**2. Photodynamic Therapy (PDT): Mechanism of action**

398 Advanced Aspects of Spectroscopy

terminal amine groups [40]. It is believed that for other compounds the results may be the

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 n-

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

The use of these dyes as singlet oxygen photosensitizer in PDT, as well as tumor cells

presence of photosensitizer dyes, the tumor cells undergo necrosis or apoptosis and the rate

50]. It is known that under laser irradiation in the

48, 49,

same due to the similarity in the chemical structure of such molecules.

alcohols; the dielectric friction must be included.

higher and the relaxation lifetime is longer [46].

removal are being investigated [47,

Selective tumor destruction without damaging surrounding healthy tissues can be reached by using PDT, which is treatment, activated by light, which requires the combination of three elements: a photosensitizer, visible or near-infrared light, and oxygen [62, 63, 64, 65, 66, 67, 68, 69]. However, the precise mechanisms of PDT are not yet fully understood but two general mechanisms of photoinduced damage in biomolecules have been proposed: Type I and Type II [62,70, 71]. Type I is the photodynamic mechanism in which the excited molecule induces radical formation that causes damage to biological targets (membranes, proteins and DNA), and an electron transfer event is the initial step [16]. In Type I mechanism, the photosensitizer in the excited state interacts directly with a neighbor molecules, preferentially O2, producing radicals or radical ions through reactions of hydrogen or electron transfer [72]. Frequently, these radicals react immediately with the O2 generating a complex mixture of reactive oxygen species (ROS), such as hydrogen peroxide, superoxide radical and hydroxyl radical, which are capable to promote oxidation a great number of biomolecules [62].

It is believed that 1O2 produced through type II reaction is primarily responsible for cell death. It is known that several factors including the PS, the subcellular localization, the substrate and the presence of O2 contribute to this process [71]. The lifetime of 1O2 is very short (approximately 10-320 nanoseconds), limiting its diffusion to only approximately 10 nm to 55 nm in cells [73]. Type I photoreaction of some PSs are primarily responsible for sensitization through radical formation under hypoxic conditions. In the presence of oxygen, 1O2 mediates photosensitization process, but the supplemental role of H2O2, OH• and O2• must also to be considered. Only substrates situated very close to the places of ROS generation will be firstly affected by the photodynamic treatment because the half-life of 1O2 in biological systems is under 0.04 μs and its action radius being lower than 0.02 μm [71]. This assumption is due to the fact that ROS are highly reactive and present a very short halflife. Type II is the photodynamic mechanism in which the photooxidation is mediated by singlet oxygen (1O2), where an energy transfer reaction from the photoexcited molecule to molecular oxygen is the initial step [16,62]. The process involves the excitation of the photosensitizer from a ground singlet state to an excited singlet state, where intersystem crossing to a longer-lived excited triplet state will occur. It is also important to point out that molecular oxygen is present in tissue with a ground triplet state. When the photosensitizer and an oxygen molecule are in proximity, an energy transfer can take place that allows the photosensitizer to relax to its ground singlet state, and create an excited singlet state oxygen molecule. Additionally, energy is transferred from triplet protoporphyrin IX to triplet oxygen, resulting in singlet ground state protoporphyrin IX and excited singlet oxygen, which reacts with biomolecules, which can damage some cells in the treatment area. Singlet oxygen is the usual name associated to the three possible excited electronic states immediately superior to the ground state of molecular oxygen (triplet oxygen) [3].

Due to the short half-life and diffusion distance of singlet oxygen in aqueous media, PDT can be considered a highly selective form of cancer treatment, as only the irradiated areas are affected, provided that the photosensitizer is nontoxic in the absence of light [74]. This combination of light/photosensitizer/oxygen as a mode of disease treatment has expanded from an initial focus on cancer tumors to include application in certain non-neoplastic diseases including age-related macular degeneration (AMD), coronary heart disease, periodontal diseases, and microbial infections [75].

Singlet oxygen is a very aggressive chemical species and will very rapidly react with any nearby biomolecules, being that the specific targets depend directly on the physicalchemistry properties of the photosensitizer used in the photodynamic process, which will result in no desired side effects, such as destructive reactions that will kill cells through apoptosis or necrosis. Therefore, depending on whether Type-I or Type-II mechanisms take place, the therapeutic efficiency of PDT may be completely altered. Therefore, the ratio of apoptotic versus necrotic cell death in tumors treated with PDT may depend on the competition between electron and energy transfer in the reaction site [16].

Oxidative stress generated by the photodynamic action occurs because in biological systems the singlet oxygen presents significantly low lifetimes, where the lifetimes of the singlet oxygen is lower than 0.04 μs, implying that its radius of action is also reduced, being usually lower than 0.02 μm [3]. Reactive oxygen species (e.g. hydroxyl radicals or superoxide) are their high reactivity and low specificity with a broad spectrum of organic substrates [76]. Various methods have been employed for the generations of hydroxyl radicals such as O3/UV, H2O2/UV, TiO2 photo-catalysis and photo assisted Fe(III)/H2O2 reaction.
