**4. Aggregation of photosensitizers and its influence in PDT**

406 Advanced Aspects of Spectroscopy

applications [145,

oxygen in PDT applications [139,

146, 147,

monocations with hydrogen ions [153].

works done on its interaction with DNA [155,

tumors [136] and can retard tumor growth [137,

Nile blue (RNB) and Meldola's Blue. It has been found to be localized selectively in animal

stain for Escherichia coli in flow cytometry [143], as a DNA probe [144] and many other

solvatochromism, they have been used as stains and imaging agents. These dyes present relatively low solubility in aqueous medium as well as their fluorescence is reduced significantly in in the presence of polar medium, which opens up new possibilities to develop aqueous analogues of these benzophenoxazines [149]. Together with the increase of the solubility in water, it is believed that the self-assembly process to form aggregates can be

NB shows thermochromic and solvatochromic behavior in its ultraviolet/visible spectra [151]. The variation in the absorption spectrum is due to the equilibrium between the monocation and the neutral molecule, where the monocationic form is the more stable in most solvents. In strong basic conditions the neutral form is observed, where in strong acidic conditions the dicationic and tricationic forms can be observed [152]. The fast decay processes study can be used to get information on the effect of medium condition, basic and acidic, on determining the excited state lifetime on the picosecond scale. It was shown that the reason for the faster decay in acidic conditions results from the formation of dications by reaction of excited state

Despite the photophysics of NB in pure solvents is well characterized in literature, the NB interaction with microheterogeneous systems, such as micelles, reverse micelles (RMs) and DNA is still not well understood. Electrochemical studies have shown that NB-DNA duplexes modified microelectrode can be used as a rapid and sensitive method to detect TATA binding to DNA in the presence of other proteins [154]. However, there are no many

> 156, 157,

NB with biomimicking self-organized assemblies (SDS micelles and AOT reverse micelles) and a genomic DNA (extracted from salmon sperm) (SS DNA), it has been shown that there are two different binding modes of NB with genomic DNA, electrostatic and intercalative modes [144]. There was no explanation for the mechanism related to these interaction modes. The electrostatic mode is believed to be responsible for electron transfer between the probe and DNA, which may result in a quenching process of the NB fluorescence emission intensity when in the presence of low concentration of DNA. The intercalative mode is believed to be the subsequent release of quenching due to the intercalation of the dye in DNA base pairs. In another study, it was shown that binding affinity of the probe is higher with SDS micelles than with the DNAs within its structural integrity in presence of the micelles. The complex rigidity of NB with various DNAs and its fluorescence quenching

with DNAs has shown a strong recognition mechanism between NB and DNA [159].

NB was immobilized in two different surfaces, a nonreactive surface (SiO2), with its conduction band at much higher energies, and a reactive surface (SiO2), with a conduction band situated at lower energies. The former is used to directly probe the excited-state dynamics of the dye undisturbed by other competing processes. The latter is used to study

disrupted resulting in an enhancement of the fluorescence intensity [150].

138]. NB has been used as a photosensitizer for

158]. In a work done on the interaction of

142], as a

140], in processes that depend on solvent polarity [141,

148]. Due to their high fluorescence quantum yield together with their

Most of these dyes form aggregates in the ground state [161, 162, 163], even when the dye concentration is low (approximately 10-6 M) and in the presence of salts and aggregation inducing agents, such as anionic micelles, heparin, polyelectrolyte, liposome and vesicles. The planar structure of such dyes is a key factor that contributes to the approaching and dimerization of the dyes [164, 165].

The presence of hydrophobic ligands in the dye structure facilitates the aggregate formation in polar medium. The effects of the planar structure of the dye, hydrophobicity and the interaction with cell membranes were observed in photosynthetic systems II of plants [166] and other systems [167]. Some studies have shown that the interaction among phenothiazines and cyclodextrins results in the aggregate formation with different sizes depending on the cyclodextrins cavity size [162,168].

Studies that were conducted previously have shown that methylene blue molecules form aggregates and the photophysical behavior changes depending on the ground state aggregation. It results in a decreasing of the fluorescence intensity and on the singlet oxygen formation [49]. These studies have shown that the interaction with micelles is responsible for the dimerization process and not the interaction with monomer of the surfactant, as it has been postulated in some works [169]. In this stage of the work it is important to study the nature of the aggregates formed in different negatives interfaces and in biological systems, more specifically in micelles, vesicles and mitochondria.

It is well known that dimerization and medium composition effects changes the energy transfer process among triplets species and molecular oxygen and other triplet suppressors [170, 171, 172, 173, 174, 175, 176, 177, 178]. Some studies carried out using thionine and MB have shown some of these effects [179]. Azure A, azure B, thionine e MB are dimerized with different dimerization constants.

The aggregation of ionic dyes cannot be assigned to a specific type of chemical interaction. There is a significant contribution of several influences, such as van der Waals interactions, intermolecular hydrogen bounds and pi-electrons interactions, being that, frequently, it is not trivial to evaluate the specific contribution of each one of these interactions [180].

The quantum behavior of extended aggregates of atomic and molecular monomers, containing from a just a few up to thousands of subunits, is attracting increasing attention in chemistry and physics, being that proeminent examples are aggregates of large dye molecules, chromophore assemblies describing the photosynthetic unit of assemblies of ultra-cold atoms [181].

According to their structure, dyes, such as phenotiazinium, exhibit J- or H-aggregates, which present very typical J- or H-absorption bands [182]. The aggregate absorption band is red-shifted in relation to the monomer absorption. These are the J-aggregates showing a very narrow band whose position is well-predicted by a theory ignoring intramolecular vibrations. By contrast, other dyes showed a shift towards the blue (i.e. higher absorption energies) and were termed H-aggregates (hypsochromic shift). Unlike the J-band, the line shape of the H-band generally shows a rich vibrational structure and has a width of the order of the monomeric band [183]. The J-band is polarized parallel to the rods, while the Hband is polarized perpendicularly to the rod long-axis [184].

Self-organized J-aggregates of dye molecules, known for over 60 years, are emerging as remarkably versatile quantum systems with applications in photography, opto-electronics, solar cells, photobiology and as supramolecular fibres [185].
