Mauricio S. Baptista

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+).

Protoporphyrin IX Nanoparticle Carrier: Preparation, Optical Properties, and Singlet Oxygen Generation

The present study is focused on developing a nanoparticle carrier for the photosensitizer protoporphyrin IX for use in photodynamic therapy. The entrapment of protoporphyrin IX (Pp IX) in silica spheres was achieved by modification of Pp IX molecules with an organosilane reagent. The immobilized drug preserved its optical properties and the capacity to generate singlet oxygen, which was detected by a direct method from its characteristic phosphorescence decay curve at near-infrared and by a chemical method using 1,3-diphenylisobenzofuran to trap singlet oxygen. The lifetime of singlet oxygen when a suspension of Pp IX-loaded particles in acetonitrile was excited at 532 nm was determined as 52 micros, which is in good agreement with the value determined for methylene blue in acetonitrile solution under the same conditions. The Pp IX-loaded silica particles have an efficiency of singlet oxygen generation (eta Delta) higher than the quantum yield of free porphyrins. This high efficiency of singlet oxygen generation was attributed to changes on the monomer-dimer equilibrium after photosentisizer immobilization.

Photodynamic Efficiency: From Molecular Photochemistry to Cell Death

Photodynamic therapy (PDT) is a clinical modality used to treat cancer and infectious diseases. The main agent is the photosensitizer (PS), which is excited by light and converted to a triplet excited state. This latter species leads to the formation of singlet oxygen and radicals that oxidize biomolecules. The main motivation for this review is to suggest alternatives for achieving high-efficiency PDT protocols, by taking advantage of knowledge on the chemical and biological processes taking place during and after photosensitization. We defend that in order to obtain specific mechanisms of cell death and maximize PDT efficiency, PSes should oxidize specific molecular targets. We consider the role of subcellular localization, how PS photochemistry and photophysics can change according to its nanoenvironment, and how can all these trigger specific cell death mechanisms. We propose that in order to develop PSes that will cause a breakthrough enhancement in the efficiency of PDT, researchers should first consider tissue and intracellular localization, instead of trying to maximize singlet oxygen quantum yields in in vitro tests. In addition to this, we also indicate many open questions and challenges remaining in this field, hoping to encourage future research.

Giant vesicles under oxidative stress induced by a membrane-anchored photosensitizer.

We have synthesized the amphiphile photosensitizer PE-porph consisting of a porphyrin bound to a lipid headgroup. We studied by optical microscopy the response to light irradiation of giant unilamellar vesicles of mixtures of unsaturated phosphatidylcholine lipids and PE-porph. In this configuration, singlet oxygen is produced at the bilayer surface by the anchored porphyrin. Under irradiation, the PE-porph decorated giant unilamellar vesicles exhibit a rapid increase in surface area with concomitant morphological changes. We quantify the surface area increase of the bilayers as a function of time and photosensitizer molar fraction. We attribute this expansion to hydroperoxide formation by the reaction of the singlet oxygen with the unsaturated bonds. Considering data from numeric simulations of relative area increase per phospholipid oxidized (15%), we measure the efficiency of the oxidative reactions. We conclude that for every 270 singlet oxygen molecules produced by the layer of anchored porphyrins, one eventually reacts to generate a hydroperoxide species. Remarkably, the integrity of the membrane is preserved in the full experimental range explored here, up to a hydroperoxide content of 60%, inducing an 8% relative area expansion.

Major determinants of photoinduced cell death: Subcellular localization versus photosensitization efficiency

We present a study on whether and to what extent subcellular localization may compete favorably with photosensitization efficiency with respect to the overall efficiency of photoinduced cell death. We have compared the efficiency with which two cationic photosensitizers, namely methylene blue (MB) and crystal violet (CV), induce the photoinduced death of human cervical adenocarcinoma (HeLa) cells. Whereas MB is well known to generate singlet oxygen and related triplet excited species with high quantum yields in a variety of biological and chemical environments (i.e., acting as a typical type II photosensitizer), the highly mitochondria-specific CV produces triplet species and singlet oxygen with low yields, acting mostly via the classical type I mechanism (e.g., via free radicals). The findings described here indicate that the presumably more phototoxic type II photosensitizer (MB) does not lead to higher degrees of cell death compared to the type I (CV) photosensitizer. In fact, CV kills cells with the same efficiency as MB, generating at least 10 times fewer photoinduced reactive species. Therefore, subcellular localization is indeed more important than photochemical reactivity in terms of overall cell killing, with mitochondrial localization representing a highly desirable property for the development of more specific/efficient photosensitizers for photodynamic therapy applications.

Solvent and concentration effects on the visible spectra of tri-para-dialkylamino-substituted triarylmethane dyes in liquid solutions.

We have characterized the spectroscopy properties of crystal violet (CV+) and ethyl violet (EV+) in liquid solutions as a function of the solvent type and dye concentration. The analysis of how solvent properties and dye concentration affects the electronic spectra of these tri-para-dialkylamino substituted tryarylmethane (TAM+) dyes was performed on the basis of two spectroscopic parameters, namely the difference in wavenumber (deltanu) between the maximum and the shoulder that appears in the short-wavelength side of the respective maximum visible band (deltanu = 1/lambda(shoulder)-1/lambda(max) cm(-1)), and the wavelength of the maximum absorption (lambda(max)). The solvent and the concentration effects on lambda(max) and deltanu have indicated that both solute/solute (ion-pairing and dye aggregation) and solute/solvent (H-bonding type) interactions modulate the shape of the visible electronic spectra of these dyes in solution. In solvent with small dieletric constant (epsilon < approximately 10), the formation of ion-pairs represents a major contribution to the shaping of these spectra. Upon increasing dye concentration the formation of ion-pairs was characterized by an increase in deltanu observed concomitantly with a red shift in lambda(max) In chloroform and chlorobenzene the ion-pair association constant of CV+ and EV+ with Cl- ions were found to be in the order of 10(6) and 10(5) M(-1), respectively. In trichloroethylene the association constant for the CV+Cl- pair was 10(8) M(-1). In water, dye aggregation instead of ion-pairing represents a major contribution to the shaping of the visible spectra of CV+ and EV+. Dye aggregation was indicated by an increase in deltanu observed concomitantly with a blue shift in lambda(max) upon increasing dye concentration. The distinct behavior of deltanu for dye aggregation and ion-pairing as a function of dye concentration can therefore assist in the characterization of these two distinct phenomena. The solute/solvent interactions were studied in a series of polar solvents in which solute/solute interactions do not occur in any detectable extent. The dependence found for deltanu as a function of the Kamlet-Tafts solvatochromic parameters (alpha, beta and pi*) is in keeping with previous inferences indicating that the splitting in the overlapped absorption band of CV+ and EV+ in hydroxilated solvents arises from a perturbation in the molecular symmetry induced by hydrogen bonding (donor-acceptor) type interactions with solvent molecules. A distinction between the effects of solute/solute and solute/solvent interactions on the visible spectra of these dyes is provided.

Chlorin photosensitizers sterically designed to prevent self-aggregation.

The synthesis and photophysical evaluation of new chlorin derivatives are described. The Diels-Alder reaction between protoporphyrin IX dimethyl ester and substituted maleimides furnishes endo-adducts that completely prevent the self-aggregation of the chlorins. Fluorescence, resonant light scattering (RLS) and (1)H NMR experiments, as well as X-ray crystallographic have demonstrated that the configurational arrangement of the synthesized chlorins prevent π-stacking interactions between macrocycles, thus indicating that it is a nonaggregating photosensitizer with high singlet oxygen (Φ(Δ)) and fluorescence (Φ(f)) quantum yields. Our results show that this type of synthetic strategy may provide the lead to a new generation of PDT photosensitizers.