RESUMEN

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.

Asunto(s)

Ácidos Grasos/química , Fármacos Fotosensibilizantes/síntesis química , Fármacos Fotosensibilizantes/farmacología , Cloruro de Tolonio/análogos & derivados , Estructura Molecular , Fármacos Fotosensibilizantes/química , Oxígeno Singlete/química , Espectrometría de Fluorescencia , Cloruro de Tolonio/síntesis química , Cloruro de Tolonio/farmacología

RESUMEN

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.

RESUMEN

Comparable intracellular concentrations (≈30pmol/10(6) cells) of bovine serum albumin-ZnPc (BSA) adduct outperformed dipalmitoyl-phosphatidyl-choline (DPPC) liposomes containing ZnPc at photodynamic-killing of human cervical cancer cells (HeLa) after only 15min of irradiation using red light (λ>620nm, 30W/cm(2)). This result could not be simply explained in terms of dye aggregation within the carrier, since in the liposomes the dye was considerably less aggregated than in bovine serum albumin, formulation that was capable to induce cell apoptosis upon red light exposure. Thus, using specific organelle staining, our cumulative data points towards intrinsic differences in intra-cellular localization depending on the cargo vehicle used, being ZnPc:BSA preferentially located in the near vicinity of the nucleus and in the Golgi structures, while the liposomal formulation ZnPc:DPPC was preferentially located in cellular membrane and cytoplasm. In addition to those differences, using real-time advanced fluorescence lifetime imaging of HeLa cells loaded with the photosensitizer contained in the different vehicles, we have found that only for the ZnPc:BSA formulation, there was no significant changes in the fluorescence lifetime of the photosensitizer inside the cells. This contrasts with the in situ≈two-fold reduction of the fluorescence lifetime measured for the liposomal ZnPc formulation. Those observations point towards the superiority of the protein to preserve dye aggregation, and its photochemical activity, post-cell uptake, demonstrating the pivotal role of the delivery vehicle at determining the ultimate fate of a photosensitizer.

Asunto(s)

1,2-Dipalmitoilfosfatidilcolina/química , Indoles/administración & dosificación , Indoles/farmacología , Compuestos Organometálicos/administración & dosificación , Compuestos Organometálicos/farmacología , Fármacos Fotosensibilizantes/administración & dosificación , Fármacos Fotosensibilizantes/farmacología , Albúmina Sérica Bovina/química , Animales , Transporte Biológico , Bovinos , Células HeLa , Humanos , Indoles/química , Indoles/metabolismo , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Espacio Intracelular/efectos de la radiación , Isoindoles , Liposomas , Compuestos Organometálicos/química , Compuestos Organometálicos/metabolismo , Fármacos Fotosensibilizantes/química , Fármacos Fotosensibilizantes/metabolismo , Compuestos de Zinc

RESUMEN

Indoor and outdoor endotoxin in PM2.5 was measured for the very first time in Santiago, Chile, in spring 2012. Average endotoxin concentrations were 0.099 and 0.094 [EU/m(3)] for indoor (N=44) and outdoor (N=41) samples, respectively; the indoor-outdoor correlation (log-transformed concentrations) was low: R=-0.06, 95% CI: (-0.35 to 0.24), likely owing to outdoor spatial variability. A linear regression model explained 68% of variability in outdoor endotoxins, using as predictors elemental carbon (a proxy of traffic emissions), chlorine (a tracer of marine air masses reaching the city) and relative humidity (a modulator of surface emissions of dust, vegetation and garbage debris). In this study, for the first time a potential source contribution function (PSCF) was applied to outdoor endotoxin measurements. Wind trajectory analysis identified upwind agricultural sources as contributors to the short-term, outdoor endotoxin variability. Our results confirm an association between combustion particles from traffic and outdoor endotoxin concentrations. For indoor endotoxins, a predictive model was developed but it only explained 44% of endotoxin variability; the significant predictors were tracers of indoor PM2.5 dust (Si, Ca), number of external windows and number of hours with internal doors open. Results suggest that short-term indoor endotoxin variability may be driven by household dust/garbage production and handling. This would explain the modest predictive performance of published models that use answers to household surveys as predictors. One feasible alternative is to increase the sampling period so that household features would arise as significant predictors of long-term airborne endotoxin levels.

Asunto(s)

Contaminantes Atmosféricos/química , Contaminación del Aire Interior/análisis , Ciudades , Endotoxinas/administración & dosificación , Endotoxinas/química , Exposición a Riesgos Ambientales/estadística & datos numéricos , Chile , Polvo/análisis , Humanos , Estaciones del Año

RESUMEN

This report presents evidence that ibuprofen interacts with red cell membranes as follows: a) in scanning electron microscopy (SEM) studies on human erythrocytes induced shape changes at a concentration as low as 10µM; b) in isolated unsealed human erythrocyte membranes (IUM) induced mild increase in the water content or in their molecular dynamics at the hydrophobic-hydrophilic interphase, while a corresponding ordering decrease at the deep phospholipids acyl chain level; c) at physiological temperature (37°C), 300µM ibuprofen induced a significant increase in the generalized polarization (GP) of dimyristoylphosphatidylcholine (DMPC) large unilamellar vesicles (LUV), an indication that ibuprofen molecules locate in the head polar group region of DMPC; d) X-ray diffraction studies showed that ibuprofen concentrations≥300µM induced increasing structural perturbation to DMPC bilayers; e) differential scanning calorimetry (DSC) data showed that ibuprofen was able to alter the cooperativity of DMPC phase transition in a concentration-dependent manner, to destabilize the gel phase and that ibuprofen did not significantly perturb the organization of the lipid hydrocarbon chains. Additionally, the effect on the viability of both human promyelocytic leukemia HL-60 and human cervical carcinoma HeLa cells was studied.

Asunto(s)

Antiinflamatorios no Esteroideos/farmacología , Eritrocitos/efectos de los fármacos , Ibuprofeno/farmacología , Modelos Moleculares , Rastreo Diferencial de Calorimetría , Línea Celular Tumoral , Dimiristoilfosfatidilcolina/química , Dimiristoilfosfatidilcolina/metabolismo , Eritrocitos/ultraestructura , Células HeLa , Humanos , Microscopía Electrónica de Rastreo , Temperatura , Liposomas Unilamelares/química , Liposomas Unilamelares/metabolismo , Difracción de Rayos X

RESUMEN

Riboflavin (RF) is an endogenous cell component and an efficient photosensitizer that can act by both types I and II photochemical mechanisms. Human tumor cells lines cultured in vitro, were used as model to study the effect of a photosensitizer synthesized from riboflavin, the 2',3',4',5'-riboflavin-tetrabutyrate (RTB), to increase the flavin concentration in the human promyelocytic leukemia cell line HL-60 and the human epithelial cervical cancer cell line HeLa. We demonstrate that this compound, alone or with Trp, has a toxic dose-response effect evidenced by abnormal cell morphology and a decrease in the cell proliferation rate. The mechanism of cell death was investigated and the experimental evidence indicates that it proceeds primarily via apoptosis; however, autophagy cannot be discarded. Nuclear fluorescent staining with Hoechst 33258 and transmission electron microscopy of the cells showed condensed chromatin margination at the nuclear periphery and the formation of apoptotic bodies. Furthermore, Caspase-3 activity was demonstrated in both cell lines. In addition, the characteristic apoptotic DNA ladder was observed in HL-60 cells. On the other hand, a high cytoplasmic vacuolization was observed by electron transmission and confocal microscopy. LysoTraker-red localization in the vacuoles was observed by fluorescence microscopy, and a significant decrease in the number of vacuoles and in the cell proliferation rate diminution was observed when irradiation was performed in the presence of the autophagy inhibitor 3-methyladenine. Considering that both cell death mechanisms have a dual role in the killing of tumor cells in vivo, a harmful effect that does not cause inflammation leading to tumor prophylaxis, we conclude that RTB could have potential clinical applications.

Asunto(s)

Muerte Celular/efectos de los fármacos , Muerte Celular/efectos de la radiación , Interacciones Hidrofóbicas e Hidrofílicas , Luz , Riboflavina/análogos & derivados , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Autofagia/efectos de los fármacos , Autofagia/efectos de la radiación , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Células HL-60 , Células HeLa , Humanos , Fármacos Fotosensibilizantes/química , Fármacos Fotosensibilizantes/farmacología , Fármacos Fotosensibilizantes/uso terapéutico , Riboflavina/química , Riboflavina/farmacología , Riboflavina/uso terapéutico , Enfermedades de la Piel/tratamiento farmacológico , Enfermedades de la Piel/patología

RESUMEN

Phthalocyanines are macrocyclic compounds that can be employed as photosensitizers in the treatment of various infections and diseases, as well as in photodynamic therapy. Nevertheless, a disadvantage for the clinical application of these compounds is their strong tendency to form oligomers (especially dimers), a phenomenon that reduces their efficiency as photosensitizers. In the present contribution, we have studied the photophysical and photochemical properties of ZnPc and ZnF(16)Pc in an organic solvent (THF) and liposomal formulations (DMPC, DPPC and DSPC). Our results show that dye incorporation into liposomes decreases its aggregation degree, as revealed by absorption spectra, triplet quantum yield, and singlet oxygen quantum yield measurements. Additionally, we studied the photodynamic activity of both phthalocyanines in liposomal formulation on human cervical carcinoma (HeLa) cells. For ZnF(16)Pc the photophysical behavior and phototoxicity in vitro correlate with the aggregation degree. The dimers are not photoactive and the photochemistry of ZnF(16)Pc depends of the fraction present as monomer. On the other hand, ZnPc aggregation is minimal and its photophysical and photochemical properties are similar in the three liposomes studied. Nevertheless, its phototoxicity in vitro is liposome dependent.

Asunto(s)

Indoles/química , Liposomas/química , Compuestos Organometálicos/química , Fármacos Fotosensibilizantes/química , Células HeLa , Humanos , Indoles/toxicidad , Isoindoles , Compuestos Organometálicos/toxicidad , Fotólisis , Fármacos Fotosensibilizantes/toxicidad , Teoría Cuántica , Oxígeno Singlete/metabolismo , Espectrometría de Fluorescencia , Compuestos de Zinc

RESUMEN

The photophysics and photochemistry of rose bengal (RB) and methylene blue (MB) bound to human serum albumin (HSA) have been investigated under a variety of experimental conditions. Distribution of the dyes between the external solvent and the protein has been estimated by physical separation and fluorescence measurements. The main localization of protein-bound dye molecules was estimated by the intrinsic fluorescence quenching, displacement of fluorescent probes bound to specific protein sites, and by docking modelling. All the data indicate that, at low occupation numbers, RB binds strongly to the HSA site I, while MB localizes predominantly in the protein binding site II. This different localization explains the observed differences in the dyes' photochemical behaviour. In particular, the environment provided by site I is less polar and considerably less accessible to oxygen. The localization of RB in site I also leads to an efficient quenching of the intrinsic protein fluorescence (ascribed to the nearby Trp residue) and the generation of intra-protein singlet oxygen, whose behaviour is different to that observed in the external solvent or when it is generated by bound MB.

Asunto(s)

Colorantes/química , Procesos Fotoquímicos , Albúmina Sérica/química , Sitios de Unión , Colorantes/metabolismo , Humanos , Azul de Metileno/química , Azul de Metileno/metabolismo , Modelos Moleculares , Conformación Proteica , Rosa Bengala/química , Rosa Bengala/metabolismo , Albúmina Sérica/metabolismo , Oxígeno Singlete/química

RESUMEN

Rose bengal (RB) readily binds to human serum albumin (HSA). At low RB concentrations, 90% of the dye is associated to the protein (5 microM), This association takes place in specific binding sites I and/or II. At higher RB concentrations, unspecific binding takes place with up to 10 RB molecules bound per protein molecule. The behavior of excited RB molecules bound to HSA is widely different to that observed in aqueous solution. Furthermore, the data also show that the behavior of bound RB molecules changes with the average number of dye molecules per protein (n). In particular, when n is large, the fluorescence yield is significantly reduced and no measurable long-lived triples and free singlet oxygen formation from bound dyes is detected. These results are related to self-quenching of the singlet and, most likely, excited triplets. All results point to the relevance of intra-protein generated singlet oxygen. However, when the dye is bound to the protein, at low oxygen concentrations such as those prevailing in vivo, trapping by oxygen of the triplet becomes inefficient and type I processes could contribute to the observed photoprocesses.

Asunto(s)

Fármacos Fotosensibilizantes/metabolismo , Rosa Bengala/metabolismo , Albúmina Sérica/metabolismo , Sitios de Unión , Fenómenos Biofísicos , Humanos , Fotoquímica , Fármacos Fotosensibilizantes/química , Unión Proteica , Rosa Bengala/química , Albúmina Sérica/química , Oxígeno Singlete/metabolismo

RESUMEN

Zinc phthalocyanine (ZnPc) is a well known Type II (singlet oxygen mediated) hydrophobic photosensitizer with potential use in PDT. We have found that the presence of bovine serum albumin diminishes the aggregation degree of ZnPc in aqueous solution, indicating that albumins could be potentially useful carriers for this type of photosensitizer in PDT. In order to explore the photochemical and photophysical behavior of ZnPc associated to the protein, we have evaluated triplet excited state lifetime and yield, dye bleaching, oxygen consumption, formation of carbonyls and peroxides, and the spontaneous chemiluminescence emitted after photolysis. The results show that dye association to BSA modifies the photophysics and photochemistry of ZnPC. In particular the decreased yield of long lived triplets suggests singlet state and/or static triplet quenching of the bound dye by the host protein.

Asunto(s)

Indoles/química , Albúmina Sérica Bovina/química , Indoles/efectos de la radiación , Rayos Infrarrojos , Isoindoles , Fotoquímica/métodos , Complejo de Proteína del Fotosistema II/química , Complejo de Proteína del Fotosistema II/efectos de la radiación , Albúmina Sérica Bovina/efectos de la radiación , Espectrofotometría , Spinacia oleracea/fisiología , Spinacia oleracea/efectos de la radiación , Rayos Ultravioleta

RESUMEN

This study describes the effect of novel 6-Arylbenzimidazo[1,2-c]quinazoline derivatives as tumor necrosis factor alpha (TNF-alpha) production inhibitors. The newly synthesized compounds were tested for their in vitro ability to inhibit the lipolysaccharide (LPS) induced TNF-alpha secretion in the human promyelocytic cell line HL-60. The compound 6-Phenyl-benzimidazo[1,2-c]quinazoline, coded as Gl, resulted as the most potent inhibitor and with no significant cytotoxic activity. Thus, 6-Arylbenzimidazo[1,2-c]quinazoline derivatives may have a potential as anti-inflammatory agents.

Asunto(s)

Antiinflamatorios/farmacología , Quinazolinas/farmacología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Antiinflamatorios/química , Células HL-60 , Humanos , Lipopolisacáridos/farmacología , Quinazolinas/química , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Acetato de Tetradecanoilforbol/análogos & derivados , Acetato de Tetradecanoilforbol/farmacología , Factor de Necrosis Tumoral alfa/biosíntesis

RESUMEN

The distribution of urocanic acid (UCA) isomers between aqueous solutions and n-octanol, egg yolk phosphatidylcholine (eggPC) liposomes or bovine serum albumin (BSA) has been evaluated. Regarding its partitioning between water and n-octanol, the behaviour of both isomers is very similar, and the amount incorporated to the organic solvent is mostly determined by the fraction of the compound that, in the aqueous phase, is present as uncharged species. This implies that the highest hydrophobicity occurs near the isoelectric point. cis- and trans-UCA are readily incorporated into eggPC unilamellar liposomes. A simple pseudophase treatment of ultrafiltration data renders a binding constant of 0.20+/-0.04mL/mg for the trans isomer at pH 7.4. The binding constant decreases, by a factor two, at pH 5.0, suggesting that the negatively charged species is more favourably bound to the liposomes than the neutral species, which is mostly present as zwitterions. The cis-isomer, at both pHs, is less incorporated to the bilayers. trans-UCA and cis-UCA readily bind to BSA at pH 7.4, with binding constants of 3400M(-1) and 6900M(-1), respectively. This result suggests that, as in the octanol/water partitioning, hydrophobic interactions predominate and the degree of binding is determined by the fraction present as uncharged species. A smaller binding constant at pH 5.0 indicates that the charge of the protein is also plying a relevant role.

Asunto(s)

1-Octanol/química , Albúmina Sérica Bovina/química , Ácido Urocánico/química , Animales , Bovinos , Concentración de Iones de Hidrógeno , Isomerismo , Liposomas/química , Estructura Molecular , Soluciones , Agua/química

RESUMEN

This study describes the effect of novel 6-Arylbenzimidazo [1,2-c] quinazoline derivatives as tumor necrosis factor alpha (TNF-á) production inhibitors. The newly synthesized compounds were tested for their in vitro ability to inhibit the lipolysaccharide (LPS) induced TNF-á secretion in the human promyelocytic cell line HL-60. The compound 6-Phenyl-benzimidazo [1,2-c] quinazoline, coded as Gl, resulted as the most potent inhibitor and with no significant cytotoxic activity. Thus, 6-Arylbenzimidazo [1,2-c] quinazoline derivatives may have a potential as anti-inflammatory agents.

Asunto(s)

Humanos , Antiinflamatorios/farmacología , Quinazolinas/farmacología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Antiinflamatorios/química , Lipopolisacáridos/farmacología , Quinazolinas/química , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Acetato de Tetradecanoilforbol/análogos & derivados , Acetato de Tetradecanoilforbol/farmacología , Factor de Necrosis Tumoral alfa/biosíntesis

RESUMEN

We have investigated the riboflavin (RF)-sensitized inactivation of glucose 6-phosphate dehydrogenase (G6PD) in the presence and absence of trans-urocanic acid (UCA). The inactivation of the enzyme results from its direct oxidation by the excited triplet RF in a Type-I-photosensitized reaction whose efficiency increases at low oxygen concentration. The addition of histidine to the system produced no change in the inactivation rate, discarding the participation of singlet oxygen in the reaction. On the other hand, the presence of UCA results in its bleaching, with a significant enhancement of RF-mediated inactivation of G6PD. Both the consumption of UCA and G6PD are faster at low oxygen concentrations. UCA also produced a decrease in the sensitizer photodecomposition yield. These results indicate that the enhancement of the RF-mediated G6PD inactivation observed in the presence of UCA is not a singlet oxygen-mediated process. It is proposed that UCA consumption and its effect on G6PD inactivation are due to a complex reaction sequence initiated by a direct oxidation of UCA by the excited sensitizer triplet. The oxidation of the semireduced flavin gives rise to reactive oxygen species (ROS) responsible for the increased rate of the process. This is supported by the protection afforded by several additives with ROS removal capacity: benzoate, superoxide dismutase and catalase.

Asunto(s)

Glucosafosfato Deshidrogenasa/metabolismo , Riboflavina/farmacología , Ácido Urocánico/farmacología , Oxidación-Reducción , Fotoquímica

RESUMEN

Time-resolved photolysis studies of riboflavin (RF) were carried out in the presence and absence of alpha-, betaH- and betaL-crystallins of bovine eye lens. The transient absorption spectra, recorded 5 micros after the laser pulse, reveal the presence of the absorption band (625-675 nm) of the RF neutral triplet state (tau = 42 micros) accompanied by the appearance of a long-lived absorption (tau = 320 micros) in the 500-600 nm region due to the formation of the semireduced RF radical. The RF excited state is quenched by the crystallin proteins through a mechanism that involves electron transfer from the proteins to the flavin, as shown by the decrease of the triplet RF band with the concomitant increase of the band of its semireduced form. Tryptophan loss on RF-sensitized photooxidation of the crystallins when irradiated with monochromatic visible light (450 nm) in a 5% oxygen atmosphere was studied. A direct correlation was found between the triplet RF quenching rate constants by the different crystallin fractions and the decomposition rate constants for the exposed and partially buried tryptophans in the proteins. The RF-sensitized photooxidation of the crystallins is accompanied by the decrease of the low molecular weight constituents giving rise to its multimeric forms. A direct correlation was observed between the initial rate of decrease of the low molecular weight bands corresponding to the irradiated alpha-, betaH- and betaL-crystallins and the quenching constant values of triplet RF by the different crystallins. The correlations found in this study confirm the importance of the Type-I photosensitizing mechanism of the crystallins, when RF acts as a sensitizer at low oxygen concentration, as can occur in the eye lens.

Asunto(s)

Cristalinas/farmacología , Cristalino/química , Riboflavina/efectos de la radiación , Animales , Bovinos , Fotólisis , Riboflavina/química , Espectrofotometría , alfa-Cristalinas/farmacología , Cadena A de beta-Cristalina/farmacología , Cadena B de beta-Cristalina/farmacología

RESUMEN

The photoconversion of tryptophan and tyrosine sensitized by riboflavin, was studied in aerobic and anaerobic atmospheres. Tyrosine photodegradation occurs by a radical mechanism (Type I). In contrast, the tryptophan photoconversion shows characteristics of both Type I and Type II ((1)O2 mediated) mechanisms. The main tyrosine photoproduct was dityrosine while for tryptophan a complex mixture of indolic, flavinic and indolic-flavinic type aggregates was obtained. The products of the thermolysis of 2,2'-azobis(2-amidinopropane) hydrochloride in the presence of each amino acid were of the same type as those of the photochemical reactions. The photoproducts of both amino acids are toxic when added to mouse tumoral cells in culture. A more deleterious effect was observed when anaerobic conditions were used, and also when tryptophan photoproducts were added.

Asunto(s)

Fármacos Fotosensibilizantes , Riboflavina , Triptófano , Tirosina , Especies Reactivas de Oxígeno , Radicales Libres