exc = �emis) was used to characterize the formation of

10 D.S. Pellosi et al. / Journal of Photochemistry and Photobiology A: Chemistry 247 (2012) 8– 15

SDS

FSC: X = H; Y = H

EOS: X = Br; Y = H

ERY: X = I; Y = H

RBB: X = I; Y = Cl

CTAB

O

X

X

X

O

X

HO

Y

Y

Y

Y

COOH

OCH2CH 2 CHH 2 CHCHO 2CH 2O

CH3

Hx

y

x

P-123: EO20PO70EO20

in B (E

se

2

st0

3

wiCpa0t

a7E1

3

T

Tatwaa

conversion processes; these contacts diminish inside the micellepseudo-phase increasing fluorescence yields [47]. Therefore, theobserved wavelength spectral shifts (�max), despite small, are asso-ciated with the partitioning of the dyes from water onto the micelle.

Table 2Partition coefficient (Kp) of dyes in water/octanol (1:1, v/v) at 30.0 ◦C.

Dyesa FSC EOS ERY RBB

POEO

Fig. 1. Xanthenes: Fluorescein (FSC), Eosin Y (EOS), Erythros

elf-aggregates of the dyes [39]. The experimental conditionsmployed were the same as the fluorescence measurements.

.2.7. Critical micellar concentration (CMC)The CMC for CTAB and SDS were obtained by surface ten-

ion measurements (DuBois Tensiometer) at 30.0 ◦C in water withhe pH 7.25 (McIlvaine buffer) and the ionic strength fixed with.10 mol L−1 of NaCl.

. Results and discussion

The physical–chemical characterization of xanthene dyes inater and aqueous surfactants solutions of SDS, CTAB and Pluron-

cs P-123 were investigated at physiological pH conditions. TheMC of the surfactants obtained in this conditions at 30.0 ◦C in theresence of [NaCl] = 0.10 mol L−1 were 1.10 × 10−3 mol L−1 for SDSnd 2.00 × 10−4 mol L−1 for CTAB. For P-123 the literature value is.9 × 10−5 mol L−1 [40]. The absorption and fluorescence spectra ofhe xanthenes dyes are depicted in Fig. 2.

The wavelengths corresponding to absorbance and the molarbsorption maxima (�max, ε�max) at pH = 7.25 are: FSC (490 nm,6.9 × 103 L mol−1 cm−1); EOS (517 nm, 97.1 × 103 L mol−1 cm−1);RY (532 nm, 96.6 × 103 L mol−1 cm−1) and RBB (543 nm,09.0 × 103 L mol−1 cm−1) [36].

.1. Partition coefficient (Kp) in octanol/water phases

The distribution of the dyes represented by Log Kp is listed inable 2.

The observed decrease in Log Kp values (RBB > ERY > EOS > FSC,able 2) follows the decrease in the volume of the substituenttoms (Fig. 1). As established in the literature [41] photosensitizer

hat presents Log Kp < 0 exhibits hydrophilic characteristics (FSC)hile Log Kp > 1.5 lipophilic characteristics. For values between 0

nd 1.5, the compounds are assigned as having amphiphilic char-cter, which is the case for EOS, ERY and RBB. Despite dianionic,

EO

RY), Rose Bengal B (RBB). Surfactants: SDS, CTAB and P-123.

these halo-xanthenes present high external surface areas (highvolumes) due to the presence of the halogen substituents, whichin turn induces high polarizability effects. Additionally the largeelectron cloud affects the �-conjugation system, delocalizing thenegative charge of the phenolate anion that can spread to the entirexanthene ring resulting in a charge density decrease [32]. These fea-tures turn halo-xanthenes less hydrophilic than a simple dianion.

3.2. Interaction of xanthene dyes with surfactants

In Fig. 3A and B the fluorescence spectra of RBB as the surfactantconcentration is increased are illustrated.

There is a red shift in the spectra when the media changedfrom water to micelles, especially with RBB. For all xanthenesinvestigated in the presence of surfactant the �emis shifted around8–10 nm in P-123 and 12–15 nm in CTAB (the range depends onthe dye). This emission peak displacement was not detected inSDS. Similar fluorescence red shifts were already reported for xan-thenes in the presence of micelles [31,42–45] and organic solvents[46]. These red shifts are explained by the loss of the stabilizationof the fundamental state of the dye in protic media by hydro-gen bonding and consequent diminution of the electronic gap[45]. In addition, the hydrogen bonds in water media sustainseveral xanthene–water molecules contacts, which favor internal

Log Kp −0.32 0.18 0.46 0.59

a Water phase at pH 7.25 with McIlvaine buffer ([Na2HPO4] = [citricacid] = 7.5 × 10−3 mol L−1) and [NaCl] = 0.10 mol L−1. [Dyes] = 3.00 × 10−6 mol L−1.

D.S. Pellosi et al. / Journal of Photochemistry and Photobiology A: Chemistry 247 (2012) 8– 15 11

750700650600550500

0

100

200

300

400

500

600

700

800

Em

issio

n (

a. u

.)

Wavelength (nm)

(B) a

bc

d

700650600550500450400350

0.0

0.1

0.2

0.3

0.4

0.5

0.6

Ab

so

rba

nce

Wavelength (nm)

(A)

a

b

c d

Fig. 2. Spectra of xanthene dyes in water, pH 7.25: (A) absorption (5 × 10−6 mol L−1) and (B) fluorescence emission (5.0 × 10−7 mol L−1). (a) FSC (�exc = 460 nm); (b) EOS(�exc = 490 nm); (c) ERY (�exc = 500 nm); and (d) RBB (�exc = 510 nm). Monochromator slits (exc/emis) for FSC (5/5, nm/nm) and for the halo-xanthenes (10/5, nm/nm).

700650600

0.0

0.2

0.4

0.6

0.8

1.0R

ela

tive

Em

issio

n

Wavelength (nm)

(B)

700650600550

0.0

0.2

0.4

0.6

0.8

1.0

Re

lative

Em

issio

n

Wavelenght (nm)

(A)

Fig. 3. Relative fluorescence emission spectra of RBB (5.0 × 10−7 mol L−1) during titimetric addition of: (A) P-123 and (B) CTAB. The normalization was done using RBB/CTAB(Fig. 3B) system as reference. pH 7.25 ([Na2HPO4] = [citric acid] = 7.5 × 10−3 mol L−1), [NaCl] = 0.10 mol L−1 at 30.0 ◦C and �exc = 510 nm. The arrow indicates the intensity assurfactant was added.

Fig. 4. Normalized emission intensity as a function of surfactants addition for: (A) FSC and (B) RBB. The normalization was done using RBB/CTAB system(Fig. 4B) as reference. [Dyes] = 5.0 × 10−7 mol L−1. pH 7.25 ([Na2HPO4] = [citric acid] = 7.5 × 10−3 mol L−1) and [NaCl] = 0.10 mol L−1 at 30.0 ◦C. (CMCCTAB = 2.00 × 10−4 mol L−1;CMCSDS = 1.10 × 10−3 mol L−1; CMCP-123 = 0.9 × 10−5 mol L−1). The full lines are related with theoretical fittings using Eq. (2).

12 D.S. Pellosi et al. / Journal of Photochemistry and Photobiology A: Chemistry 247 (2012) 8– 15

0.0 1.0x10-4 2.0x10

-43.0 x10

-44.0x 10

-4

0.0

0.2

0.4

0.6

0.8

1.0

Fluorescence

RLS signal

Re

lative

em

issio

n

CTAB (mol L-1)

Fig. 5. Variations of fluorescence for RBB (5.0 × 10−7 mol L−1) as a function ofC(A

tdt

nmwt

udv

tadsp

tctCwctntrrc“ss

ttmaRs(d

Table 3Dye/surfactant binding constant (Kb) calculated by Eq. (2) for aqueous CTAB andP-123 systems.

Kba FSC EOS ERY RBB

P-123 (103 L mol−1) 2.8 (0.991)b 8.0 (0.999) 13.3 (0.999) 14.3 (0.998)CTAB (105 L mol−1) 1.9 (0.997) 8.5 (0.993) 13.3 (0.992) 16.2 (0.999)

ent CMC” exhibits values lower than the CMC. Similar resultswere obtained for methylene blue and SDS system [53]. Addi-tionally the order of the decrease in this “apparent CMC” follows,also again, the sequence of the hydrophobic character of the

Table 4Values of “apparent CMC” and N resulted from the titimetry of the xanthene dyesby CTAB, applying Eq. (2).

FSa Apparent CMC (10−4 mol L−1) N

FSC 1.3 ± 0.1 1.6 ± 0.4

TAB addition (Fig. 4 in more detail to CTAB system): fluorescence intensity�emis = 571 nm) and RLS signal monitored at 560 nm. The arrow indicates the CMC.t 30.0 ◦C and pH 7.25.

The curves of fluorescence intensity for FSC and RBB as a func-ion of surfactant concentrations are exhibited in Fig. 4. These twoyes are representative of the behavior of the xanthenes here inves-igated.

It is observed that the addition of SDS (even above its CMC) doot change the emission intensities and the wavelength emissionaxima suggesting that the xanthene derivatives do not interactith this micelle, specially at the head group region probably due

o charge repulsion.In P-123 and CTAB, RBB showed the highest intensity variation

pon surfactant addition (Fig. 4B), which is evidence of significantye–surfactant interaction while FSC (Fig. 4A) have the smallestariations in both surfactants.

The overall behavior of the dyes among these surfactants showshat there are no variations in SDS, variations in P-123 and high vari-tions in CTAB micelles. The electrostatic attractive forces mainlyrive the interaction with CTAB, while for P-123 (non-chargedurfactant) by hydrophobic forces. However, for CTAB system therofile was not so simple (Fig. 5).

For xanthenes at the beginning of CTAB titimetry it is observedhat fluorescence emission decreases. The presence of oppositeharges in the surfactant monomer and dyes drives the interac-ion. A “minimum intensity” is observed well below the CMC of theTAB, followed by an increased fluorescence as more surfactantas added (Fig. 5) characterizing a biphasic interaction. Similar

hanges were observed by other researchers working with xan-henes and cationic micelles below their CMC, which region isamed as pre-micellar region [48,49]. According to these authors,his region is dominated by “pre-micellar aggregates” or “dye-ich-micelles” species formed by [X2−(S+)n], where X2− and S+

epresents the xanthene and CTA+ molecules, respectively. Thelose proximity of xanthene molecules inside these probably tinypre-micellar aggregates” (present as dimers, trimers and othersmall dye self-aggregate) decreased the fluorescence emission byelf-suppression processes [50,51].

Monitoring the dye–CTAB system by RLS at low CTAB concen-ration, it is observed a peak at 560 nm. This RLS signal is due tohe resonance among their transition dipole moment of each chro-

ophore unit that composes the self-aggregates (or “pre-micellarggregates”) [39,52]. At the beginning of the titimetry followed by

LS, the signal goes through maxima with increasing [CTAB] at theame region of the “minimum intensity” in the emission spectraFig. 5). The subsequent addition of surfactant leads to the RLS signalecrease and at the same time the fluorescence emission increases,

a [Dyes] = 5.0 × 10−7 mol L−1, pH 7.25 and [NaCl] = 0.10 mol L−1 at 30.0 ◦C.b Linear regression coefficients.

which reflects the dye–dye monomerization processes by inclu-sion of surfactant molecules in these pre-micelles. This pre-micellarregion appears in surfactant concentrations well below the normalCMC of CTAB (10−4 mol L−1) evidently caused by the formation of[X2−(S+)n] species that acts as a nucleation sites for the formationof micelles [53–55].

The decrease magnitude in the fluorescence intensities fol-lows the order FSC < EOS < ERY < RBB. This sequence parallels thehydrophobic characteristics of the dyes (Table 2). Thus, the pres-ence of the conjugate species [X2−(S+)n] evidently is dependent notonly on the charge effect but also on the xanthenes HLB parameter.

The binding constants were estimated from the minimum inthe fluorescence intensity towards its maximum using Eq. (2). Theresults are presented in Table 3 [54,56].

The model represented by Eq. (2) resulted in good adjustmentsof the data (Table 3 and Fig. 4). Although the dianionic xanthenesare soluble in water, they show relatively high affinity to P-123 andCTAB micelles. The Kb found are only 12–4 times smaller than thevalues obtained for chlorophyll derivatives (very high hydrophobicmolecules) measured under similar conditions in micellar media[4]. The relative low Kb values for FSC in both micellar systemsreflect its low hydrophobicity (Table 3). The order of Kb values con-firms the same sequence of the xanthenes HLB parameter (Table 2,Kp).

The Kb of CTAB are about 2 orders of magnitude higher thanthose values obtained in P-123 (non-charged micelle), which isjustified by the electrostatic attraction dye–surfactant with the for-mation of pre-micelles. Values of Kb in the same order of magnitudewere obtained for the interaction of rhodamines (cationic xanthenedye) with vesicles of anionic and zwitterionic DPPS and DPPG [56].

For P-123 system, all fitting (Eq. (2)) had the value of N around1, thus it is necessary one micelle to stabilize one dye molecule.However in CTAB micelles attempts to fix N = 1 (which represents asimple balance) and/or to impose its true CMC leads to poor fitting,which pointed out the complexity of this system. Similar interpre-tation was reported by Caetano and Tabak [54] that studied biphasicinteraction of chloropromazine and trifluorperazine in aqueousSDS. In fact, the presence of hydrophobic nucleation species (such asthe [X2−(S+)n] species) induces the micellization process and leadsto an “apparent CMC” obtained from Eq. (2) (Table 4).

For CTAB, the experimentally measured CMC by surface tensionis 2.00 × 10−4 mol L−1. However, as it is expected the “appar-

EOS 0.9 ± 0.1 1.7 ± 0.5ERY 0.8 ± 0.1 1.7 ± 0.4RBB 0.6 ± 0.2 1.8 ± 0.5

a [Dyes] = 5.0 × 10−7 mol L−1, pH = 7.25, 30.0 ◦C and [NaCl] = 0.10 mol L−1.

D.S. Pellosi et al. / Journal of Photochemistry and Photobiology A: Chemistry 247 (2012) 8– 15 13

Table 5KSV values and the (correlation coefficient) results. It is included the percentage difference between the KSV values obtained in water (reference) against in surfactantssolutions (�).

FS KSV (L mol−1) water KSV (L mol−1) SDSa KSV (L mol−1) P-123b KSV (L mol−1) CTABc

FSC 8.52 (0.999) 8.44 (0.998) � 1% 8.07 (0.998) � 5% 36.75 (0.992) � 331%EOS 2.84 (0.999) 2.71 (0.998) � 5% 2.22 (0.998) � 22% 23.09 (0.993) � 713%ERY 0.68 (0.998) 0.14 (0.998) � 79% 0.34 (0.997) � 50% 21.62 (0.997) � 3079%RBB 1.92 (0.999) 0.44 (0.997) � 77% 0.36 (0.998) � 81% 30.26 (0.995) � 1476%

[ rfactanc

[CMC

xi

(ef

3

itd

cie(f

fmicmawitpi(httm

fKe

wtEwfsnwt(pbme

effective presenting lower coupling efficiency.Results of ˚�

1O2 (Table 6) showed that for EOS the loca-tion inside the micelle diminishes the 1O2 formation, ˚�

1O2:

Table 6Fluorescence (˚F) and singlet oxygen (˚�

1O2) quantum yield for xanthene dyes.

FS ˚Fa ˚�

1O2b

Water SDSc P-123d CTABe Water SDSc P-123d CTABe

FSC 0.79 0.76 0.82 0.92 – – – –EOS 0.20 0.21 0.38 0.57 0.59 0.54 0.38 0.33ERY 0.02 0.03 0.08 0.07 0.62 0.64 0.67 0.58RBB 0.02 0.02 0.17 0.14 0.92b 0.76 0.81 0.75

Dyes] = 5.0 × 10−7 mol L−1, pH 7.25, [NaCl] = 0.10 mol L−1 and 30.0 ◦C. Su[CTAB] = 4.00 × 10−3 mol L−1. For SDS and CTAB the concentrations were around 5×

anthenes, reinforcing its important contribution in dye–surfactantnteraction.

The values of CMC for P-123 systems, also extracted from Eq.2), are similar with the literature that pointed out that low pres-nce of the dye molecules in the solution does not affect its micelleormation process for this polymeric surfactant.

.3. The KSV determinations

Iodide ion was chosen as a water species quencher. In Fig. 6 it isllustrated the Stern–Volmer plot for the systems investigated. Allhe Stern–Volmer Quenching Constants KSV (from Eq. (3) and Fig. 6ata) are listed in Table 5.

The data in Fig. 6 show linear dependence upon the quencheroncentration except for CTAB system where a small curvatures observed (Fig. 6D, experiments where low iodide amount wasmployed). The correlation coefficients from Stern–Volmer plotsTable 5) confirm the linearity for all investigated systems exceptor CTAB (again an especial case).

Clearly, the suppression of energy of the excited state by iodideor FSC is more effective. This dye (highest ˚F among xanthenes)

ostly remains in water media instead inside the micelle, exceptn CTAB. The literature [57] pointed out that the suppression pro-ess in xanthenes is due to dynamic quenching (collisions) in wateredia. Our experiments demonstrated that the KSV values increase

s the temperature increases in SDS and P-123 media (not shown),hich reinforce the suggestion of dynamic quenching. However,

n the especial case of CTAB there was a decrease in KSV values ashe temperature is raised, which pointed out a static quenchingarticipation probably due to the electrostatic attraction of both,

odide and xanthenes anions, to the positive charged CTAB micellesprobably by a sphere of action mechanism). As consequence, muchigher values of KSV were observed in CTAB than in the other sys-ems (Table 5). So the lack of linearity and the high values of KSV onhe CTAB media probably are due to the participation of the static

echanism allied to the dynamic quenching.For all systems investigated the KSV decrease as: FSC > EOS > ERY

ollowing the order of Kp and Kb except for RBB, in which highSV values probably are due to the chlorine atoms presence (stronglectron-withdrawing substituent).

The close values of KSV for FSC (and EOS) in SDS compared inater confirmed no significant dye–micelle interaction (no more

han � = 5%, Table 5), as seen in Kb experiments. However, forRY and RBB the KSV in SDS is much lower than the values inater, respectively, � 79 and 77% (∼4.5× times folder). There-

ore, both ERY and RBB are partially localized inside the dodecylulfate micelles, however, the binding constant experiments wereot capable to detect it. Probably the dye–SDS interaction, despiteeak, is enough to keep ERY and RBB molecules in the Stern layer of

he micelle partially protecting these dyes from the iodide approachby iodide–SDS electrostatic repulsion). The dye–SDS interaction

robably is due to the presence of large iodine substituents inoth dyes turning the negative charges spread all over the entireolecule (charge density decrease) allied to high polarizability

ffects (large van der Walls forces) [32].

t concentration: a[SDS] = 5.00 × 10−3 mol L−1; b[P-123] = 1.70 × 10−3 mol L−1;], while for P-123 was 180× [CMC].

In the case of P-123 micelles the excited dyes also suffer lowerquenching by the iodide compared in water. This decrease in �(Table 5) follows: FSC < EOS < ERY < RBB, which result matches withthe Kp (Table 2) and Kb (Table 3) sequence. So that, while FSC isalmost not affected by P-123, the other xanthenes are positionedinside these micelles.

3.4. The photophysical properties

Photophysical properties as fluorescence and singlet oxygen for-mation are essential parameters to photodynamic evaluation. Theexperimentally obtained values of fluorescence quantum yield (˚F)and quantum yield of singlet oxygen (˚�

1O2) in water and aqueousmicellar systems are listed in Table 6.

Due to the extremely short lifetime of excited FSC, the inter-system crossing is very unlikely [53]. Substitution of hydrogensby halogens in aromatic molecules, such as in halo-xanthenes,favors the inter-system crossing through the spin–orbit coupling[44–46]. Usually high values of ˚�

1O2 imply in ˚F diminution(Table 6).

The value obtained by us for ˚F (0.79) of FSC in water at pH7.25 was lower than the ˚F of the FSC used as standard at pH 12(˚F = 0.92 [38]). The reason is that at pH 12 all molecules of FSC areas dianionic form, the most fluorescent species [58], while at pH7.25 part of the molecules are present as less fluorescent monoan-ionic form (FSC pKa = 6.1 in water media [33]). The same behavioris observed for FSC in SDS micelles.

In more hydrophobic environment such as in micelle core, thexanthene dyes exhibit higher ˚F (Table 6) as previously discussed[38]. In P-123 the ˚F was smaller than those in CTAB, but higherthan in SDS (and water) for FSC and EOS. For ERY and RBB ˚Fs weresimilar in P-123 and CTAB systems.

In water and in SDS surfactant system, the resulted ˚F and˚�

1O2 were approximately equal, once the dianionic speciesexhibit low interaction to anionic micelles. Among the xanthenesthe most efficient 1O2 photo-generator are ERY and mainly RBB(iodine atoms as substituent) while EOS (bromine atoms) is less

Measurements at pH 7.25, [NaCl] = 0.10 mol L−1 at 30.0 ◦C. a˚F obtained using asstandard the same dye in water at pH 9.2 [53]. bRBB used as standard, mea-surement of 1O2 phosphorescence at 1270 nm. Aqueous surfactant concentration(all above their CMC): c[SDS] = 5.00 × 10−3 mol L−1; d[P-123] = 1.70 × 10−3 mol L−1;e[CTAB] = 4.00 × 10−3 mol L−1.

14 D.S. Pellosi et al. / Journal of Photochemistry and Photobiology A: Chemistry 247 (2012) 8– 15

0.180.150.120.090.060.030.00

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

F0/F

Iodide (mol L-1)

FSC

EOS

ERY

RBB

(C)

0.0 5.0x10-4

1.0x10-3

1.5x10-3

2.0x10-3

1.00

1.02

1.04

1.06

1.08

F0/F

Iodide (mol L-1)

FSC

EOS

ERY

RBB

(D)

0.180.150.120.090.060.030.00

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

F0/F

Iodide (mol L-1 )

FSC

EOS

ERY

RBB

(A)

0.180.150.120.090.060.030.00

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

F0/F

Iodide (mol L-1)

FSC

EOS

ERY

RBB

(B)

Fig. 6. Stern–Volmer plots for xanthenes (5.0 × 10−7 mol L−1) in aqueous pH 7.25 ([Na2HPO4] = [citric acid] = 7.5 × 10−3 mol L−1) and [NaCl] = 0.10 mol L−1 at 30.0 ◦C in: (A)w

woimiti

4

brhobsotfaT˚tca

ater; (B) SDS; (C) P-123; and (D) CTAB.

ater > SDS > P-123 > CTAB which sequence follows the same orderf Kb. For ERY and RBB, still high values of ˚�

1O2 are obtainedn SDS, CTAB and especially in P-123, showing that the new

icroenvironment inside these micelles does not affect thentersystem crossing to triplet state. Therefore, 1O2 produc-ion remains high for ERY and RBB in all surfactant solutionsnvestigated.

. Conclusions

The ability of the dianionic protolytic form of xanthene dyes toe positioned into micelle is dependent on their HLB parameter rep-esented by Kp, which was dependent on the number and size of thealogen atom bounded to the xanthene ring. In fact, the extensionf the dye–micelle interaction not only depends on the hydropho-icity of the xanthene but also the micellar surface potential. Theequence of Kb in P-123 and CTAB micelles obeys exactly the samerder as Kp. The KSV data support the localization of the dye withinhe micelle. The intensity of the dye–micelle interaction increasedrom SDS, P-123 and finally CTAB surfactants due to the chargesnd among the photosensitizers, the hydrophobic characteristics.he experimentally obtained values of ˚F are consistent with the

�

1O2. Inside the micelles, ERY and RBB still are very efficient pho-osensitizers. The results in P-123 suggest that this micellar systeman be applied as drug delivery systems with ERY and RBB in PDTnd/or PDIMO.

Acknowledgments

This work was sponsored by the Brazilian funding agenciesFundac ão Araucária and CNPq (processes: 481804/2010-2 and400618/2004-4 Rede NbioNet).

References

[1] F.I. Simplicio, F. Maionchi, N. Hioka, Terapia Fotodinâmica: AspectosFarmacológicos Aplicac ões e Avanc os Recentes no Desenvolvimento deMedicamentos, Quimica Nova 25 (2002) 801–807.

[2] J.C. Kennedy, Introduction, in: R. Pottier, B. Krammer, H. Stepp, R. Baumgartner(Eds.), Photodynamic Therapy with ALA: A Clinical Handbook, RSC Publishing,Cambridge, 2006.

[3] L.S. Peloi, C.E.G. Biondo, E. Kimura, M.J. Politi, M.V.C. Lonardoni, S.M.A. Aris-tides, R.C.C. Dorea, N. Hioka, T.G.V. Silveira, Photodynamic therapy for Americancutaneous leishmaniasis: the efficacy of methylene blue in hamsters experi-mentally infected with Leishmania (Leishmania) amazonensis, ExperimentalParasitology 128 (2011) 353–356.

[4] A.P. Gerola, A. Santana, P.B. Franc a, T.M. Tsubone, H.P.M. de Oliveira, W.Caetano, E. Kimura, N. Hioka, Effects of metal and the phytyl chain on chloro-phyll derivatives: physicochemical evaluation for photodynamic inactivationof microorganisms, Photochemistry and Photobiology 87 (2011) 884–894.

[5] C.S. Foote, E.L. Clennan, Properties and reactions of singlet dioxygen, in: ActiveOxygen in Chemistry, Chapman & Hall, London, 1995.

[6] T.S. Mang, Lasers and light sources for PDT: past, present and future, Photodi-agnosis and Photodynamic Therapy 1 (2004) 43–48.

[7] E.J. Dennis, D.E. Dolmans, D. Fukumura, R. Jain, Photodynamic therapy for can-cer, Nature 3 (2003) 380–387.

[8] D. Gabrielli, E. Belisle, D. Severino, A. Kowaltowski, M.S. Baptista, Binding,aggregation and photochemical properties of methylene blue in mitochondrialsuspensions, Photochemistry and Photobiology 79 (2004) 227–232.

y and

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

D.S. Pellosi et al. / Journal of Photochemistr

[9] T.N. Demidova, M.R. Hamblin, Photodynamic therapy targeted to pathogens,International Journal of Immunopathology and Pharmacology 17 (2004)245–254.

10] J.R. Perussi, Photodynamic inactivation of microorganisms, Quimica Nova 30(2007) 988–994.

11] E.D. Sternberg, D. Dolphin, C. Brückner, Porphyrin-based photosensitizers foruse in photodynamic therapy, Tetrahedron 54 (1998) 4151–4202.

12] A. Chakrabarty, P. Das, A. Mallick, N. Chattopadhyay, Effect of surfactant chainlength on the binding interaction of a biological photosensitizer with cationicmicelles, Journal of Physical Chemistry B 112 (2008) 3684–3692.

13] J.P. Tardivo, A.D. Giglio, C.S. Oliveira, D.S. Gabrielli, H.C. Junqueira, D.B. Tada,D. Severino, R.F. Turchiello, M.S. Baptista, Methylene blue in photodynamictherapy: from basic mechanisms to clinical applications, Photodiagnosis andPhotodynamic Therapy 2 (2005) 175–191.

14] G.L. Know, Polymeric Drug Delivery Systems, Taylor & Francis Group, BocaRaton, FL, 2005.

15] B.A. Previdello, F.R. Carvalho, A.L. Tessaro, V.R. Souza, N. Hioka, O pKa de indi-cadores ácido-base e os efeitos de sistemas coloidais, Quimica Nova 29 (2006)600–606.

16] C.A. Suchetti, E.N. Durantini, Monomerization photodynamic activity of Zn(II)tetraalkyltetrapyridinoporphyrazinium derivatives in AOT reverse micelles,Dyes and Pigments 74 (2007) 630–635.

17] H. Waterbeemd, Physico-chemical approaches to drug absorption, in: H. Water-beemd, H. Lennernas, P. Artursson (Eds.), Drug Bioavailability Estimationof Solubility, Permeability, Absorption and Bioavailability, Wiley, Weinheim,2004.

18] J.H. Fendler, Membrane Mimetic Chemistry, Wiley-Interscience, New York,1982.

19] B.M. Aydin, M. Acar, M. Arik, Y. Onganer, The fluorescence resonance energytransfer between dye compounds in micellar media, Dyes and Pigments 81(2009) 156–160.

20] M. Firczuk, D. Nowis, J. Golab, PDT-induced inflammatory and host responses,Photochemistry and Photobiology Science 10 (2011) 653–663.

21] V.P. Torchilin, Structure and design of polymeric surfactant based drug deliverysystems, Journal of Controlled Release 73 (2001) 137–172.

22] C.J.F. Rijcken, O. Soga, W.E. Hennink, C.F. Nostrum, Triggered destabilization ofpolymeric micelles and vesicles by changing core polarity: a new tool for drugdelivery, Journal of Controlled Release 120 (2007) 131–148.

23] T. Tsai, Y.T. Yang, T.H. Wang, H.F. Chien, C.T. Chen, Improved photodynamicinactivation of gram-positive bacteria using hematoporphyrin encapsulated inliposomes and micelles, Lasers in Surgery and Medicine 41 (2009) 316–322.

24] H. Ding, B.D. Sumer, C.W. Kessinger, Y. Dong, G. Huang, D.A. Boothman, J.Gao, Nanoscopic micelle delivery improves the photophysical properties andefficacy of photodynamic therapy of protoporphyrin IX, Journal of ControlledRelease 151 (2011) 271–277.

25] M. Yokoyama, Polymeric micelles for the targeting of hydrophobic drugs, in:Polymeric Drug Delivery Systems, Taylor & Francis Group, Boca Raton, FL, 2005.

26] W. Loh, Block Copolymer Micelles, Encyclopedia of Colloid and Surface Science,New York, 2002.

27] A.V. Kabanov, J. Zhu, Pluronic block copolymers for drug and gene delivery, in:Polymeric Drug Delivery Systems, Taylor & Francis, Boca Raton, 2005.

28] G.K. Walkup, S.C. Burdette, S.J. Lippard, R.Y. Tsien, A new cell-permeable flu-orescent probe for Zn2+, Journal of the American Chemical Society 122 (2000)5644–5645.

29] A. Saha, S.K. Basiruddin, R. Sarkar, N. Pradhan, N.R. Jana, Functionalizedplasmonic-fluorescent nanoparticles for imaging and detection, Journal ofPhysical Chemistry C 113 (2009) 18492–18498.

30] D.C. Neckers, R. Bengal, Journal of Photochemistry and Photobiology A 47 (1989)1–29.

31] A. Song, J. Zhang, M. Zhang, T. Shen, J. Tang, Spectral properties and structureof fluorescein and its alkyl derivatives in micelles, Colloids and Surfaces 167(2000) 253–262.

32] X. Zhang, I. Zhang, L. Liu, Photophysics of halogenated fluoresceins: involve-ment of both intramolecular electron transfer and heavy atom effect in thedeactivation of excited states, Photochemistry and Photobiology 86 (2010)492–498.

33] V.R. Batistela, J.C. Cedran, H.P.M. Oliveira, I.S. Scarminio, L.T. Ueno, A.E.H.Machado, N. Hioka, Protolytic fluorescein species evaluated using chemometryand DFT studies, Dyes and Pigments 86 (2010) 15–24.

34] T. Miura, Y. Urano, K. Tanaka, T. Nagano, K. Ohkubo, S. Fukuzumi, Rational designprinciple for modulating fluorescence properties of fluorescein-based probes

[

[

Photobiology A: Chemistry 247 (2012) 8– 15 15

by photoinduced electron transfer, Journal of the American Chemical Society125 (2003) 8666–8671.

35] E. Gandin, Y. Lion, A. Van de Vorst, Quantum yield of singlet oxygen productionby xanthene derivatives, Photochemistry and Photobiology 37 (1983) 271–278.

36] V.R. Batistela, D.S. Pellosi, F.D. Souza, W.F. Costa, S.M.O. Santin, V.R. Souza,H.P.M. Oliveira, I.S. Scarminio, N. Hioka, pKa determinations of xanthenederivates in aqueous solutions by multivariate analysis applied to UV–Vis spec-trophotometric data, Spectrochimica Acta, Part A 79 (2011) 889–897.

37] W. Caetano, M. Tabak, Interaction of chlorpromazine and trifluoperazine withionic micelles: electronic absorption spectroscopy studies, SpectrochimicaActa, Part A 55 (1999) 2513–2528.

38] G.R. Fleming, A.W.E. Knight, J.M. Morris, R.J.S. Morrison, G.W. Robinson, Picosec-ond fluorescence studies of xanthene dyes, Journal of the American ChemicalSociety 99 (1977) 4306–4311.

39] R.F. Pasternack, C. Bustamante, P.J. Collings, A. Giannetto, E.J. Gibbs, Porphyrinassemblies on DNA as studied by a resonance light scattering technique, Journalof the American Chemical Society 115 (1993) 5393–5399.

40] P. Alexandridis, J.F. Holzwarth, T.A. Hatton, Micellization of poly(ethyleneoxide)–poly(propylene oxide)–poly(ethylene oxide) triblock copolymers inaqueous solutions: thermodynamics of copolymer association, Macro-molecules 27 (1994) 2414–2425.

41] H. Waterbeemd, Pharmacokinetics and Metabolism in Drug Design, Wiley-VCHVerlag GmbH & Co. KGaA, Weinheim, 2001.

42] M. Chakraborty, A.K. Panda, Spectral behaviour of Eosin Y in different solventsand aqueous surfactant media, Spectrochimica Acta, Part A 81 (2011) 458–465.

43] C.C. Chang, Y.T. Yang, J.C. Yang, H.D. Wu, T. Tsai, Absorption and emission spec-tral shifts of Rose Bengal associated with DMPC vesicles, Dyes and Pigments 79(2008) 170–175.

44] B.B. Bhowmik, P. Ganguly, Photophysics of xanthene dyes in surfactant solution,Spectrochimica Acta, Part A 61 (2005) 1997–2003.

45] J. Kibblewhite, G.G. Drummond, F. Grieser, P.J. Thistlethwaite, Lipoidal Eosinand fluorescein derivatives as probes of the electrostatic characteristics of self-assembled surfactant/water interfaces, Journal of Physical Chemistry 93 (1989)7464–7473.

46] N.O. Mchedlov-Petrossyan, O.N. Tychina, T.A. Berezhnaya, V.I. Alekseeva, L.P.Savvina, Ionization and tautomerism of oxyxanthene dyes in aqueous butanol,Dyes and Pigments 43 (1999) 33–46.

47] N. Martin, Hydrogen bond effects on radiationless electronic transitions in xan-thene dyes, Chemical Physics Letters 35 (1975) 105–111.

48] P. Bilski, R. Dabestani, C.F. Chignell, Influence of cationic surfactant on the pho-toprocesses of Eosin and Rose Bengal in aqueous solution, Journal of PhysicalChemistry 95 (1991) 5784–5791.

49] P. Bilski, R.N. Holt, C.F. Chignell, Premicellar aggregates of Rose Bengal withcationic and zwitterionic surfactants, Journal of Photochemistry and Photobi-ology A 110 (1997) 67–74.

50] N. Micali, V. Villari, A. Romeo, M.A. Castriciano, L.M. Scolarro, Light scatteringenhancement in an aqueous solution of spermine-induced fractal j-aggregatecomposite, Physical Review E 76 (2007), n.011404-1R.

51] M. Deumié, M. Elbaraka, Self-aggregation of R110 and R123 rhodamines withsurfactants and phospholipid vesicles of negative charge: a qualitative fluores-cence study, Journal of Photochemistry and Photobiology A 74 (1993) 255–266.

52] P.J. Collings, E.J. Gibbs, T.E. Starr, O. Vafek, C. Yee, L.A. Pomerance, R.F.Pasternack, Resonance light scattering and its application in determining thesize, shape, and aggregation number for supramolecular assemblies of chro-mophores, Journal of Physical Chemistry B 103 (1999) 8474–8481.

53] H.C. Junqueira, D. Severino, L.G. Dias, M.S. Gugliotti, M.S. Baptista, Modulationof methylene blue photochemical properties based on adsorption at aqueousmicelle interfaces, Physical Chemistry Chemical Physics 4 (2002) 2320–2328.

54] W. Caetano, M. Tabak, Interaction of chlorpromazine and trifluoperazine withanionic sodium dodecyl sulfate (SDS) micelles: electronic absorption and fluo-rescence studies, Journal of Colloid and Interface Science 225 (2000) 69–81.

55] N. Hioka, M.J. Politi, H. Chaimovich, Kinetic demonstration of pre-micellaraggregation. The alkaline hydrolysis of N-hexadecyl-4-cyanopyridinium bro-mide, Tetrahedron Letters 30 (1989) 1051–1054.

56] M. Deumié, P. Lorente, D. Morizon, Quantitative binding and aggregation ofR123 and R6G rhodamines at the surface of DPPG and DPPS phospholipid

vesicles, Journal of Photochemistry and Photobiology A 89 (1995) 239–245.

57] W.J. Svirbely, N.E. Sharpless, The quenching of the fluorescence of the eosin ion,Journal of the American Chemical Society 76 (1954) 1404–1409.

58] R. Sjöback, J. Nygren, M. Kubista, Absorption and fluorescence properties ofFluorescein, Spectrochimica Acta, Part A 51 (1995) L7–L21.