**1. Introduction**

Oscar Raab demonstrated, in 1900, that the light incidence on dyes can induce cell death [1]. A photosensitizer is a chemical compound that is activated by light of a specific wavelength that leads to tumor destruction [2]. Indeed, Photodynamic Therapy (PDT) is considered to have its origin in 1900 with the classical experiments by the german scientist Oscar Raab. Raab noticed that the exposure of *Paramecium caudatum* to acridine orange and later subjection to light resulted in death of this organism. Raab and his supervisor Hermann von Tappeiner later coined the term "photodynamic therapy" and applied PDT successfully for the treatment of cutaneous tumors using eosin. From that concept, photodynamic therapy (PDT) [3, 4, 5, 6], as we known today, was founded. Since then, the development of other studies, culminating with those performed by Dougherty and coworkers resulted in a non-invasive technique for cancer treatment and other diseases [7, 8]. In fact, precancerous cells, certain types of cancer cells and microbial infections can be treated this way.

Interesting data regarding the application of PDT against several diseases have been reported, since the employment of this therapy in different diseases has increased significantly. In fact, PDT has been used with phenotiazinium [methylene blue (MB) and toluidine blue] as photosensitizers against AIDS-related Kaposi's sarcoma, promoting complete sarcoma remission with excellent cosmetic results [9]. PDT with MB (and LED as light source), which is a very inexpensive system, has been applied against *Leishmania*,

© 2012 Moreira et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

promoting significant reduction in the size of the lesions, diminishing the parasitic load in the draining lymph node and healing the lesions in hamsters experimentally infected with *L. amazonensis* [10]. This therapeutic alternative is very interesting due to the resistance of this organism to pentavalent antimonials (SbV), which constitutes the mainstay pharmacological alternative for leishmaniasis, due to emergence of drug resistance [11].

Tumor, which is also called neoplasm or blastoma, is the abnormal growth of tissues. Sick cells with genetic disturb develop more rapidly than the normal cells, which provokes the development of the tumor (that can be malign or non-malign cells). When the growth of the tumor is a very fast and chaotic process, with tendency to arrive in other organs, generally is a malign tumor [4]. Cancer is the general name of all malign tumors. This term cancer is originated from latin and means "crab". This name is due to the tendency of the tumor to be fixed in several biological tissues, which is correlated to the ability of the crab to be fixed in various surfaces [4].

Interestingly, the PDT procedure is easily performed in a physician's office or outpatient setting, which favors the application of this therapy in several environments, since PDT does not need great structural pre-requisites. In this context, it is important to notice that multicenter randomized controlled studies have demonstrated high efficacy and superior cosmetic outcome over standard therapies [12]. In fact, several cosmetic methodologies have been developed with PDT, such as resurfacing. For many non-oncologic dermatological diseases, such as *acne vulgaris*, viral warts and localized scleroderma, case reports and small series have confirmed the potential of PDT [12]. After the development of topical photosensitizers 5-aminolevulinic acid (ALA) or its methyl ester (MAL), PDT has gained worldwide popularity in dermatology, since these drugs do not induce prolonged phototoxicity as the systemic photosensitizing hematoporphyrin derivatives do [12]. PDT has essentially three steps. First, a light-sensitizing liquid, cream, or intravenous drug (photosensitizer) is applied or administered. Second, there is an incubation period of minutes to days. Finally, the target tissue is then exposed to a specific wavelength of light that activates the photosensitizing medication.

More than one million cases of skin cancer were diagnosed during 2008 in the U.S.A. and its worldwide incidence has risen throughout the last four decades. Squamous cell carcinoma (SCC) is the second most frequent skin cancer, only after basal cell carcinoma (BCC) [13]. In the 20th century, SCC was mainly linked to occupational sun exposure, whereas in the last decades the strongest link has been to ultraviolet (UV) radiation. On one hand, UVB exposure leads to direct DNA damage by pyrimidine dimer formation. On the other hand, UVA induces formation of reactive oxygen species which indirectly also cause DNA damage. Other factors such as the phototype, the genetic predisposition or the immune response are also involved in the carcinogenic process [13].

It is also important to notice that photoantimicrobial agents, that is, chemical compounds that exhibit increased inactivation of microorganisms when exposed to light, have been known also for over a century [14]. While there are several studies regarding the use of photosensitizers against bacterial and viral targets, the clinical use of photosensitizers in antimicrobial therapy has been developed very slowly through small scale trials. This is particularly a surprise considering the efficacy exhibited, especially by cationic photosensitizers, against pathogenic drug-resistant bacteria such as methicillin-resistant *Staphylococcus aureus* and vancomycinresistant *Enterococcus faecium* [14]. Furthermore, the exponentially increasing threat of microbial multidrug resistance has highlighted antimicrobial photodynamic inactivation (APDI) as a promising alternative treatment for localized infections [15]. APDI involves the direct application of the PS to the infected tissue rather than being injected intravenously, as the usual procedure for cancer treatment with PDT [15].

394 Advanced Aspects of Spectroscopy

various surfaces [4].

that activates the photosensitizing medication.

response are also involved in the carcinogenic process [13].

promoting significant reduction in the size of the lesions, diminishing the parasitic load in the draining lymph node and healing the lesions in hamsters experimentally infected with *L. amazonensis* [10]. This therapeutic alternative is very interesting due to the resistance of this organism to pentavalent antimonials (SbV), which constitutes the mainstay pharmacological

Tumor, which is also called neoplasm or blastoma, is the abnormal growth of tissues. Sick cells with genetic disturb develop more rapidly than the normal cells, which provokes the development of the tumor (that can be malign or non-malign cells). When the growth of the tumor is a very fast and chaotic process, with tendency to arrive in other organs, generally is a malign tumor [4]. Cancer is the general name of all malign tumors. This term cancer is originated from latin and means "crab". This name is due to the tendency of the tumor to be fixed in several biological tissues, which is correlated to the ability of the crab to be fixed in

Interestingly, the PDT procedure is easily performed in a physician's office or outpatient setting, which favors the application of this therapy in several environments, since PDT does not need great structural pre-requisites. In this context, it is important to notice that multicenter randomized controlled studies have demonstrated high efficacy and superior cosmetic outcome over standard therapies [12]. In fact, several cosmetic methodologies have been developed with PDT, such as resurfacing. For many non-oncologic dermatological diseases, such as *acne vulgaris*, viral warts and localized scleroderma, case reports and small series have confirmed the potential of PDT [12]. After the development of topical photosensitizers 5-aminolevulinic acid (ALA) or its methyl ester (MAL), PDT has gained worldwide popularity in dermatology, since these drugs do not induce prolonged phototoxicity as the systemic photosensitizing hematoporphyrin derivatives do [12]. PDT has essentially three steps. First, a light-sensitizing liquid, cream, or intravenous drug (photosensitizer) is applied or administered. Second, there is an incubation period of minutes to days. Finally, the target tissue is then exposed to a specific wavelength of light

More than one million cases of skin cancer were diagnosed during 2008 in the U.S.A. and its worldwide incidence has risen throughout the last four decades. Squamous cell carcinoma (SCC) is the second most frequent skin cancer, only after basal cell carcinoma (BCC) [13]. In the 20th century, SCC was mainly linked to occupational sun exposure, whereas in the last decades the strongest link has been to ultraviolet (UV) radiation. On one hand, UVB exposure leads to direct DNA damage by pyrimidine dimer formation. On the other hand, UVA induces formation of reactive oxygen species which indirectly also cause DNA damage. Other factors such as the phototype, the genetic predisposition or the immune

It is also important to notice that photoantimicrobial agents, that is, chemical compounds that exhibit increased inactivation of microorganisms when exposed to light, have been known also for over a century [14]. While there are several studies regarding the use of

alternative for leishmaniasis, due to emergence of drug resistance [11].

The photodynamic process involves photophysical and photochemical steps, which can be applied with several aims, such as therapies against cancer or infections. PDT light sources include laser, intense pulsed light, light-emitting diodes (LEDs), blue light, red light, and many other visible lights (including natural sunlight). Photosensitizer drugs may become activated by one or several types of light. The optimal light depends on the ideal wavelength for the particular drug used and target tissue.

Electron and energy transfer in the excited state govern the efficiency of a variety of photoinduced processes, including photosynthesis, light to energy conversion in semiconductor devices, cell damage induced by solar exposition and photodynamic action [16, 17, 18]. It is well reported that photophysical behavior of a dissolved dye depends on the nature of its environment, i.e., the solvent influences the spectra characteristics of the solute molecules [19]. Several factors influence the visible spectral behavior of dissolved dye molecules, especially the solvent's polarity and its hydrogen-bond donor/acceptor capacities [19]. The properties can be determined by the solvent dielectric constant, ε, and solvatochromic parameters. The strong solvatochromic behavior can be observed for dye molecules with large dipole moment changes during transitions between two electronic states. The solvent can differentially stabilize the ground and/or the excited state in polar and non-polar solvents [19].

The series of phenothiazine [thionine, methylene blue (MB), azure A (AZA) and azure B (AZB)] derivatives (Fig. 1) are positive dyes used as a model for phototherapeutic agent as well as for dye sensitized solar energy converter [20, 21] due to their appropriate biological, chemical, photochemical and photophysical properties [22, 23, 24].

The intersystem cross quantum yield and the singlet oxygen formation for MB is 0.52 [25, 26, 27, 28, 29], the triplet lifetime is higher, approximately 3.0 μs, in air saturated aqueous solution, and up to 50 μs in nitrogen saturated aqueous solution. The singlet excited state has a lower lifetime, approximately 1,400 ps (Table 1), and it is due to the higher internal conversion and triplet formation, with a fluorescence quantum yield of 0.04 in methanol [30, 31, 32, 33]. In addition, MB and and MB derivatives that have been used as photosensitizers in PDT showed a good biocompatibility (appropriate citotoxicity and phototoxicity) when used *in vitro* to attack key organelles in cells [14,21].


**Figure 1.** Thionine derivatives.


**Table 1.** Values of lifetime () of some dyes at 25C.

The Fluorescence decays of dyes were obtained by single-photo-counting technique. The excitation source was a Tsunami 3950 Spectra Physics titanium-sapphire laser, pumped by a Millenia X Spectra Physics solid state laser. The laser was tuned that a third harmonic generator BBO crystal (GWN-23PL Spectra Physics) gave the 292 nm excitation pulses that were directed to an Edinburgh FL900 spectrometer. The spectrometer was set in L-format configuration, the emission wavelength was selected by a monochromator (680 nm), and emitted photons were detected by a refrigerated Hamamatsu R3809U microchannel plate photomultiplier. The software provided by Edinburgh Instruments was used to analyze the individual decays. The quality of the fit was judged by the analysis of the statistical parameters reduced-2 and Durbin-Watson, and by the inspection of the residuals distribution.

396 Advanced Aspects of Spectroscopy

**Figure 1.** Thionine derivatives.

Toluidine blue Methylene blue

Toluidine blue Methylene blue

**Table 1.** Values of lifetime () of some dyes at 25C.

medium: water

azure A azure B

N

R4=H

N S N

Cl

Methylene blue

R1 = R2 = R3 = R4 = H

R1 = R2 = R3= R4 = CH3

thionine

R3

R4

medium: ethanol

The Fluorescence decays of dyes were obtained by single-photo-counting technique. The excitation source was a Tsunami 3950 Spectra Physics titanium-sapphire laser, pumped by a Millenia X Spectra Physics solid state laser. The laser was tuned that a third harmonic generator BBO crystal (GWN-23PL Spectra Physics) gave the 292 nm excitation pulses that were directed to an Edinburgh FL900 spectrometer. The spectrometer was set in L-format configuration, the emission wavelength was selected by a monochromator (680 nm), and

2179,65 (66,72%) e 358,03 (33,28%) 328,84

R1 = R2 = R3 = CH3

643,89 465,96

Azure B 1268,56 (48,89%) e 306,00 (51,11%)

Dye (ps) Thionine 314,40 Nile blue 372,75 Azure A 421,28

R1= R2 = CH3 R3 = R4 = H

R1

R2

Dye (ps) Thionine 848,84 Nile blue 1170,03 Azure A 776,43 Azure B 724,78

The dyes stock solutions were prepared in ethanol (6.0 x 10-5 M) and aliquots of these stock solutions were added, via a calibrated Hamilton microsyringe, to volumetric flasks containing water or ethanol, and the solutions were stirred for 30 minutes. The final concentrations of dyes were 1.0 x 10-6 M. All measurements were performed at 25°C using a cuvette with 0.2 cm of optical path.

The excited state lifetime depends on the solvent [34, 35]. The dependence of the lifetime on the viscosity and solvent dielectric constant indicates that the dye excited state deactivation process is slow as the medium viscosity increases. This effect is related to the partial inhibition or the higher friction on the dye substitute groups rotation, such as –CH3, -NH2, -N(CH3)2 and –N(CH2CH3)2 [36]. The lifetime values are in agreement with the results reported in the literature. Lee and Mills [37] showed the lifetime values for methylene blue aqueous solutions (358 ± 20 ps). Grofcsik et al [34] measured the lifetime of Nile blue excited state and oxazine 720 in different solvents at 20 oC. The thionine dye photophysics is well known [38]. In an aqueous solution, thionine has a fluorescence lifetime of 320 ± 60 ps when excited at 610 nm [37,39]. In organic solvent, the increase of the thionine fluorescence lifetime (450 ps in ethanol and 760 ps in terc-butilic alcochol) results in a increasing of the fluorescence quantum yield [38]. The thionine lifetime differences observed in an aqueous medium and ethanol is quite high, which shows the effect of microenvironment polarity on the excited state decaying [38]. In our experiments, in an aqueous medium thionine has a useful lifetime of 314.4012 ps, which is in agreement with the results presented in the literature.

The Nile blue lifetime in ethanol and water are 1420 and 418 ps, respectively. These results are higher than those that we found in our work. However, it should be taken into account that the temperatures used in our experiments are different from those whose results are different. It was shown that the lifetime of Nile blue depends on the temperature due to the intermolecular charge transfer [34,35,36]. This charge transfer process is facilitated by the presence of NH2 groups in molecule structure, such as on the Nile blue structure, which may change the lifetime values. Grofcsik et al [34] studied these probes in different solvents where it was observed that there is a relationship between the solvent permissivity and the excited state lifetime. It was shown that the lifetime is higher in nonpolar solvents, where protic solvents decreases the excited state lifetime. This behavior was observed in both dye molecules that were studied, which have a similar chemical structure.

Grofcsik et al [34,35] have shown that there is a relationship between the excited state lifetime of Nile blue and Oxazine 720 with the acidity of the medium. As the hydrogen ion concentration increases it is observed a decrease of the excited state lifetime [40]. It was also observed for methylene blue, azure A, azure B and azure C [41]. The reason for the rapid decay in acid medium is due to the formation of dications from the monocations reaction in the excited state with hydrogen ions. These results indicate that the reaction in the excited state the additional protons are located on the nitrogen atom of the ring and not on the terminal amine groups [40]. It is believed that for other compounds the results may be the same due to the similarity in the chemical structure of such molecules.

Dutt et al [42] studied the fluorescence lifetime of cresila violet, Nile blue, oxazine 720 and Nile red, using different solvents, such as alcohols, polyalcohols, amides and some aprotic solvents. The authors showed that the lifetime values for these dyes are approximately 3.5 ns for n-alcohols, which are higher than that for the Nile blue (1.62 ns in ethanol). This result is in agreement with our studies. When it is considered the behavior of bipolar solute in polar solvents, the hydrodynamic and dielectric contribution must be taken into account [42]. However, it is not well known how to measure these hydrodynamic and dielectric contributions individually. In the case of the four dyes, when in the presence of amides and aprotic solvents, as described above, the contributions are reasonably described by the hydrodynamic friction, where to describe the rotating relaxation in the presence of nalcohols; the dielectric friction must be included.

Chen et al [43] studied the quantum yield of the methylene blue singlet oxygen as a function of the medium pH values. The authors showed that the protonated acid (3MBH2+) triplet state is similar to the base (3MB+) triplet state, and the quantum yield of the singlet oxygen formed is much higher in basic medium than that in acidic medium. The singlet oxygen formation increases as the pH of the medium is increased, while the singlet state lifetime decay the triplet state formation do not depend on the pH changes. It can be explained by the population decay rate of the singlet state due to the internal conversion to the fundamental state, and the intersystem crossing to the triplet state, which are much higher that the protonation rate [43,44]. Also [43] studied the behavior of methylene blue, 1,9 dimethyl-methylene blue and toluidine blue in aqueous medium and methanol. The triplet state formation and the singlet oxygen quantum yield in water were very similar to that for methylene blue and for 1,9-dimethyl-methylene blue. The kinetic studies results for the singlet state decay of methylene blue in water and in methanol were 0.37 and 0.62 ns, respectively, where for toluidine blue the results were 0.28 and 0.40 ns, respectively. In the case of methylene blue the decay useful life of the singlet excited state in methanol is approximately two times higher than in water. The authors showed that there is no influence of the solution concentration on the singlet state lifetime, where the differences on the lifetime decays that were observed in water and methanol are not related to the methylene blue dimerization in water. The methylene blue lifetime decay decreases with the increase of the dielectric constant of protic solvents due to the interaction of the methylene blue with the polar solvent [45]. In protic alcohols and in aqueous solutions the methylene blue excited state lifetime is higher than of the fundamental state. Therefore, the differences between the singlet and triplet states decrease as the relaxation rate is increased. In the presence of aprotic solvents, such as acetone, acetonitrile, and dimethyl sulfoxide, the dipole excited state is lower in the fundamental state, where the energy differences observed is higher and the relaxation lifetime is longer [46].

The use of these dyes as singlet oxygen photosensitizer in PDT, as well as tumor cells removal are being investigated [47, 48, 49, 50]. It is known that under laser irradiation in the presence of photosensitizer dyes, the tumor cells undergo necrosis or apoptosis and the rate of tumor cell removal through apoptosis increases [51, 52, 53]. This behavior has been related to the presence of singlet oxygen in the tumor cells [54, 55]. The increase of cell removal through apoptosis is of great importance in the PDT treatment [50,56]. There are no side effects in the cell removal through apoptosis because it is a controlled cell removal process, where there is no inflammation of the laser irradiated tissue. In some cases changes in the PDT mechanism has been observed, type I via free radical and type II via oxygen singlet, which could be related to the interaction among the dyes and the cellular system [57, 58, 59, 60]. These changes involve the aggregation of two or more dye molecules in the same site [61].
