**3.1. Phenothiazinium dyes**

The phenothiazinium dyes were first synthesized in the late 19th century—e.g. both Methylene Blue (Caro) and Thionin (Lauth) in 1876—during what might be considered to be a ''gold rush'' period of chemical experimentation after the discovery of the first aniline dyes [77]. Among photobactericidal compounds, the phenothiazinium photosensitizers methylene blue (MB) and toluidine blue O (TBO) have often been used as lead structures, being effective photosensitizers with singlet oxygen quantum yields of approximately 0.40 and exhibiting low toxicity levels in mammalian cells [14]. Members of the phenothiazine class are known to cross the blood-brain barrier and to be relatively nontoxic [78, 79].

400 Advanced Aspects of Spectroscopy

site [16].

**3. Photosensitizers** 

**3.1. Phenothiazinium dyes** 

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

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,

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

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

The phenothiazinium dyes were first synthesized in the late 19th century—e.g. both Methylene Blue (Caro) and Thionin (Lauth) in 1876—during what might be considered to be a ''gold rush'' period of chemical experimentation after the discovery of the first aniline dyes [77]. Among photobactericidal compounds, the phenothiazinium photosensitizers methylene blue (MB) and toluidine blue O (TBO) have often been used as lead structures, being effective photosensitizers with singlet oxygen quantum yields of approximately 0.40

O3/UV, H2O2/UV, TiO2 photo-catalysis and photo assisted Fe(III)/H2O2 reaction.

immediately superior to the ground state of molecular oxygen (triplet oxygen) [3].

periodontal diseases, and microbial infections [75].

The biomedical use of phenothiazinium dyes has begun with specimen staining for microscopy by various medical scientists, among whom were famous scientists such as Romanovsky, Koch and Ehrlich. The idea of structure—activity relationships in stains developed in this era, particularly by Paul Ehrlich, laid the foundations for modern medicinal chemistry, and these principles should be followed by those attempting the properly organized photosensitizer synthesis [77]. Cellular uptake is determined by a combination of charge type/distribution and lipophilicity, both of which characteristics may be controlled by informed synthesis. Due to the expansion of PDT into the antimicrobial milieu, a far greater scope for photosensitizer design exists now. For example, in the field of blood product disinfection, an ideal candidate photosensitizer would be effective in the inactivation of bacteria, viruses, yeasts and protozoan, but would remain non-toxic and nonmutagenic in a human recipient. It is hardly surprising that none of the currently available agents fits all of these criteria [77].

Phenothiazinium dyes are cationic compounds with high redox potential that interacts with visible light inactivating several kinds of pathogenic agents in fresh plasma. Phenothiazinium dyes present great reactivity with the proteins and lipoproteins (cell membranes) and nucleic acids. These cationic compounds have limited capability to permeate the cell membrane as function of their elevated hydrophilic character [80]. Phenothiazinium dyes present significant action against encapsulated virus and some virus without capsule, such as parvovirus B19. As function of its genotoxic action, the employment of phenothiazinium dyes is prohibited in several countries, such as Germany80. On the other hand, the Methylene Blue is a highly hydrophobic compound with higher chemical affinity to the nucleic acids, which denotes its potential to application against virus.

Phenothiazinium dyes are photocytotoxic, and can cause photoinduced mutagenic effects [81]. In living systems, DNA acts as an important target for phenothiazinium dyes. It has been proved that these dyes can photosensitize biological damage. Azure B (AZB) is an easy available phenothiazinium dye, and has been widely employed both in metal determination and DNA staining detection. Owing positive charges on its molecular structure, AZB can bind to the DNA polyanion in living systems through electronic interactions. So, the study of the interaction of AZB with DNA in vitro is of importance.

Methylene Blue, MB (Figure 2) is a phenothiazinic dye current applied in PDT as therapeutic agent or photosensitizing compound. MB has a recognized antimicrobial effect in the dark (citotoxicity property) which can be increased, at oxygenated environment, by the incidence of light with a wavelength corresponding to its electronic absorption band [82, 83].

Methylene Blue is a well-known photochemical oxidant. The photoreduction reaction of this dye by various types of electron donors has been studied quite often, and in most cases an electron transfer mechanism was proposed for explaining the observed results [84].

**Figure 2.** Structure of Methylene Blue

This molecule is particularly interesting for application in PDT due to its known physical chemical properties. For example, MB is a positive charged dye with three aromatic rings (6 members) very soluble in ethanol. It is already used clinically in humans for the treatment of metahemoglobinemia, without significant side effects. Besides these characteristics, MB presents a quantum yield of singlet oxygen formation around 0.5, with a low reduction potential, intense light absorption in the region of 664 nm in water (within the phototherapeutic window). Also, it displays a high photodynamic efficiency causing apoptosis of cancer cells, by mono or polychromatic light excitation). Currently, MB is used by several european agencies for disinfection of blood plasma, due to its efficiency in photodynamic inactivation of microorganisms such as viruses [85], including HIV, hepatitis B and C [86, 87].

MB has been clinically used as a photosensitizer drug for PDT in the treatment of different types of tumors [88]. Phototherapeutic application examples include treatment of bladder cancer, inoperable esophagus tumors, skin virulence, psoriasis and adenocarcinomas [89]. Additionally, an important point to be considered is the extremely low cost of a treatment based on this dye compared with other available photo-drugs.

Although MB possesses a positive charge and the planar structure with delocalized charge, it has a tendency to form dimers, trimers or type H aggregated systems in the presence of certain additives, cell organelles or solvents, for example, water [90, 91, 92, 93]. The development of self-aggregates compromises its photodynamic activity, impairing the production of singlet oxygen, principal phototoxic species in PDT. In self-aggregated states autoquenching processes occur where the excited monomers have the energy suppressed by collisions with other monomers that constitute the aggregate [94, 95, 96, 97, 98].

Often, treatment protocols require unusual preparation methods, or conditions that may have many distinct characteristics of the most ideal conditions. One example is that the MB in diluted aqueous solution, with concentration around 2x10-5 mol L-1, is found in monomeric form. However, its uses in topical treatments require concentrations higher than 6x10-2 mol L-1, where self-aggregation and its consequences are significant [82].

Therefore, it is important to investigate the phenomena of MB self-aggregation present in solvent mixtures and / or interaction with biomolecules [90]. This study aims to investigate changes in MB spectroscopic properties caused by self-aggregate formation induced by solvent mixtures.

402 Advanced Aspects of Spectroscopy

**Figure 2.** Structure of Methylene Blue

B and C [86,

87].

This molecule is particularly interesting for application in PDT due to its known physical chemical properties. For example, MB is a positive charged dye with three aromatic rings (6 members) very soluble in ethanol. It is already used clinically in humans for the treatment of metahemoglobinemia, without significant side effects. Besides these characteristics, MB presents a quantum yield of singlet oxygen formation around 0.5, with a low reduction potential, intense light absorption in the region of 664 nm in water (within the phototherapeutic window). Also, it displays a high photodynamic efficiency causing apoptosis of cancer cells, by mono or polychromatic light excitation). Currently, MB is used by several european agencies for disinfection of blood plasma, due to its efficiency in photodynamic inactivation of microorganisms such as viruses [85], including HIV, hepatitis

MB has been clinically used as a photosensitizer drug for PDT in the treatment of different types of tumors [88]. Phototherapeutic application examples include treatment of bladder cancer, inoperable esophagus tumors, skin virulence, psoriasis and adenocarcinomas [89]. Additionally, an important point to be considered is the extremely low cost of a treatment

Although MB possesses a positive charge and the planar structure with delocalized charge, it has a tendency to form dimers, trimers or type H aggregated systems in the presence of

of self-aggregates compromises its photodynamic activity, impairing the production of singlet oxygen, principal phototoxic species in PDT. In self-aggregated states autoquenching processes occur where the excited monomers have the energy suppressed by

Often, treatment protocols require unusual preparation methods, or conditions that may have many distinct characteristics of the most ideal conditions. One example is that the MB in diluted aqueous solution, with concentration around 2x10-5 mol L-1, is found in monomeric form. However, its uses in topical treatments require concentrations higher than

Therefore, it is important to investigate the phenomena of MB self-aggregation present in solvent mixtures and / or interaction with biomolecules [90]. This study aims to investigate

6x10-2 mol L-1, where self-aggregation and its consequences are significant [82].

91, 92,

95, 96, 97, 98].

93]. The development

based on this dye compared with other available photo-drugs.

certain additives, cell organelles or solvents, for example, water [90,

collisions with other monomers that constitute the aggregate [94,

The MB is an oxazinic dye soluble in water or alcohol. It presents a quantum yield of oxygen singlet formation of about 0.5 and low reduction potential [25]. It is a dye with low toxicity, which absorb in the UV-visible light (máx = 664 nm; solvent: water) and shows good photodynamic efficiency to kill cancer cells, which can be excited by monochromatic and polychromatic light within the therapeutic window [82]. It is a hydrophobic dye, which forms aggregates when in the presence of aggregation agents such as polyelectrolyte, or when in the presence of solvents that induces the aggregation process. The aggregate formation changes photosensitization efficiency, decreasing the amount of singlet oxygen produced by light stimulation. The most important application of methylene blue (MB) is its use in PDT as a photosensitizer agent, in oncology and potentially in the treatment of other diseases, such as Leishmaniosis.

Teichert et al.[99] used *Candida albicans* strains that are resistant to the conventional treatment of *Candida* infections, which were collected from HIV-positive patients. These strains were inoculated in the oral cavity of rats that, subsequently, were submitted to the topic application of 1 mL of Methylene Blue at concentrations of 250, 275, 300, 350, 400, 450 and 500 μg mL-1. After 10 minutes of dye application, the authors employed the diode laser with wavelength of 664 nm with potency of 400 mW (687.5 seconds), resulting in an energetic density of 275 J/cm2 [100]. After one unique application, it was realized microbial culture exam of the respective samples and the individuals were sacrificed to the histological analysis of the tongue. The results obtained in this procedure demonstrated a complete elimination of the microorganisms, when the dye concentrations of 450 e 500 μg mL-1were employed. In the histological analysis, the rats that were treated with PDT had no inflammatory signals. The tongues of the control group rats presented high level of infection by *Candida* which was located in the keratin layers [100]. The respective authors concluded that the PDT is a potential alternative to the treatment of the fungi infection, emphasizing, as advantages of this technique, its topic character, simple methodology and, mainly, the unspecific characteristic of PDT, i.e., the possibility of to be applied to a great number of microorganisms. Moreover, PDT can be applied several times without risk of selection of resistant yeasts [100].

Azures A, B and C, are examples of photosensitizer agents, which have the cationic derivatives, such as the Azure Bf4. The organic ions can interfere through fluorescent radiation absorption that is emitted by excited molecules, resulting in a photobactericidal effect on the *Staphylococcus aureus* and *Enterococcus faecium* colonies. This behavior is related to the light stimulation wavelength because the organic compounds present in the system absorb electromagnetic radiation. However, only organic compounds that present double bond conjugated system, such as azure A, B or C, are capable to absorb the visible light radiation.

It was observed that red visible light (600-700 nm) and nearinfrared are the wavelengths that can penetrate the human skin. The phenothiazinic dyes, such as Azures, absorb light in such wavelengths with high intensity. They show the formation of aggregates due to the presence of aggregation agents such as polyelectrolytes, or due to the presence of solvents that favors the aggregate formation, such as water. The aggregate formation changes the photosensitization process efficiency, decreasing the amount of singlet oxygen produced by the light stimulus. The self-aggregation phenomenon can be minimized by adding charged groups in the dye structure, which results in an electrostatic repulsion interaction, increasing the hydrophilic behavior of the dye, such as Azure B and Azure BF4.

Azures are phenotiazine compounds. This class of dye has low toxicity in the dark, constant composition, being synthesized with high yield. Azures present great selectivity to the tumor cells and significant photo stability, being not maintained in the body for long interval of time. These dyes can be applied through endovenous and topic ways. Azures present high bactericide ability, being very auspicious compounds to be applied as photosensitizes in PDT, especially due to their favorable photodynamic properties and low cost [101, 102]. Azure dyes (including Azure B) are recalcitrant compounds used in the textile industry. For instance, Azure B has been used in a selective assay for detecting lignin peroxidase, the oxidative enzyme with the highest redox potential produced by white-rot fungi [103, 104].

Azure B is a very sensitive dye and extremely susceptible to detect slight alterations in its chemical environment, presenting significant solvatochromic processes. Physico-chemical properties of Azure B have motivated the employment of Azure B as a chromogenic reagent for the spectrophotometric determination of several compounds, which are relevant to biological and environmental chemistry such as periodate [105]. This cited method is simple and rapid, offering advantages of sensitivity and wide range of determinations, without involvement of any stringent reaction conditions, being successfully applied to the determination of periodate in solution and in several river water samples. In its time, Azure-C (AZC), and related phenothiazine compounds has been widely used for accelerating the oxidation of NADH, but not in connection to the NAD+ reduction process.

Thionine has been a subject of many studies, as for example in a photochemical and electrochemical biosensor [106, 107, 108, 109, 110] and in photovoltaic cells [111]. Thionine is a positively charged tricyclic heteroaromatic molecule, which has been investigated for its photoinduced mutagenic actions [112, 113], toxic effects, damage on binding to DNA [114] and photoinduced inactivation of viruses [115]. Thionins consist of 45–47 residues bound by three to four disulfide bonds, which includes α1-purothionin, βPTH, and β-hordothionin (βHTH) [116, 117, 118].

It has the ability to immobilize proteins and DNA and act as molecular adhesive [119]. Biophysical and calorimetric studies with three natural DNAs of varying base compositions, have shown the intercalative binding and high affinity of thionine to GC rich DNAs [120]. Thionine presented a high preference to the alternating GC sequences followed by the homo GC sequences contained in different synthetic polynucleotides [121]. AT polynucleotides presented a lower binding affinities but the alternating AT sequences had higher affinity compared to the homo stretches. The intercalation and the sequence of specific intercalative binding of thionine were shown by fluorescence, viscosity experiments and circular dichroic studies, respectively [121].

404 Advanced Aspects of Spectroscopy

cost [101,

fungi [103,

[116, 117, 118].

104].

electrochemical biosensor [106,

photoinduced mutagenic actions [112,

wavelengths with high intensity. They show the formation of aggregates due to the presence of aggregation agents such as polyelectrolytes, or due to the presence of solvents that favors the aggregate formation, such as water. The aggregate formation changes the photosensitization process efficiency, decreasing the amount of singlet oxygen produced by the light stimulus. The self-aggregation phenomenon can be minimized by adding charged groups in the dye structure, which results in an electrostatic repulsion interaction, increasing

Azures are phenotiazine compounds. This class of dye has low toxicity in the dark, constant composition, being synthesized with high yield. Azures present great selectivity to the tumor cells and significant photo stability, being not maintained in the body for long interval of time. These dyes can be applied through endovenous and topic ways. Azures present high bactericide ability, being very auspicious compounds to be applied as photosensitizes in PDT, especially due to their favorable photodynamic properties and low

industry. For instance, Azure B has been used in a selective assay for detecting lignin peroxidase, the oxidative enzyme with the highest redox potential produced by white-rot

Azure B is a very sensitive dye and extremely susceptible to detect slight alterations in its chemical environment, presenting significant solvatochromic processes. Physico-chemical properties of Azure B have motivated the employment of Azure B as a chromogenic reagent for the spectrophotometric determination of several compounds, which are relevant to biological and environmental chemistry such as periodate [105]. This cited method is simple and rapid, offering advantages of sensitivity and wide range of determinations, without involvement of any stringent reaction conditions, being successfully applied to the determination of periodate in solution and in several river water samples. In its time, Azure-C (AZC), and related phenothiazine compounds has been widely used for accelerating the

Thionine has been a subject of many studies, as for example in a photochemical and

positively charged tricyclic heteroaromatic molecule, which has been investigated for its

photoinduced inactivation of viruses [115]. Thionins consist of 45–47 residues bound by three to four disulfide bonds, which includes α1-purothionin, βPTH, and β-hordothionin (βHTH)

It has the ability to immobilize proteins and DNA and act as molecular adhesive [119]. Biophysical and calorimetric studies with three natural DNAs of varying base compositions, have shown the intercalative binding and high affinity of thionine to GC rich DNAs [120]. Thionine presented a high preference to the alternating GC sequences followed by the homo GC sequences contained in different synthetic polynucleotides [121]. AT polynucleotides presented a lower binding affinities but the alternating AT sequences had higher affinity compared to the homo stretches. The intercalation and the sequence of specific intercalative

110] and in photovoltaic cells [111]. Thionine is a

113], toxic effects, damage on binding to DNA [114] and

oxidation of NADH, but not in connection to the NAD+ reduction process.

107, 108, 109,

102]. Azure dyes (including Azure B) are recalcitrant compounds used in the textile

the hydrophilic behavior of the dye, such as Azure B and Azure BF4.

Studies based on absorbance, fluorescence, circular dichroic spectroscopy, viscosity, thermal melting and calorimetric techniques were used to understand the binding of thionine, with deoxyribonucleic acids of varying base composition, where strong binding of thionine to the DNAs were shown. Strong hypochromic and bathochromic effects and quenching of fluorescence were observed that showed strong binding of thionine to the DNAs [97]. The binding process is exothermic, which is associated to a large positive entropy changes and a negative enthalpy, and it showed that nonelectrostatic contributions are very important for the association of thionine to DNA. Studies on the interaction of thionine with sodium dodecylsulfate (SDS) micelles have shown that thionine binding affinity to SDS micelles was decreased with increasing temperature due to the thermal agitation [122].

The spectroscopic characteristics of thionine aggregates have shown that it depends on the concentration of thionine and on the chemical nature of the solvent [123]. Two peaks can be observed, at 597 nm and at the lower wavelength side of the 597 nm peak, and they related to the monomeric species and to the aggregate formation, respectively [124]. The understanding of the thionine aggregation process is very important for some application, such as in photovoltaic cells, where the reverse homogeneous redox reaction can be inhibited due to the presence of a surfactant in the system. The presence of a surfactant interferes in the thionine aggregation and polymerization process [125].

Several works about sensors have shown that the changing of spectroscopic and electrochemical properties of organic molecules, such as Toluidine Blue O (TBO), a phenothiazine dye, may indicate that there are some interaction with mediators and biological molecules [126, 127, 128]. Photochemical and electrochemical properties of TBO have been used to develop new photovoltaic devices for energy conversion and storage [129]. The aggregation behavior of such dyes in phase solution can be studied by using several optical rotation and circular dichroism techniques, as it can be seen in some studies of the interaction of TBO molecules on the DNA surface [130]. It was suggested that both intercalative and electrostatic interactions of TBO with DNA, where it was pointed out that the electrostatic interaction play an important role on the formation of the bridged structure of TBO with DNA [131].

TB can also be used as an oxygen radical inactivation, biological sensitizer and complexing agent in biological systems avoiding pathological changes [132]. Due to its low toxicity and high water solubility in salt form, which has an intense absorption peak in the visible region [133], it has been used in pharmaceutical formulations for cancer treatment [134]. Studies on the micellar solutions have shown that the aggregation properties and distribution behavior of toluidine blue in the presence of surfactant depend on the electrostatic interaction. In the case of surfactin, a natural surfactant, TB molecules can be located in the palisade of surfactin micelle [135].

Nile blue (NB) belongs to a class of molecules whose basic framework is that of a benzophenoxazine, a class which also includes Nile red, a phenoxazinone, here termed red Nile blue (RNB) and Meldola's Blue. It has been found to be localized selectively in animal tumors [136] and can retard tumor growth [137, 138]. NB has been used as a photosensitizer for oxygen in PDT applications [139, 140], in processes that depend on solvent polarity [141, 142], as a stain for Escherichia coli in flow cytometry [143], as a DNA probe [144] and many other applications [145, 146, 147, 148]. Due to their high fluorescence quantum yield together with their 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 disrupted resulting in an enhancement of the fluorescence intensity [150].

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 monocations with hydrogen ions [153].

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 works done on its interaction with DNA [155, 156, 157, 158]. In a work done on the interaction of 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 the charge injection process from the excited dye into the semiconductor nanocrystallites, acting as an electron acceptor. The transient absorption measurements of NB adsorbed on SiO2 colloids (inert support) show that the NB aggregates have a relatively short-lived excitonic singlet state ( = 40 ps) (Table 1). The lifetime of the excited singlet of the monomer in aqueous solution is 390 ps. NB aggregates that were immobilized on reactive surface also inject electrons into SnO2, resulting in the formation of the the cation radical, (NB)2 +, of the NB aggregates and by the trapping of electrons the in SnO2 nanocrystallites. The monophotonic dependence of the formation of (NB)2 + on SnO2 surface supports the charge transfer from NB aggregates to SnO2. The rate constant for this heterogeneous electron transfer process is 3.3.108 s-1 [160].
