Phenothiazine Dyes Research Articles

LED antimicrobial photodynamic therapy with phenothiazinium dye against Staphylococcus aureus: An in vitro study

The objective of this study was to evaluate, in vitro, the bactericidal effect of AmPDT on Staphylococcus aureus (ATCC 25923) using different concentrations (100, 50, 25, 12.5 and 6.25μg/mL) of phenothiazine compound combined with LED light (λ632±2nm) using varied energy densities (12, 9.6, 7.2, 4.8 and 2.4J/cm2). The experiments were carried out in triplicate and the samples were divided into groups: Control, Irradiated (treated only with light at different energy densities), Photosensitizer (treated only in the presence of the dye), AmPDT (treatment with light associated with dye). Counts of the colony forming units and the data obtained were statistically analyzed (ANOVA, Tukey's test, p<0.05). The results showed no difference between irradiated and Control groups. However, using the photosensitizer alone caused significant increased cytotoxicity and consequent reduction on the CFU counts (12.5μg/mL (p<0.001), 25μg/mL, 50μg/mL and 100μg/mL (p<0.0001). When AmPDT was used significant inhibition above 70% were detected for all concentrations of the photosensitize (p<0.0001) except for 6.25μg/mL. The results indicate a dose-response dependent when the photosensitizer is used alone but not for the sole use of the light is used. It is concluded that, a single application of AmPDT, using energy density of 12J/cm2 associated either to 12.5 (81.52%) or 25μg/mL (91.57%) resulted in higher in vitro inhibition of S. aureus.

Additive-induced aggregate changes of two structurally similar dyes in aqueous solutions: A comparative photophysical study

Absorption and emission spectral characteristics of the two structurally similar phenothiazine dyes, azure B and toluidine blue, in aqueous solutions of the two sets of molecular additives (ureas and monosaccharides) were studied as a function of the dye and additive concentrations. The absorption spectra of the dyes were also studied in pure tetramethylurea with an aprotic nature. The spectral data were analyzed using DECOM Program. The dimer structure of the interacting molecules in these dyes was discussed using the exciton model. The urea class of additives was found to act as water structure-breakers over the range of studied concentration. The carbohydrate additives were found to act as water structure-breakers at low concentrations. However, the water structure breaking process may be disfavored by the additive-additive interactions at higher concentrations. It can be concluded that at low additive concentrations, the main driving force for breaking the dye association is water-additive interaction, which disrupts the water hydrogen bonds induced by the additives. However, at the high additive concentrations, the different phenomena including additive-additive and additive-dye interactions can change the structure, strength, and aggregative properties of the dyes. Finally, the urea in water induces noticeably fluorescence quenching in emission spectra of both the dyes.

Dual-stimulus-responsive crosslinking chitosan as a gene vector

Absorption and emission spectral characteristics of the two structurally similar phenothiazine dyes, azure B and toluidine blue, in aqueous solutions of the two sets of molecular additives (ureas and monosaccharides) were studied as a function of the dye and additive concentrations. The absorption spectra of the dyes were also studied in pure tetramethylurea with an aprotic nature. The spectral data were analyzed using DECOM Program. The dimer structure of the interacting molecules in these dyes was discussed using the exciton model. The urea class of additives was found to act as water structure-breakers over the range of studied concentration. The carbohydrate additives were found to act as water structure-breakers at low concentrations. However, the water structure breaking process may be disfavored by the additive-additive interactions at higher concentrations. It can be concluded that at low additive concentrations, the main driving force for breaking the dye association is water-additive interaction, which disrupts the water hydrogen bonds induced by the additives. However, at the high additive concentrations, the different phenomena including additive-additive and additive-dye interactions can change the structure, strength, and aggregative properties of the dyes. Finally, the urea in water induces noticeably fluorescence quenching in emission spectra of both the dyes.

International, prospective haemovigilance study on methylene blue-treated plasma.

Methylene blue is a phenothiazine dye, which in combination with visible light has virucidal and bactericidal properties, disrupting the replication of a broad range of enveloped viruses and some non-enveloped viruses. The study objective was to collect data on adverse reactions occurring with methylene blue plasma administered in a routine clinical practice environment and document their characteristics and severity. This was an open label, multicentre, non-controlled, non-randomized, non-interventional study. Patients who receive a methylene blue plasma transfusion were observed for any signs and symptoms (adverse reactions) within 24 h safter the start of the transfusion, in different hospitals for a study duration of at least 1 year. A total of 19 315 methylene blue plasma units were transfused. There were eight patients with adverse reactions recorded during the study, one of them serious. Two had more than one reaction (two and four, respectively). Three patients had previous transfusions with methylene blue plasma only. Methylene blue plasma has a very acceptable safety profile with a rate of serious adverse reactions of 0·5/10 000 units.

Phenothiazine dyes photosensitize protein damage through electron transfer and singlet oxygen generation

Photosensitized protein damage is an important process in medical applications of photochemical reaction, such as photodynamic therapy. A similar method has been applied to photodynamic antimicrobial chemotherapy (PACT), which can treat sinusitis and periodontal disease. For example, methylene blue is used as a photosensitizer for PACT. However, the mechanism of PACT has not been clarified completely. In this study, protein damage photosensitized by methylene blue and its analogues was examined. The scavenger effect on protein photodamage by dyes and fluorescence measurements suggests the contribution of the electron transfer mechanism to this photosensitized protein damage as the singlet oxygen generation mechanism. The quantum yields of protein damage by these photosensitizers could be determined. The electron transfer mechanism is the direct electron abstraction from the biomolecule to the photoexcited photosensitizer. Because this mechanism does not necessarily require oxygen in vivo, the mechanism of PACT, such as in sinusitis treatment by methylene blue under a hypoxic condition, might be explained by the electron transfer.

Comparison of photoinactivation of T. rubrum by new methylene blue (NMB) and indocyanine green (EmunDo®)

BackgroundSuperficial mycotic skin infections which are predominantly caused by Trichophyton rubrum, poorly responsd to conventional therapies. A great amount of attention has focused on finding more effective treatments. The current work is aimed to compare the effectiveness of phoinactivation of Trichophyton rubrum by two relatively new photosensitizers: a phenothiazinium dye(New methylene blue) and Indocyanine green (EmunDo®). Materials and methodsA Final inoculum of T. rubrum which corresponded to 106 colony forming unit per milliliter (CFUml−1) was prepared. Antimicrobial Photodynamic treatment (aPDT) of T. rubrum was carried out by either EmunDo® (1mg/ml, Infra-red laser (IRL, λ=810nm, Energy Density 55J/cm2)) or NMB (10μM, Red laser (RL), λ=630nm, Energy Density of 5J/cm2). The suspensions thereafter were subcultured on Sabouraud dextrose agar (SDA) and were counted on due time. based on colony-forming unit per milliliter (CFU/ml). ResultsaPDT with either EmunDo® (E) or NMB (N) considerably diminished the viability of inoculated T. rubrum with respective reduction of 0.64 log and 0.4 log compared to the control group (P<0.001). No significant difference was found between two laser only groups (P=0.79) and two aPDT groups (P=0.73), however significant reduction of T. rubrum in red laser only group (P=0.04) and EmunDo® only group (P=0.04) was found as compared to the control group (P<0.05). ConclusionThe study provides evidence regarding satisfactory photodynamic inactivation of T. rubrum with EmunDo® or NMB as photosensitizers. Irradiation by only red laser source was found superior to only infra-red laser source. Dark toxicity of EmunDo® was more successful than new methylene blue dye.

A comparative spectroscopic study of thiourea effect on the photophysical and molecular association behavior of various phenothiazine dyes

This paper describes the role of a structure breaking additive (thiourea) on the photophysical and molecular association of a series of phenothiazine dyes in aqueous media using the absorption and fluorescence spectroscopic methods for the first time. The studied dyes were thionine, azure A, azure B, toluidine blue, and methylene blue. The spectral data were analyzed using DECOM program. Relevant spectral parameters in the dye solutions were estimated and discussed based on the chemical structure of the additive and excitonic treatment. The observation of spectral changes in the spectral data of the (water-additive-dye) system indicates the possible structure formation between the dye and additive molecules. However, it is found that in the high concentrations of thiourea the dimer geometries (H- and J-type dimers) are influenced by the dye-additive interactions. As a result, a competition between the dye-additive and dye-dye interactions was also observed.

A Self‐Powered Ethanol Biosensor

AbstractWe describe the fabrication of a self‐powered ethanol biosensor comprising a β‐NAD+‐dependent alcohol dehydrogenase (ADH) bioanode and a bienzymatic alcohol oxidase (AOx) and horseradish peroxidase (HRP) biocathode. β‐NAD+ is regenerated by means of a specifically designed phenothiazine dye (i. e. toluidine blue, TB) modified redox polymer in which TB was covalently anchored to a hexanoic acid tethered poly(4‐vinylpyridine) backbone. The redox polymer acts as an immobilization matrix for ADH. Using a carefully chosen anchoring strategy through the formation of amide bonds, the potential of the TB‐based mediator is shifted to more positive potentials, thus preventing undesired O2 reduction. To counterbalance the rather high potential of the TB‐modified polymer, and thus the bioanode, a high‐potential AOx/HRP‐based biocathode is suggested. HRP is immobilized in a direct‐electron‐transfer regime on screen‐printed graphite electrodes functionalized with multi‐walled carbon nanotubes. The nanostructured cathode ensures the wiring of the iron‐oxo complex within oxidized HRP, and thus a high potential for the reduction of H2O2 of about +550 mV versus Ag/AgCl/3 M KCl. The proposed biofuel cell exhibits an open‐circuit voltage (OCV) of approximately 660 mV and was used as self‐powered device for the determination of the ethanol content in liquor.

A comparison of antibacterial and antibiofilm efficacy of phenothiazinium dyes between Gram positive and Gram negative bacterial biofilm

BackgroundAntimicrobial photodynamic therapy (APDT) is a process that generates reactive oxygen species (ROS) in presence of photosensitizer, visible light and oxygen which destroys the bacterial cells. We investigated the photoinactivation efficiency of phenothiazinium dyes and the effect of ROS generation on Gram positive and Gram negative bacterial cell as well as on biofilm. Material and methodsEnterococcus faecalis and Klebsiella pneumonia were incubated with all the three phenothiazinium dyes and exposed to 630nm of light. After PDT, colony forming unit (CFU) were performed to estimate the cell survival fraction. Intracellular reactive oxygen species (ROS) was detected by DCFH-DA. Crystal violet (CV) assay and extracellular polysaccharides (EPS) reduction assay were performed to analyze antibiofilm effect. Confocal laser electron microscope (CLSM) scanning electron microscope (SEM) was performed to assess the disruption of biofilm. Results8log10 reduction in bacterial count was observed in Enterococcus faecalis while 3log10 in Klebsiella pneumoniae. CV and EPS reduction assay revealed that photodynamic inhibition was more pronounced in Enterococcus faecalis. In addition to this CLSM and SEM study showed an increase in cell permeability of propidium iodide and leakage of cellular constituents in treated preformed biofilm which reflects the antibiofilm action of photodynamic therapy. ConclusionWe conclude that Gram-positive bacteria (Enterococcus faecalis) are more susceptible to APDT due to increased level of ROS generation inside the cell, higher photosensitizer binding efficiency and DNA degradation. Phenothiazinium dyes are proved to be highly efficient against both planktonic and biofilm state of cells.

Synthesis of a dibenzo-BODIPY-incorporating phenothiazine dye as a panchromatic sensitizer for dye-sensitized solar cells

The introduction of dibenzo-BODIPY into a phenothiazine dye led to the production of a panchromatic sensitizer for dye-sensitized solar cells, a cell with the dye showing a PCE (η) value of 7.69%.

Pseudocapacitive-dye-molecule-based high-performance flexible supercapacitors.

Currently, the development of novel, cheap and high-performance electrode materials for flexible energy-storage devices is being intensively pursued. Organic dyes are regarded as one of the most common water pollution sources, and their discharge not only damages the ecological balance but also wastes a lot of valuable chemical raw materials. With intrinsic redox functional groups, two types of widely used dyes including anthraquinone derivatives and phenothiazine dyes can be effectively removed from their effluent by a facile galvanostatic polymerization method, and resource utilized as electrochemical energy-storage materials. Based on this, a new kind of flexible supercapacitor based on dye molecules has been successfully fabricated, and exhibits excellent electrochemical performance, demonstrating a wise strategy for simultaneous wastewater treatment and preparation of functional devices.

Exploring the interaction of phenothiazinium dyes methylene blue, new methylene blue, azure A and azure B with tRNAPhe: spectroscopic, thermodynamic, voltammetric and molecular modeling approach

This study focuses on the understanding of the interaction of phenothiazinium dyes methylene blue (MB), new methylene blue (NMB), azure A (AZA) and azure B (AZB) with tRNAPhe with particular emphasis on deciphering the mode and energetics of the binding. Strong intercalative binding to tRNAPhe was observed for MB, NMB and AZB, bound by a partial intercalative mode. AZA has shown groove binding characteristics. From spectroscopic studies binding affinity values of the order of 105 M-1 were deduced for these dyes; the trend varied as MB > NMB > AZB > AZA. The binding was characterized by an increase of thermal melting temperatures and perturbation in the circular dichroism spectrum of tRNA. All the dyes acquired optical activity upon binding to tRNA. The binding was predominantly entropy driven with a favorable enthalpy term that increased with temperature in all the cases. Dissection of the Gibbs energy to polyelectrolytic and non-polyelectrolytic terms revealed a major role of the non-electrostatic forces in the binding. The small but significant heat capacity changes and the observed enthalpy-entropy compensation phenomenon confirmed the involvement of multiple weak non-covalent forces driving the interaction. The mode of binding was confirmed from quenching, viscosity and cyclic voltammetric results. Using density functional theory, ground state optimized structures of the dyes were calculated to provide insight into theoretical docking studies to correlate the experimental approaches. The modeling results verified the binding location as well as the binding energy of complexation. The results may provide new insights into the structure-activity relationship useful in the design of effective RNA targeted therapeutic agents.

The photodynamic efficiency of phenothiazinium dyes is aggregation dependent

Effectiveness increased in the order of Azure A &lt; Azure B &lt; Methylene Blue while aggregation increased in the order of Methylene Blue &lt; Azure B &lt; Azure A.

Binding interaction of phenothiazinium dyes with double stranded RNAs: Spectroscopic and calorimetric investigation

RNA targeting through small molecules is an emerging and promising therapeutic route that necessitates identification of small molecules that can selectively target specific RNA structures. In this context a comparative study of the interaction of two phenothiazinium dyes thionine (THN) and toluidine blue O (TBO) with three double stranded RNA polynucleotides (ds RNAs) viz. poly(I).poly(C), poly(A).poly(U) and poly(C).poly(G) was conducted by various biophysical techniques. A higher binding of THN with poly(I).poly(C) over poly(A).poly(U) and poly(C).poly(G) was observed. The intercalative binding and RNA induced fluorescence quenching of the dyes through a static mechanism was confirmed by viscosity studies and steady state polarization anisotropy experiments. Binding induced structural perturbation in the RNA polynucleotides was confirmed from circular dichroism spectroscopy. DSC and thermal melting experiments confirmed that the binding resulted in strong thermal stabilization. The binding affinity of THN with poly(I).poly(C) was the highest followed by that to poly(A).poly(U) and poly(C).poly(G). The trend was the same for TBO also, but THN bound stronger than TBO. The binding of the dyes was characterized by strong negative enthalpy changes with minimum positive entropy changes indicating typical intercalative interaction. The results presented here may be useful to design new types of RNA binding antitumor, antibacterial and anticancer agents.

ChemInform Abstract: Recent Advances in Phenothiazine‐Based Dyes for Dye‐Sensitized Solar Cells

AbstractReview: 119 refs.

Attenuation of synaptic toxicity and MARK4/PAR1-mediated Tau phosphorylation by methylene blue for Alzheimer's disease treatment.

Alzheimer’s disease (AD) is a neurodegenerative disease characterized by genotypic and phenotypic heterogeneity. Critical components of the two AD pathological pathways, Aβ-amyloidosis and Tauopathy, have been considered as therapeutic targets. Among them, much effort is focused on aberrant Tau phosphorylation and targeting Tau-phosphorylating kinases. Methylene blue (MB), a phenothiazine dye that crosses the blood-brain barrier, has been shown to hit multiple molecular targets involved in AD and have beneficial effects in clinical studies. Here we present evidence that microtubule affinity-regulating kinase (MARK4) is a novel target of MB. MB partially rescued the synaptic toxicity in Drosophila larva overexpressing PAR1 (MARK analog). In 293T culture, MB decreased MARK4-mediated Tau phosphorylation in a dose dependent manner. Further studies revealed a two-fold mechanism by MB including down-regulation of MARK4 protein level through ubiquitin-proteasome pathway and inhibition of MARK4 kinase activity in vitro. This study highlights the importance of MARK4 as a viable target for Tauopathy and provides fresh insight into the complex mechanism used by MB to treat AD.

Effectiveness of antimicrobial photodynamic therapy (AmPDT) on Staphylococcus aureus using phenothiazine compound with red laser

The aim of this study was to evaluate, in vitro, the bactericidal effect of antimicrobial photodynamic therapy (AmPDT) using phenothiazinium dyes (Toluidine Blue O and methylene blue, 1:1) using different concentrations (100, 50, 25, 12.5, and 6.25μg/mL) associated to red laser with different energy densities (2.4, 4.8, 7.2, 9.6, and 12J/cm2) on a strain of Staphylococcus aureus (ATCC 23529). On this study, tests were performed in triplicate and the samples were distributed into 36 test groups: Control and bacterial suspensions were irradiated with the different energy densities, respectively, in the absence of photosensitizer, bacterial suspensions were irradiated with the laser in the different concentrations of the photosensitizer, and finally bacterial suspensions only in the presence of phenothiazinium dye. The pre-irradiation time was 5min. Therefore, we analyzed the potential of the AmPDT by counting colony-forming units. The logarithm of CFU/mL (log10 CFU/mL) was calculated and the data was analyzed statistically (ANOVA, Tukey's test, p < 0.05). The results showed that the association 50 and 100μg/mL with 12J/cm2 showed the highest percentage of inhibition (100%). Based upon the present results, it may be concluded that the AmPDT was able to enhance the antimicrobial effect of phenothiazines and both concentration of the compound and energy density are important factors for greater effectiveness of therapy.

Enhanced photovoltaic performances of dye-sensitized solar cells sensitized with D-D-π-A phenothiazine-based dyes

Two new D-D-π-A phenothiazine-based metal free organic dyes (WY5, WY6) were designed, synthesized, and applied in dye-sensitized solar cells (DSSCs) by introducing of benzil-based auxiliary electron donors into simple D-π-A phenothiazine dye to improve the related photovoltaic performances. In the WY6-based solar cell, 4,4′-dimethoxybenzil acts as electron donor was used to improve the electron donating ability. So, the WY6-based solar cell finally obtains a better photovoltaic performance than that of WY5-based one. Furthermore, with the addition of benzil-based auxiliary donor, the absorption properties turn better, which directly results in the enhanced short-circuit photocurrent density (from 8.78 (T0) to 10.58 (WY5) and 11.50mAcm−2 (WY6)) and thus the enhanced power conversion efficiency (from 4.24 (T0) to 4.78 (WY5) and 5.52% (WY6)). In a word, the addition of benzil-based auxiliary electron donors effectively improved the performances of DSSCs.

Spectroscopic, calorimetric, cyclic voltammetric and molecular modeling studies of new methylene blue-polyadenylic acid interaction and comparison to thionine and toluidine blue O: Understanding self-structure formation by planar dyes

Molecules capable of inducing self-structure in poly(A) may arrest protein synthesis by preventing poly(A) chain elongation thereby inhibiting the function of mRNA. We examined whether new methylene blue can induce such structural organization in single stranded poly(A) and compared the results with those of the phenothiazinium dyes thionine and toluidine blue O. Strong association between the dyes and poly(A) was revealed from the spectroscopy and cyclic voltammetry. Thionine has a higher affinity compared to toluidine blue O and new methylene blue at 50 mM and 100 mM, but at 200 mM [Na+] new methylene blue exhibited the highest binding affinity. Negative enthalpy and positive entropy changes, and enthalpy-entropy compensation behaviour were characteristic features for the complexation. Involvement of hydrophobic forces in the binding process is correlated to the negative heat capacity changes. Differential scanning calorimetry and circular dichroism results revealed that the dyes bind poly(A) and induce a stable secondary structure with a melting temperature of 333.15 K. The binding constants increased in the range of 50–200 mM [Na+]. Molecular docking results revealed that not only van der Waals forces of attraction but hydrogen bonding also plays a crucial role in dye-poly(A) binding. Recognition and binding of small molecules to single stranded poly(A) may present a new avenue for therapeutic intervention.

Spectroscopic investigation on interaction of toluidine blue/ AOT/ γ-cyclodextrin ternary system

Interaction of toluidine blue (TB), a biologically potent cationic phenothiazinium dye, with anionic surfactant, sodium bis(2-ethylhexyl) sulfosuccinate (AOT) have been thoroughly studied employing absorption and emission spectroscopy. A completely distinct spectral behavior of TB has been observed corresponding to pre-micellar and post-micellar region of AOT. Steady-state fluorescence anisotropy measurement has been carefully undertaken to rationalize the spectroscopic results. Effect of γ-cyclodextrin (γ-CD) on the spectral properties of TB has also been encountered for understanding of binding interaction between them. Molecular docking study has been accomplished to enlighten the probable orientation of TB inside the γ-CD core. Here particular interest has been focused on a mixed system, composed of AOT pre-micelles and γ-CD. A remarkable diminution of both absorption and emission intensities of TB has been observed in AOT pre-micelle with a simultaneous colorimetric change of TB solution from dark blue to lavender, and subsequent addition of γ-CD results in enhancement of intensities with dramatic reversal of the lavender colored solution to the original dark blue color. The emission characteristics of TB in the presence of AOT and γ-CD may prove as promising for an ‘IMPLICATION’ logic gate which may perform a significant role in the field of molecular electronics.