Photodegradation in Micellar Aqueous Solutions of Erythrosin Esters ...

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Strong light absorption and high levels of singlet oxygen produc- tion indicate erythrosin B as a viable candidate as a photosensitizer in photodynamic therapy or ...

Photodegradation in Micellar Aqueous Solutions of Erythrosin Esters Derivatives Leandro Silva Herculano,a,* Gustavo Vinicius Bassi Lukasievicz,b,* Elizandra Sehn,c Wilker Caetano,d Diogo Silva Pellosi,d Noboru Hioka,d Nelson Guilherme Castelli Astrath,e Luis Carlos Malacarnee a

Universidade Tecnolo´gica Federal do Parana´, Santa Helena, PR 85892-000, Brazil Universidade Tecnolo´gica Federal do Parana´, Toledo, PR 85902-490, Brazil c Universidade Tecnolo´gica Federal do Parana´, Medianeira, PR 85884-000, Brazil d Universidade Estadual de Maringa´, Departamento de Quı´mica, Maringa´, PR 87020-900, Brazil e Universidade Estadual de Maringa´, Departamento de Fı´sica, Maringa´, PR 87020-900, Brazil b

Strong light absorption and high levels of singlet oxygen production indicate erythrosin B as a viable candidate as a photosensitizer in photodynamic therapy or photodynamic inactivation of microorganisms. Under light irradiation, erythrosin B undergoes a photobleaching process that can decrease the production of singlet oxygen. In this paper, we use thermal lens spectroscopy to investigate photobleaching in micellar solutions of erythrosin ester derivatives: methyl, butyl, and decyl esters in low concentrations of non-ionic micellar aqueous solutions. Using a previously developed thermal lens model, it was possible to determine the photobleaching rate and fluorescence quantum efficiency for dye–micelle solutions. The results suggest that photobleaching is related to the intensity of the dye–micelle interaction and demonstrate that the thermal lens technique can be used as a sensitive tool for quantitative measurement of photochemical properties in very diluted solutions. Index Headings: Thermal lens; Erythrosin; Photodynamic therapy; Thermo-optical properties.

INTRODUCTION Photodynamic therapy (PDT) is a minimally invasive therapeutic modality specially applied for cancer treatment.1–4 Photodynamic therapy requires a photosensitizing drug (PS), light, and oxygen. The PS is activated by exposure to the light of a specific wavelength and interacts with oxygen molecules to produce reactive singlet oxygen.1–5 The singlet oxygen reacts readily with essential components of various biological systems, mainly biological membranes. The high quantum yields of singlet oxygen, the selectivity to the target, and the low aggregation in aqueous environments, are important prerequisites to the PSs that are drug candidates for PDT.1–5 The xanthene dyes, especially erythrosin B (ERY), have high molar absorption 6,7 (e 532nm = 96.6 3 103 L mol1 cm1) and high singlet oxygen quantum yield (0.62 in water) at physiological pH.6–9 Erythrosin B has been successfully tested as a photosensitizer in dental plaque treatment by photodynamic inactivation of microorganisms.9 Received 8 January 2015; accepted 19 February 2015. * Author to whom correspondence should be sent. E-mail: [email protected]; [email protected] DOI: 10.1366/15-07865

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Although erythrosine B has excellent photodynamic properties, its high hydrophilic characteristic causes a low interaction with biological membranes. Biological membranes are recognized preferential sites for photodynamic action; the photosensitizer–membrane interaction depends on the hydrophobic and electrostatic characteristics of the PS and the membrane.1,2 To overcome this problem and to increase the biological effects of the ERY, in a previous paper,7 the authors synthesized ester derivatives of ERY: methyl (ERYMET), butyl (ERYBUT), and decyl (ERYDEC) esters by the insertion of the alkyl group chain.6 The insertion of the alkyl group increases the hydrophobicity and cell interaction and does not affect the light absorption process.6,7 Although ERY ester derivatives can be strong candidates for use in PDT, the mechanisms of photodegradation or photobleaching presented in ERY aqueous or micellar solutions are far from being simple and, most often, unknown. The interest in the photobleaching of dyes has appeared over the past years with the advances of science and technology, and it is relevant in photomedicine and environmental protection.1–4 Time-resolved thermal lens (TL) spectroscopy has been demonstrated to be a highly sensitive technique to investigate photochemical and mass diffusion processes in hydrocarbon fuels, dyes, and micellar solutions and in the determination of thermal and optical properties in solid and liquid materials.10–19 The remote and nondestructive characteristics of TL make this technique an important tool for studying thermal, optical, and photophysical properties of low concentration solutions during light irradiation.16 In this paper, we apply the TL method to investigate the photobleaching processes of erythrosin B and its ester derivatives at Pluronics F-127 micellar solutions at low concentration. The polymeric surfactant Pluronics consists of hydrophilic ethylene oxide and hydrophobic propylene oxide block arranged in the triblock manner. They are often used as solubilizers, emulsifiers, or coating agents.20–22 These polymers are non-ionic, stable, and biocompatible. It was reported that drug encapsulation in Pluronics micelles can increase drug solubility and distribution and significantly enhanced the antifungal and antibacterial drugs.20 We used a TL theoretical model13 for quantitative determination of fluorescence quantum efficiency and

0003-7028/15/6907-0883/0 Q 2015 Society for Applied Spectroscopy



photobleaching reaction rate of low concentration dyes in micellar aqueous solutions.

THEORY The dual beam mode-mismatched TL configuration was used in this research. A continuous Gaussianshaped excitation laser beam is employed to produce a radial distribution of temperature inside the sample with the temporal evolution described by the thermal diffusion equation. The radial and temporal distribution of temperature induces a time-dependent thermal lens in the sample that acts as an optical element, causing a phase shift in the probe beam. The TL effect is monitored by measuring the intensity variation of the central portion of a Gaussian probe beam with the detector positioned in a far-field region. The theory describing the photobleaching effect in the TL experiments was recently developed.13 During laser excitation, the absorbed energy from the excitation beam can induce a chemical reaction, creating a timedependent optical absorption coefficient. In a very diluted solution, the time-dependent optical absorption coefficient can be expressed in terms of species concentration as b(r,t) = eRCR(r,t) þ ePCP(r,t), where eR and eP are the molar optical absorption coefficients of the reactants and products, respectively. The time-dependent concentration of reactant species is given by the solution of the differential equation @ 2 2 CR ðr ; tÞ  Dm r2 CR ðr ; tÞ ¼ Ke 2r =x0 e CR ðr ; t Þ @t

@ 2 2 T ðr ; t Þ  Dth r2 T ðr ; t Þ ¼ Q 0 ½ð1  eÞe KT t þ ee 2r =x0e @t ð2Þ in which Dth is the thermal diffusivity coefficient and Q0 ¼ 2Pe u=qcpx20e . Here, Pe is the optical power of excitation laser beam, q is the mass density, c is the specific heat, and u is the fraction of the absorbed energy available for conversion to heat. Using the integral transform method to solve Eq. 2, the tempera-

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þ ð1  eÞe KT t




0 1 2  x2r2 e KT s 0e A d s ð3Þ [email protected] 1 þ 2s 1 þ 2s tc tc

The characteristic thermal time constant is tc ¼ x20e =4Dth . The temperature distribution induces a thermal lensing that acts as an optical element, causing a phase-shift U to the probe beam. For a fluid, the phase shift is given in terms of the temperature gradient by U(r,t) = (2pL/kP)[(dn/dT)T(r,t)  (dn/dCR)CR(r,t)].15,17 Here, (dn/dT) and (dn/dCR) are the temperature and concentration coefficients of the refractive index at the probe beam wavelength (kP), and L is the sample thickness. Based in the on–off transient analysis with different pumping wavelengths, the concentration gradient contribution on the induced phase shift could be neglected; detailed discussion is provided in Refs. 13– 15. Using Eq. 3, the phase-shift can be described as Uðg; t Þ ¼


where Dm is the mass diffusion coefficient, K is the photobleaching rate, and x0e is the radius of excitation laser beam inside the sample. We assume the reaction process as a first- or pseudo-first-order reaction, in which the photoreaction process is CR(r,0) þ hv ! CR(r,0) CP(r,0), with the constraint that the sum of the concentration of the reactant and the concentration of the product are constant in time, CR(t) þ CP(t) = C0. An analytical solution to Eq. 1 is obtained assuming a spatial average of each term.13 This approximation results in a simple first-order equation. The timedependent optical absorption coefficient can be written as bðr ; t Þ ¼ b½ð1  eÞeKT t þ e, with b as the optical absorption coefficient of the sample at t = 0; KT represents the average rates of mass diffusion and photobleaching and e = eR/eP the ratio between the molar absorptivities of the reactant (eR) and product (eP). For a homogeneous, isotropic, and weakly absorbing material, the distribution of the temperature is described by the heat diffusion equation


ture distribution can be written as 0 1 2  x2r2 1 0e Ad s [email protected] T ðr ; t Þ ¼ Q0 1 þ 2s 1 þ 2s tc tc

h tc


Z 0


1  e 2mg=ð1þ2s=tc Þ ds 1 þ 2s tc


eÞe KT t



e KT s 0

! 1  e 2mg=ð1þ2s=tc Þ ds 1 þ 2s tc


where m = (x1P/x0e)2, x1P is the radius of the probe beam laser at the sample, g = (r/x1P)2, and h is defined as   Pe bL dn u ð5Þ h¼ k kP dT The intensity at the center of the probe beam in a far field can be calculated using the Fresnel diffraction theory14 by Z ‘ 2   ð1þiV ÞgiUðg;t Þ  e dg  ð6Þ Iðt Þ ¼  0

Here, V = Z1/Zc þ Zc/Z2[1 þ (Z1/Zc)2], where Zc is the confocal distance of the probe beam, Z1 is the distance from the probe beam waist to the sample, and Z2 is the distance from the sample position to the photodetector. Also, m and V are parameters of the experimental setup that were previously determined. Further, h, tc, e, and KT are obtained from regression analysis of the experimental data by using Eq. 6.

EXPERIMENTAL Figure 1 illustrates the thermal lens experimental setup used in this paper. The probe and excitation

FIG. 1. Schematic diagram of the time-resolved TL experiment apparatus. Mi, Li, and Pi denote mirrors, lenses, and photodiodes, respectively. The experimental parameters used in the TL experiment were: xe = 70.0 lm, m = 43.2, and V = 9.3.

beams are both continuous with TEM00 Gaussian intensity distribution. An argon ion laser at ke = 514.5 nm (Coherent, Model INNOVA 90) and a He–Ne laser kP = 632.8 nm (Melles Griot, Model 25-LHR-151-249) were used to pump and probe the samples, respectively. A mechanical shutter (Thorlabs, Model SH05) controlled the exposure time of the excitation laser beam at the sample. The excitation beam was focused on the sample using a lens with focal length L1 = 30.0 cm. The probe beam was focused by a lens with focal length L2 = 20.0 cm and passed through the sample almost collinear with the excitation beam. The intensity at the center of the probe beam was measured by a pinhole-photodiode assemble P1 (Thorlabs, Model DET100A/M) positioned at distance Z2 = 4 m. The signal from P1 was recorded by a digital oscilloscope (Tektronix, Model TDS1001B), which was triggered by a photodiode P2 (Thorlabs, Model PDA10A). The erythrosin ester derivatives were synthesized according to the procedure described in Amat-Guerri et al.23, and the experiments are performed in aqueous micellar solutions using the non-ionic surfactant Pluronics F-127 at pH 7.25 (Mcllavine, [Na2HPO4] = [citric acid] = 7.5 mM).6 The liquid samples were contained in a L = 5 mm quartz cuvette placed in a furnace connected to a temperature controller (Lake Shore, Model 340). The concentrations of dyes were measured by an ultraviolet– visible spectrometer (Perkin Elmer, Model Lambda 1050), and the emission spectrum was measured using a fluorescence spectrometer (Perkin Elmer, Model LS45) at an excitation wavelength. Thermal lens experiments were performed at 25 8C, and five different excitation powers were used. For each excitation power, several transients were obtained. In addition, the temperature coefficient of the refractive index of F-127 aqueous solution was measured using an optical interferometer, as described in Steimacher et al.24

FIG. 2. (a) Optical absorption and (b) emission spectra of erythrosin B and the erythrosin ester derivatives in F-127 micellar solutions at 250 nM.

RESULTS AND DISCUSSION Figure 2a shows the optical absorption coefficient spectra of erythrosin B and its three ester derivatives in F-127 micellar solutions. The optical absorption coefficient spectra of the ERYMET, ERYBUT, and ERYDEC are similar to ERY, with an intense absorption band around 544 nm, showing that the insertion of the alkyl group does not affect the light absorption characteristics of the xanthene dyes. Small variations observed in the absorption and emission spectra are associated with the micelle environment and the different hydrophobicity level of each ester. The small increase of the optical absorption and a red shift are reported in ionic and non-ionic micelles.2,25 Figure 2b shows the emission spectra of dyes with mean emission wavelength of hkemi = 554 nm for ERY, 566 nm for ERYMET and ERYBUT, and 571 nm for ERYDEC. The observed red shift in the absorption spectra and the increase of intensity in the emission clearly show that the esters interact with the micelles. These effects are beneficial for photodynamic therapy and result from changes of the fundamental state of the dye in protic media by a hydrogen bonding as the dye is displaced into the micellar environment.6,25,26



FIG. 3. Normalized TL transient for F-127 (open circles) and ERY (open squares) micellar solutions. Solid lines represent curve fits using Eq. 6. For the F-127 sample, KT = 0 was used in Eq. 6.

Figure 3 shows normalized TL transients I(t)/I(0) for dye-free F-127 aqueous micellar solution and ERY. The results for F-127 resemble transients where only thermal effects are present, a decreasing signal arising from a defocusing the TL created in the sample. On the other hand, low concentration of erythrosin B (ERY sample) in the micellar solution alters considerably the transient behavior. Initially, the amplitude of the TL signal decreases when the excitation laser irradiates the dye solution. Mainly thermal effects are supposed to occur for t , 50 ms. Thereafter, photobleaching takes place, decreasing the amplitude of the thermal lens signal by reducing the concentration of absorbing species. The TL signal reaches a steady state after photobleaching and mass diffusion reach equilibrium. Studies in photobleaching of PDT sensitizers have reported that the processes are oxidative, and there is evidence that they involve singlet oxygen.5 However, the photobleaching process in micellar solutions of xanthene dyes is often unknown or not completely described. The slow process of photobleaching of erythrosin and its ester derivatives in micellar solutions can be quantitatively investigated using the TL theoretical model to fit the experimental transients. For very low concentration of the dyes, the thermal properties of the solutions can be approximated by the values of the pure aqueous solution of F-127 surfactant. Lines in Fig. 3 show the fits. For F-127 aqueous solution, regression analysis was performed using Eq. 6 with KT = 0, yielding the thermal diffusivity Dth = (1.62 6 0.05) 3 103 cm2/s. This value was fixed during the regressions for the dye solutions. Here, h/Pe was also obtained from regression analysis yielding h/Pe = (0.050 6 0.001) W1. Using u = 1 in Eq. 5, the optical absorption coefficient of F-127 aqueous solution was calculated as bF-127 = (0.036 6 0.002) m1, in which (dn/dT) = (1.07 6 0.01) K1 was measured with an optical interferometer,24 and the thermal conductivity k = (0.061 6 0.03) W/mK obtained from thermal diffusivity, mass density, and specific heat.


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FIG. 4. Open circles represent normalized TL transients for the (a) ERY, (b) ERYMET, (c) ERYDEC, and (d) ERYBUT micellar solutions under five different excitation powers from 74 to 313 mW. Solid lines represent the curve fits using Eq. 6.

Similar to the erythrosin B, its dye derivatives presented photobleaching as the samples were irradiated by the pump laser, as shown in Fig. 3 for ERY, ERYMET, ERYBUT, and ERYDEC. Figure 4 shows the TL transients for dye– micellar solutions with different excitation powers and the corresponding fitting with Eq. 6. Although all the transients exhibit similar trends, the photobleaching effect in each sample has different intensity and evolution time, which can be quantitatively described by the parameter KT obtained from the fits. The results for the photobleaching rate KT, given in Fig. 5a, show apparently no correlation with the hydrophobic characteristics of the dyes (ERY , ERYMET , ERYBUT , ERYDEC).6,7 Despite this unexpected behavior, the ester derivatives ERYMET, ERYBUT, and ERYDEC show an increasing resistance to photodegradation when compared with ERY. It is important to note that ERY is a dianionic hydrophilic dye and has a small binding interaction with Pluronics surfactant (F-127).6 On the other hand, the decrease in charge density and the presence of the alkyl group increases the dye–micelle interaction for the ester derivatives. The dye–micelle interaction properties of ester derivatives could explain the increase of the photostability in comparison with ERY. In fact, previous papers showed that the binding constant is 30 times larger for ERYDEC when compared to ERY.6,7 For the dye solutions, u = 1  gke/kem, in which g is the fluorescence quantum efficiency. Using Eq. 5 for F127 and the dye solutions and assuming the same values of dn/dT, L, kP, and k for the diluted dye–micelle solutions as for F-127, we can write hdye Pe hF-127 Pe

  bdye ke ¼ 1g bF-127 hkem i


Figure 5b shows the fluorescence quantum efficiency for the dye–micelle solutions. The values are within the range of the reported values for xanthene dyes.27–30

photo-induced chemical reaction, in addition to the spectrophotometric and fluorimetric method, the photobleaching rate and the fluorescence quantum efficiency were determined for low dye concentration. The dependence of the dye–micelle interaction seems to be the rule of photobleaching and fluorescence process. The results demonstrate the potential of the TL method for quantitative study of photosensitizer compounds at low concentration, providing thermo-optic and photobleaching behaviors at real-time as a function of different physical parameters. ACKNOWLEDGMENTS This work was sponsored by the Brazilian funding agencies Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nivel Superior (CAPES), Conselho Nacional de Desenvolvimento Cientı´ fico e Tecnolo´gico (CNPq), and Fundac¸a˜o Arauca´ria (process: 076/2014-40453).

FIG. 5. (a) KT and (b) fluorescence quantum efficiency obtained from regression analysis for erythrosin B and its ester derivatives. Solid lines are guides to the eye.

As for the photobleaching behavior, the fluorescence quantum efficiency does not follow the hydrophobicity of dyes. Among the xanthene esters, the lack of correlation of KT and g with the hydrophobicity could be due to the location of each dye inside the micelle of F-127 or the PS self-aggregation—in small amount, mainly with the decyl and butyl derivatives, even in micelle presence. In the self-aggregated state occur the self-quenching processes that deactivate the PS excited state. The difference between the values of ERY and the esters could be associated with the dye– micelle interaction. A complete evaluation of the photobleaching behavior in a photosensitizer is a complex problem, and further studies are needed for the identification of the influence of the dye–micellar interaction in the photobleaching kinetics. Although complex, the photobleaching process in dye–micelle solutions can be investigated using thermal lens spectroscopy.

CONCLUSIONS In summary, we have presented a quantitative study of the photobleaching process in micellar aqueous solutions of erythrosin and its ester derivatives. The results showed that the thermal lens signal is strongly affected by the photobleaching process. By using a generalized theoretical model for the thermal lens effect caused by

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23. F. Amat-Guerri, M.M.C. Lo´pez-Gonza´lez, R. Martı´ nez-Utrilla, R. Sastre. ‘‘Synthesis and Spectroscopic Properties for New Rose Bengal and Eosin Y Derivatives’’. Dyes Pigm. 1990. 12(4): 249-272. 24. A. Steimacher, A.N. Medina, A.C. Bento, J.H. Rohling, M.L. Baesso, V.C.S. Reynoso, S.M. Lima, M.N. Petrovich, D.W. Hewak. ‘‘The Temperature Coefficient of Optical Path Length as a Function Temperature in Different Optical Glasses’’. J. Non-Cryst. Solids. 2004. 348: 240-244. 25. J. Kibblewhite, G.G. Drummond, F. Grieser, P.J. Thistlethwaite. ‘‘Lipoidal Eosin and Fluorescein Derivatives as Probe of the Electrostatic Characteristics of Self-Assembled Surfactant/Water Interfaces’’. J. Phys. Chem. 1989. 93(21): 7464-7473. 26. I.J. Macdonald, T.J. Dougherty. ‘‘Basic Principles of Photodynamic Therapy’’. J. Porphyrins Phthalocyanines. 2001. 5(2): 105-129. 27. A. Chatier, J. Georges, J.M. Mermet. ‘‘Limitations of the Thermal Lens Method in Fluorescence Quantum-Yield Measurements’’. Chem. Phys. Lett. 1990. 171(4): 347-352. 28. M.M. Matin. ‘‘Hydrogen-Bond Effects on Radiationless ElectronicTransitions in Xanthene Dyes’’. Chem, Phys. Lett. 1975. 35(1): 105111. 29. J. Olmestad. ‘‘Calorimetric Determination of Absolute Fluorescence Quantum-Yield’’. J. Phys. Chem. 1979. 83(20): 2581-2584. 30. J. Shen, R.D. Snook. ‘‘Thermal Lens Measurement of Absolute Quantum Yields Using Quenched Fluorescent Samples as References’’. Chem. Phys. Lett. 1989. 155(6): 583-586.

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