On the mechanism of visible-light sensitized photosulfoxidation of toluidine blue O.

We report on the formation of toluidine blue O (TBO) sulfoxide by a self-sensitized photooxidation of TBO. Here, the photosulfoxidation process was studied by mass spectrometry (MS) and discussed in the context of photodemethylation processes which both contribute to TBO consumption over time. Analysis of solvent effects with D2O, H2O, and CH3CN along with product yields and MS fragmentation patterns provided mechanistic insight into TBO sulfoxide's formation. The formation of TBO sulfoxide is minor and detectable up to 12% after irradiation of 3 h. The photosulfoxidation process is dependent on oxygen wherein instead of a type II (singlet oxygen, 1O2) reaction, a type I reaction involving TBO to reach the TBO sulfoxide is consistent with the results. Density functional theory results point to the formation of the TBO sulfoxide by the oxidation of TBO via transiently formed peroxyl radical or thiadioxirane intermediates. We discover that the TBO photosulfoxidation arises competitively with TBO photodemethylation with the latter leading to formaldehyde formation.

Recent Photosensitizer Developments, Delivery Strategies and Combination-based Approaches for Photodynamic Therapy†.

Photodynamic therapy of cancer (PDT) is a therapeutic technique, minimally invasive, which is currently used to treat cancerous lesions and tumors that have been in the spotlight for its potential over the recent decades. Nonetheless, PDT still needs further development to become a first-option treatment for patients. This review compiles recent progress in several aspects of the current research in the constantly growing area of PDT to overcome the main challenges as an attempt to serve as a guide and reference for newcomers into this research area. This review has been prepared to highlight the use of chemical modifications on photosensitizers to improve their solubility, photostability, selectivity and phototoxicity. Additionally, the use of liposomes and cavitands as drug delivery systems to aid in the biodistribution and bioaccumulation of photosensitizers is presented. Also, the combination of PDT with chemotherapy or immunotherapy as an option to boost and improve treatment outcomes is discussed. Finally, the inhibition of antioxidant enzymes as a strategy for a synergistic effect to ameliorate the performance of the photosensitizers in PDT is presented as an alternative for future researchers.

Biosupramolecular complexes of amphiphilic photosensitizers with human serum albumin and cucurbit[7]uril as carriers for photodynamic therapy.

In the present work, we evaluated the supramolecular interactions between three photosensitizers, namely toluidine blue O (TBO, positively charged) and two fatty acid conjugates of 6 and 14 carbon atoms chain lengths (TBOC6 and TBOC14), with human serum albumin (HSA) and the macrocycle cucurbit[7]uril (CB[7]), alone or in combination within a biosupramolecular system as potential carriers of photosensitizers for Photodynamic therapy (PDT). Binding studies were carried out using photophysical and calorimetric techniques and accompanied with molecular docking simulations. Amphiphilic photosensitizers, particularly TBOC14, showed stronger binding to HSA and (CB[7]). Comparing the different delivery systems, (CB[7]) had a marginal effect on cell uptake and phototoxicity in HeLa cells, while HSA showed enhanced cell uptake with phototoxicities that depended on the photosensitizer. Despite low cell uptake, the combination of both (CB[7]) and HSA was the most phototoxic, which illustrates the potential of combining these systems for PDT applications.

Fatty Acid Conjugates of Toluidine Blue O as Amphiphilic Photosensitizers: Synthesis, Solubility, Photophysics and Photochemical Properties†.

Toluidine blue O (TBO) is a water-soluble photosensitizer that has been used in photodynamic antimicrobial and anticancer treatments, but suffers from limited solubility in hydrophobic media. In an effort to incrementally increase TBO's hydrophobicity, we describe the synthesis of hexanoic (TBOC6) and myristic (TBOC14) fatty acid derivatives of TBO formed in low to moderate percent yields by condensation with the free amine site. Covalently linking 6 and 14 carbon chains led to modifications of not only TBO's solubility, but also its photophysical and photochemical properties. TBOC6 and TBOC14 derivatives were more soluble in organic solvents and showed hypsochromic shifts in their absorption and emission bands. The solubility in phosphate buffer solution was low for both TBOC6 and TBOC14, but unexpectedly slightly greater in the latter. Both TBOC6 and TBOC14 showed decreased triplet excited-state lifetimes and singlet oxygen quantum yields in acetonitrile, which was attributed to heightened aggregation of these conjugates particularly at high concentrations due to the hydrophobic "tails." While in diluted aqueous buffer solution, indirect measurements showed similar efficiency in singlet oxygen generation for TBOC14 compared to TBO. This work demonstrates a facile synthesis of fatty acid TBO derivatives leading to amphiphilic compounds with a delocalized cationic "head" group and hydrophobic "tails" for potential to accumulate into biological membranes or membrane/aqueous interfaces in PDT applications.

Mechanism of Visible-Light Photooxidative Demethylation of Toluidine Blue O.

Experiments and theoretical calculations by density functional theory (DFT) have been carried out to examine a self-sensitized type I photooxidation of toluidine blue O (TBO+). This study attempts to build a connection between visible-light photolysis and demethylation processes of methylamine compounds, such as TBO+. We show that controlled photoinduced mono- and double-demethylation of TBO+ can be achieved. The kinetics for the appearance rate of the mono-demethylated TBO+ and the double-demethylated TBO+ were found to fit pseudo-first-order kinetics. DFT calculations have been used to examine the demethylation of TBO+ and included N, N-dimethylaniline as a model compound for TBO+. The results show an oxygen-dependent demethylation process. The mechanism for the sequential methyl loss is proposed to be due to H• or e-/H+ transfer to 3TBO+* followed by a reaction of TBO+• with O2, yielding a C-peroxyTBO+• intermediate. Instead of aminyl radical peroxyl formation, i.e., N-peroxyTBO+•, the C-centered peroxyTBO+• is favored, that upon dimerization (Russell mechanism) leads to dissociation of formaldehyde from the methylamine site.

Encapsulation of Chemotherapeutic Drug Melphalan in Cucurbit[7]uril: Effects on Its Alkylating Activity, Hydrolysis, and Cytotoxicity.

The formation of inclusion complexes between drugs and macrocycles has proven to be an effective strategy to increase solubilization and stabilization of the drug, while in several cases improving their biological activity. In this context, we explored the formation of an inclusion complex between chemotherapeutic drug Melphalan (Mel) and cucurbit[7]uril (CB[7]), and studied its effect on Mel alkylating activity, hydrolysis, and cytotoxicity. The formation of the inclusion complex (Mel@CB[7]) was proven by absorption and fluorescence spectroscopy, NMR, docking studies, and molecular dynamics simulations. The binding constant for Mel and CB[7] was fairly high at pH 1 ((1.7 ± 0.7) × 106 M-1), whereas no binding was observed at neutral pH. The Mel@CB[7] complex showed a slightly decreased alkylating activity, whereas the cytotoxicity on the HL-60 cell line was maintained. The formation of the complex did not protect Mel from hydrolysis, and this result is discussed based on the simulated structure for the complex.

Fluorescence properties of aurone derivatives: an experimental and theoretical study with some preliminary biological applications.

In this paper, we explored the fluorescence properties of eight aurone derivatives bearing methoxy groups and bromine atoms as substituents in the benzene rings. All derivatives showed strong solvatochromic absorption and emission properties in solvents of different polarities. Some of them showed high fluorescence quantum yields, which make them potential compounds for sensing applications. The position of the methoxy groups in the benzofuranone moiety and the presence of bromine atoms in the benzene ring had a strong influence on the fluorescence behaviour of the aurones. DFT calculations allowed us to explain the emission properties of aurones and their solvatochromism, which was related to an excited state with strong charge-transfer character. Aurone 4 has the most promising characteristics showing a large difference in the quantum yields and large Stokes shifts depending on the solvent polarities. These results prompted us to explore some preliminary biological applications for aurone 4 such as the sensing of hydrophobic pockets of a protein and its thermotropic behaviour in liposomes.

Photochemical behavior of biosupramolecular assemblies of photosensitizers, cucurbit[n]urils and albumins.

Biosupramolecular assemblies combining cucurbit[n]urils (CB[n]s) and proteins for the targeted delivery of drugs have the potential to improve the photoactivity of photosensitizers used in the photodynamic therapy of cancer. Understanding the complexity of these systems and how it affects the properties of photosensitizers is the focus of this work. We used acridine orange (AO+) as a model photosensitizer and compared it with methylene blue (MB+) and a cationic porphyrin (TMPyP4+). Encapsulation of the photosensitizers into CB[n]s (n = 7, 8) modified their photoactivity. In particular, for AO+, the photo-oxidation of HSA was enhanced in the presence of CB[7]; meanwhile it was decreased when included into CB[8]. Accordingly, peroxide generation and protein fragmentation were also increased when AO+ was encapsulated into CB[7]. The triplet excited state lifetimes of all the photosensitizers were lengthened by their encapsulation into CB[n]s, while the singlet oxygen quantum yield was enhanced only for AO+ and TMPyP4+, but it decreased for MB+. The results obtained in this work prompt the necessity of further investigating these kinds of hybrid assemblies as drug delivery systems because of their possible applications in biomedicine.