Divinomar Severino

Methylene blue in photodynamic therapy: From basic mechanisms to clinical applications

Methylene blue (MB) is a molecule that has been playing important roles in microbiology and pharmacology for some time. It has been widely used to stain living organisms, to treat methemoglobinemia, and lately it has been considered as a drug for photodynamic therapy (PDT). In this review, we start from the fundamental photophysical, photochemical and photobiological characteristics of this molecule and evolved to show in vitro and in vivo applications related to PDT. The clinical cases shown include treatments of basal cell carcinoma, Kaposis Sarcoma, melanoma, virus and fungal infections. We concluded that used together with a recently developed continuous light source (RL50(®)), MB has the potential to treat a variety of cancerous and non-cancerous diseases, with low toxicity and no side effects.

Binding, aggregation and photochemical properties of methylene blue in mitochondrial suspensions.

Abstract Methylene Blue (MB) has well-established photochemical properties and has been used in a variety of photochemical applications including photodynamic therapy. Despite the fact that most of MBs cytotoxic effects in cells are attributed to mitochondrial damage, the interactions of this dye with mitochondria and the consequent effects on photochemical properties have not yet been fully determined. We monitored MB binding, aggregation and its ability to release singlet oxygen (1O2) on irradiation when interacting with mitochondrial suspensions. MB actively binds to mitochondria and enters the matrix in a manner stimulated by the mitochondrial proton potential and by the increase in mitochondrial concentrations. The greater accumulation of MB in mitochondria with elevated proton potentials or those treated with high concentrations of MB results in the formation of MB dimers, previously shown to be less effective generators of 1O2. Accumulation of MB within mitochondria with high membrane potentials also results in the reduction of MB to the photochemically inactive leuco-MB. Indeed, irradiation of mitochondria with high proton potentials in the presence of MB results in the generation of approximately half the quantity of 1O2 compared with 1O2 generated in mitochondria with low proton potentials. These differences in photochemical properties should influence the cytotoxic effects of photodynamic treatment in the presence of MB.

Influence of Negatively Charged Interfaces on the Ground and Excited State Properties of Methylene Blue

Abstract Properties of the ground and excited states of methylene blue (MB) were studied in negatively charged vesicles, normal and reverse micelles and sodium chloride solutions. All these systems induce dimer formation as attested by the appearance of the dimer band in the absorption spectra (λD ∼ 600 nm). In reverse micelles the dimerization constant (KD) corrected for the aqueous pseudophase volume fraction is two–three orders of magnitude smaller than KD of MB in water, and it does not change when W0 is increased from 0.5 to 10. Differences in the fluorescence intensity as a function of dimer–monomer ratio as well as in the resonance light scattering spectra indicate that distinct types of dimers are induced in sodium dodecyl sulfate (SDS) micelles and aerosol-OT (sodium dioctyl sulfoxinate, AOT) reversed micelles. The properties of the photoinduced transient species of MB in these systems were studied by time-resolved near infrared (NIR) emission (efficiency of singlet oxygen generation), by laser flash photolysis (transient spectra, yield and decay rate of triplets) and by thermal lensing (amount of heat deposited in the medium). The competition between electron transfer (dye*–dye) and energy transfer (dye*–O2) reactions was accessed as a function of the dimer–monomer ratio. The lower yield of electron transfer observed for dimers in AOT reverse micelles and intact vesicles compared with SDS micelles and frozen vesicles at similar dimer–monomer ratios is related with the different types of aggregates induced by each interface.

Modulation of methylene blue photochemical properties based on adsorption at aqueous micelle interfaces

Methylene Blue (MB+) is a sensitizer that has been used for a variety of applications including energy conversion and photodynamic therapy (PDT). Although its photochemical properties in isotropic solution are well established, its effect in vivo and in restricted reaction environments is somewhat erratic. In order to understand its photochemical behavior when it interacts with biomolecules, in particular with membranes, MB+ properties were studied in sodium dodecyl sulfate (SDS) and cetyl trimethylammonium bromide (CTAB) solutions. Because of an electrostatic attraction, SDS and MB+ form complexes, changing the properties of both the micelles and the MB+ solutions. Surface tension measurements show that the c.m.c. of SDS decreases from ∼7 mM to ∼70 μM when the MB+ concentration increases from 0 to 45 μM. Above the c.m.c., binding of MB+ in the micelle pseudo-phase causes the formation of aggregates (mostly dimers) as attested by the increase in the absorption at 580 nm and the decrease in fluorescence emission. The extent of dimer formation is dependent on the relative concentrations of MB+ and SDS. In the presence of excess of SDS, MB+ is mainly in the monomer form and at low SDS concentration dimers are favored. Such effect, which was not observed in CTAB micelles, was modeled qualitatively by considering that MB+ molecules partition to the micelle pseudo-phase which favors or disfavors dimers as a function of its volume. MB+ transient species were characterized by laser flash photolysis and NIR emission showing the presence of triplets and subsequently singlet oxygen at high SDS concentration and semi-reduced and semi-oxidized MB+ radicals at low SDS concentration. Therefore it was shown that, depending on the ground state MB+ monomer/dimer equilibrium, induced by the micelles, the photochemical properties of MB+ can be shifted from a Type II (energy transfer to oxygen forming singlet oxygen) to a Type I mechanism (electron transfer forming the semi-reduced and the semi-oxidized radicals of MB+).

Melanin Photosensitization and the Effect of Visible Light on Epithelial Cells

Protecting human skin from sun exposure is a complex issue that involves unclear aspects of the interaction between light and tissue. A persistent misconception is that visible light is safe for the skin, although several lines of evidence suggest otherwise. Here, we show that visible light can damage melanocytes through melanin photosensitization and singlet oxygen (1O2) generation, thus decreasing cell viability, increasing membrane permeability, and causing both DNA photo-oxidation and necro-apoptotic cell death. UVA (355 nm) and visible (532 nm) light photosensitize 1O2 with similar yields, and pheomelanin is more efficient than eumelanin at generating 1O2 and resisting photobleaching. Although melanin can protect against the cellular damage induced by UVB, exposure to visible light leads to pre-mutagenic DNA lesions (i.e., Fpg- and Endo III-sensitive modifications); these DNA lesions may be mutagenic and may cause photoaging, as well as other health problems, such as skin cancer.

Spectrofluorimetric Determination of Second Critical Micellar Concentration of SDS and SDS/Brij 30 Systems

Potentially useful stead-state fluorimetric technique was used to determine the critical micellar concentrations (CMC1 and CMC2) for two micellar media, one formed by SDS and the other by SDS/Brij 30. A comparative study based on conductimetric and surfacial tension measurements suggests that the CMC1 estimated by the fluorimetric method is lower than the value estimated by these other techniques. Equivalent values were observed for SDS micelles without Brij 30 neutral co-surfactant. The use of acridine orange as fluorescent probe permitted to determine both CMC1 and CMC2. Based on it an explanation on aspects of micelle formation mechanism is presented, particularly based on a spherical and a rod like structures.

Generation and suppression of singlet oxygen in hair by photosensitization of melanin.

We have studied the spectroscopic properties of hair (white, blond, red, brown, and black) under illumination with visible light, giving special emphasis to the photoinduced generation of singlet oxygen ((1)O(2)). Irradiation of hair shafts (λ(ex)>400 nm) changed their properties by degrading the melanin. Formation of C3 hydroperoxides in the melanin indol groups was proven by (1)H NMR. After 532-nm excitation, all hair shafts presented the characteristic (1)O(2) emission (λ(em)=1270 nm), whose intensity varied inversely with the melanin content. (1)O(2) lifetime was also shown to vary with hair type, being five times shorter in black hair than in blond hair, indicating the role of melanin as a (1)O(2) suppressor. Lifetime ranged from tenths of a nanosecond to a few microseconds, which is much shorter than the lifetime expected for (1)O(2) in the solvents in which the hair shafts were suspended, indicating that (1)O(2) is generated and suppressed inside the hair structure. Both eumelanin and pheomelanin were shown to produce and to suppress (1)O(2), with similar efficiencies. The higher amount of (1)O(2) generated in blond hair and its longer lifetime is compatible with the stronger damage that light exposure causes in blond hair. We propose a model to explain the formation and suppression of (1)O(2) in hair by photosensitization of melanin with visible light and the deleterious effects that an excess of visible light may cause in hair and skin.

Rapid screening of potential autophagic inductor agents using mammalian cell lines

Recent progress in understanding the molecular basis of autophagy has demonstrated its importance in several areas of human health. Affordable screening techniques with higher sensitivity and specificity to identify autophagy are, however, needed to move the field forward. In fact, only laborious and/or expensive methodologies such as electron microscopy, dye‐staining of autophagic vesicles, and LC3‐II immunoblotting or immunoassaying are available for autophagy identification. Aiming to fulfill this technical gap, we describe here the association of three widely used assays to determine cell viability – Crystal Violet staining (CVS), 3‐[4, 5‐dimethylthiaolyl]‐2, 5‐diphenyl‐tetrazolium bromide (MTT) reduction, and neutral red uptake (NRU) – to predict autophagic cell death in vitro. The conceptual framework of the method is the superior uptake of NR in cells engaging in autophagy. NRU was then weighted by the average of MTT reduction and CVS allowing the calculation of autophagic arbitrary units (AAU), a numeric variable that correlated specifically with the autophagic cell death. The proposed strategy is very useful for drug discovery, allowing the investigation of potential autophagic inductor agents through a rapid screening using mammalian cell lines B16‐F10, HaCaT, HeLa, MES‐SA, and MES‐SA/Dx5 in a unique single microplate.

Singlet oxygen quantum yields (Φd) in water using beetroot extract and an array of LEDs

It is proposed a simple and inexpensive strategy to determine singlet oxygen (1O2) quantum yields (ΦΔ) of photosensitizers (PS) in water using beetroot extract containing betacyanin (Bc) and a set of light emitting diodes (LEDs) for excitation. Bc, a cationic natural dye, was obtained by flash chromatography purification from the red beet extract (Beta vulgaris) and employed as a convenient probe for 1O2 detection. Solutions of Bc and PS were illuminated with an array of LEDs adapted in the cuvette compartment of a commercial spectrophotometer, and the decrease in Bc absorbance was followed as a function of time. Bc photobleaching decreased in de-aerated solution and increased in D2O, indicating the involvement of 1O2. The observed photobleaching rate constant (kobs) was proportional to the LED intensity, concentration and ΦΔ of the PS. By keeping the light source constant we could estimate the overlap integral (R) between the LED emission and PS absorbance for different PS concentrations. The slope of R versus kobs is the value of the photobleaching rate constant (k), which was shown to be proportional to ΦΔ. Values of ΦΔ obtained by this method were compared with those obtained by measuring NIR (near infrared) emission for a series phenothiazine dyes.

Langmuir films of petroleum at the air-water interface.

Understanding the behavior of petroleum films at the air/water interface is crucial for dealing with oil slicks and reducing the damages to the environment, which has normally been attempted with studies of Langmuir films made of fractions of petroleum. However, the properties of films from whole petroleum samples may differ considerably from those of individual fractions. Using surface pressure and surface potential measurements and Brewster angle and fluorescence microscopy, we show that petroleum forms a nonhomogeneous Langmuir film at the air-water interface. The surface pressure isotherms for petroleum Langmuir films exhibit gas (G), liquid-expanded (LE), and liquid-condensed phases, with almost no hysteresis in the compression-decompression cycles. Domains formed upon compression from the G to the LE phase were accompanied by an increase in fluorescence intensity with excitation at 400-440 nm owing to an increase in the surface density of the chromophores in the petroleum film. The surface pressure and the fluorescence microscopy data pointed to self-assembling domains into a pseudophase in thermodynamic equilibrium with other less emitting petroleum components. This hypothesis was supported by Brewster angle microscopy images, whereby the appearance of water domains even at high surface pressures confirms the tendency of petroleum to stabilize emulsion systems. The results presented here suggest that, for understanding the interaction with water, it may be more appropriate to use the whole petroleum samples rather than its fractions.