Characterization of Photodegradation of Meta-tetra (Hydroxyphenyl)chlorin (mTHPC) in Solution: Biological Consequences in Human Tumor Cells

The photobleaching of meta -tetra(hydroxyphenyl)chlorin m THPC) (irradiation wavelength 413 nm) in protein-containing solution was evaluated by decay in absorbance in Soret band and in fluorescence (λ_exc= 423 nm, λ_em= 655 nm). Light exposure resulted in a decrease in absorption throughout the spectrum and simultaneous appearance of new absorption bands in the spectral region 325–450 nm. The rate of m THPC photodegradation, followed by decay in absorbance, was 15-fold lower than that observed in fluorescence. This fact reflects the photobleaching of presumably monomeric, fluorescing species of m THPC. In order to determine the consequences of photobleaching of fluorescing m THPC material on cellular uptake and photocytotoxicity, human HT29 colon adenocarcinoma cells were incubated with photobleached m THPC during 5 h with or without following irradiation with the fixed fluence. Surprisingly, but up to the time when the fluorescence decreased by 50%, only a slight decrease in photocytotoxicity was detected. Either aggregated forms that have been taken up undergo intracellular monomerization (but we did not observe increase in fluorescence in living cells) or the photodynamic activity is mostly due to aggregates. The discrepancy of m -THPC-photodynamic therapy (PDT) effect and fluorescence measurements may suggest that aggregated m -THPC plays an important role in m THPC-PDT.     

Rapid spectrophotometric determination of phenothiazines with molybdophosphoric acid

A simple and rapid spectrophotometric method for the determination of six phenothiazines can be based on the formation of a coloured compound between molybdophosphoric acid and phenothiazines. The i.r. and e.s.r. spectra of the coloured compounds showed that molybdophosphoric acid oxidizes phenothiazines to a radical cation with which it then forms a coloured compound. The proposed method is employed for the determination of phenothiazines in pharmaceutical preparations. Calibration graphs are linear over ranges of about 100–2000 μg of the phenothiazine.     

Utility of diphenylamine and N-bromosuccinimide for colorimetric determination of certain phenothiazine drugs

A sensitive colorimetric method for the quantitative estimation of 11 phenothiazine drugs was developed. The method was based on the interaction of phenothiazine compounds with diphenylamine in presence of N-bromosuccinimide and sulfuric acid. Most of studied phenothiazines yielded bluish green products with two absorption maxima, one in the range of 392-396 nm with higher molar absorptivity and the other in the range of 770-780 nm with lower molar absorptivity. Phenothiazine base, mepazine HCl and pericyazine yielded blue products with only one maximum at 655, 775 and 778 nm, respectively. The color was stable for at least 1 h. The reproducibility and recovery of the method were excellent. The method was applied successfully to the determination of some commercially available phenothiazines in different dosage forms. Results were comparable to those obtained by official and reported methods.     

Interaction of Chlorpromazine and Trifluoperazine with Anionic Sodium Dodecyl Sulfate (SDS) Micelles: Electronic Absorption and Fluorescence Studies

The characteristics of binding of two phenothiazine antipsychothic drugs, chlorpromazine (CPZ) and trifluoperazine (TFP), to anionic sodium dodecyl sulfate (SDS) monomers and/or micelles were investigated using electronic absorption and fluorescence spectroscopies. Binding constants K(b) and pK(a) values for the drugs in SDS micelles were estimated using the red shifts of the maximum absorption and changes in absorption upon alkalization or in the presence of surfactant. The pK(a) shift of CPZ due to its interaction with SDS micelles is about 0.7 unit to higher values, as compared to the reported value of pK(a) obtained in buffer around 9.3. For TFP the pK(a) shift is 0.4 unit to higher values compared to that in buffer, reported as 4.0. The electronic absorption spectroscopic data suggest a biphasic interaction as a function of detergent concentration which is quite dependent of the protonation states of the drugs. In the case of TFP a very strong binding takes place when the drug is fully protonated (pH 2.0) and a distinct binding takes place at stoichiometric (low) surfactant concentrations (interaction via surfactant monomers) and at higher concentrations (in the presence of micelles). Static fluorescence probe analysis using pyrene was used to study the nature of the phenothiazine-surfactant premicellar and self-aggregates. The I(3)/I(1) and I(475)/I(1) ratios associated to pyrene fluorescence vibronic bands and excimer intensities ratios, respectively, were monitored for several ratios [SDS]/[drug] and significant changes, dependent of the drug presence and its protonation state, have been observed revealing a hydrophobic microenvironment provided by TFP-SDS aggregates in comparison with CPZ both at pH 7.0 and 4.0. Static anisotropy was also used to monitor the changes of the self-aggregates and micellar packing in the presence of the phenothiazine drugs. In aqueous solutions the anisotropy of the fluorescent probe dipyridamole (DIP) is quite low, being around 0.005 at pH 7.0 and 0.025 at pH 4.0, and the addition of detergent leads to an increase in the values of anisotropy to 0.030 at pH 7.0 and 0.070 at pH 4.0. In the presence of the phenothiazine drugs, and in the premicellar detergent concentration range, the anisotropy of DIP increases to 0.134 and 0.111 (dependent on drug concentration) for CPZ and TFP, respectively, at pH 4.0. These results suggest that the presence of both phenotiazine drugs makes the premicellar aggregates more rigid by decreasing the probe mobility, and are consistent with a more polar localization of the CPZ in the micelles as compared with TFP. At pH 7.0 the anisotropy changes are smaller, suggesting a slight decrease in CMC induced by the phenothiazines. Copyright 2000 Academic Press.     

Trifluoperazine causes a disturbance in glycerophospholipid monolayers containing phosphatidylserine (PS): effects of pH, acyl unsaturation, and proportion of PS

We have studied the interaction of trifluoperazine (TFP) with monolayers of various glycerophospholipids at 37 degrees C. TFP (1-10 microM) had little effect on surface pressure/molecular area isotherms in monolayers (on pure water) of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylethanolamine but greatly increased the mean molecular area (mma) of dipalmitoylphosphatidylserine; the increment was greatest between 0 and 1 microM, and a further increase to 10 microM TFP gave only a slight increase in mma. With phosphatidylserine (PS)-containing stearoyl and varying acyls in the sn-1 and -2 positions, respectively, TFP increased the mma in a manner that depended on the number of double bonds and chain length. In mixtures of DPPC with two of these PS species the TFP-induced mma of the monolayers (on buffer, pH 7.4) increased linearly with the proportion of PS. Both PS and TFP have ionizable groups, and the TFP-induced mma increase had optima at pH 5.0 and 7.0. We conclude that the TFP-PS interaction is mainly, but not entirely, driven by electrostatic interactions between the TFP cation and PS headgroup anion, with an insertion of the phenothiazine moiety among the acyls in the monolayer that depends on the packing of the acyls.     

Synthesis and DNA interactions of a bis-phenothiazinium photosensitizer

We report the synthesis and characterization of N,N-bis[(7-dimethylamino)phenothiazin-5-ium-3-yl]-4,4-ethylenedipiperidine diiodide (3), consisting of two photosensitizing phenothiazinium rings attached to a central ethylenedipiperidine linker. At all time points (10, 30, 60 min) and all wavelengths (676, 700, 710 nm) tested, photocleavage of pUC19 plasmid DNA (22 degrees C and pH 7.0) was markedly enhanced by 1 microM of 3 in comparison to 1 microM of the parent phenothiazine methylene blue (MB). At concentrations of phenothiazine ranging from 5 to 0.5 microM, the photocleavage levels produced by compound 3 were consistently higher than the cleavage produced using approximately twice the amount of MB (e.g., 710 nm irradiation of 5 microM of 3 and 10 microM of MB cleaved the plasmid DNA in 93% and 71% yields, respectively). Scavenger assays provided evidence for the involvement of singlet oxygen and, to a lesser extent, hydroxyl radicals in DNA damage. Analysis of photocleavage products at nucleotide resolution revealed that direct strand breaks and alkaline-labile lesions occurred predominantly at guanine bases. While compound 3 and MB were both shown to stabilize duplex DNA, the DeltaTm values of calf thymus (CT) and C. perfringens DNAs were approximately three fold higher in the presence of compound 3. Finally, viscometric data indicated that CT DNA interacts with compound 3 and MB by a combination of groove binding and monofunctional intercalation, and with compound 3 by a third, bisintercalative binding mode.     

Continuous photometric method for the screening of human urines for phenothiazines

A fully automated urinary screening system for five phenothiazines has been developed. The method is based on the oxidation of phenothiazines in acid medium to colourless sulphoxides via orange or purple coloured intermediates, which are continuously monitored at 520 nm. Two innovations have been implemented versus the conventional method; first, sulphuric acid (ideal reaction medium) has been changed by nitric acid (less corrosive, and compatible with pumping tubes) and second, problems associated with the instability of phenothiazonium radical cation are eliminated as the measurements are carried out simultaneously to the formation of the coloured products in the flow system. The configuration adopted permits selective retention of phenothiazines on a LiChrolut^®-EN sorbent column before their oxidation with hexacyanoferrate(III) ions in acid medium. The proposed method allows phenothiazines determination within the interval 3-50 μM (1-20 μg ml⁻¹), with a throughput of 12 h⁻¹; an average relative S.D. of 4% (n=11) was obtained for phenothiazine concentration of 5 μM. Finally, a comprehensive study of 50 real urine samples (phenothiazines free) obtained from different individuals provided 6% of false positives and 0% false negatives for chlorpromazine concentrations of 1.5 and 3 μM, respectively.     

EQUILIBRIUM AMONG HEMATOPORPHYRIN-DERIVATIVE COMPONENTS: INFLUENCE OF THE INTERACTION WITH CELLULAR STRUCTURES

Abstract— Hematoporphyrin-derivative (HpD) is a complex mixture of porphyrins in different aggregation states. The spectral analysis of HpD in aqueous solution shows the presence of monomeric species through the fluorescence emission spectrum and of both monomeric and aggregated species through the absorption spectrum.
The interaction with biopolymers and cellular components results in the appearance of new emission bands at 630 nm and in the640–670 nm spectral region which can be evidenced under suitable excitation conditions. Correspondingly, two new decay times (∼ 0.6 ns, and ∼ 3 ns), are observable. The new fluorescent species detected can be considered as the result of the hydrophobic effect induced by cellular structures on porphyrin aggregates.     

Preparation and characterization of an anionic dye-polycation molecular films by electrostatic layer-by-layer adsorption process

This communication reports the formation and characterization of self-assembled films of a low molecular weight anionic dye amaranth and polycation poly(allylamine hydrochloride) (PAH) by electrostatic alternating layer-by-layer (LBL) adsorption. It was observed that there was almost no material loss occurred during adsorption process. The UV-vis absorption and fluorescence spectra of amaranth solution reveal that with the increase in amaranth concentration in solution, the aggregated species starts to dominate over the monomeric species. New aggregated band at 600 nm was observed in amaranth-PAH mixture solution absorption spectrum. A new broad low intense band at the longer wavelength region, in the amaranth-PAH mixture solution fluorescence spectrum was observed due to the closer association of amaranth molecule while tagged into the polymer backbone of PAH and consequent formation of aggregates. The broad band system in the 650-750 nm region in the fluorescence spectra of different layered LBL films changes in intensity distribution among various bands within itself, with changing layer number and at 10 bilayer LBL films the longer wavelength band at 710 nm becomes prominent. Existence of dimeric or higher order n-meric species in the LBL films was confirmed by excitation spectroscopic studies. Almost 45 min was required to complete the interaction between amaranth and PAH molecules in the one-bilayer LBL film.     

Flash photolysis and pulse radiolysis studies on collagen Type I in acetic acid solution

An investigation of the photochemical properties of collagen Type I in acetic acid solution was carried out using nanosecond laser irradiation. The transient spectra of collagen solution excited at 266 nm show two bands. One of them with maximum at 295 nm and the second one with maximum at 400 nm. The peak at 400 nm is assigned to tyrosyl radicals. The first peak of the transient absorption spectra at 295 nm is probably due to photoionisation producing collagen radical cation. The transient for collagen solution in acetic acid at 640 nm was not observed. It is evidence that there is no hydrated electron in the irradiated collagen solution. The reactions of hydrated electrons and (*)OH radicals with collagen have been studied by pulse radiolysis. In the absorption spectra of products resulting from the reaction of collagen with e(aq)(-) no characteristic maximum absorption in UV and visible light region has been observed. In the absorption spectra of products resulting from the reaction of the hydroxyl radicals with collagen two bands have been observed. The first one at 320 nm and the second one at 405 nm. Reaction of (*)OH radicals with tyrosine residues in collagen chains gives rise to Tyr phenoxyl radicals (absorption at 400 nm).     

Pulse radiolysis and cyclic voltammetry studies of redox properties of phenothiazine radicals

One-electron transfer equilibria between seven phenothiazines were characterized by pulse radiolysis, producing radical-cations via oxidation by Br₂^·− or (SCN)₂^·− radicals. The reduction potentials of the phenothiazine radicals were determined by cyclic voltammetry. As an independent check, the redox equilibrium between one phenothiazine and the redox indicator ABTS was investigated. The data establish phenothiazines as useful indicators for radical redox properties. However, there are potential problems of aggregation, additional reactions with Br⁻/Br₂^·− and reactivity of the radicals towards buffers or other nucleophiles.     

Post-column oxidative derivatization for the liquid chromatographic determination of phenothiazines

A first post-column chemical derivatization method for the liquid chromatographic determination of phenothiazines is presented. Peroxyacetic acid is introduced as a derivatizing agent for phenothiazines, yielding the colored radical cations or fluorescent sulfoxides, depending on reaction conditions. Both reaction products were successfully employed for the detection of the phenothiazines after their liquid chromatographic separation. The fluorescence spectroscopic detection of the sulfoxides proved to be the more robust and sensitive method. Limits of detection ranged from 4 nM for triflupromazine and trimeprazine to 300 nM for phenothiazine for the fluorescence spectroscopic detection of the sulfoxide and from 0.3 μM for phenothiazine and triflupromazine to 2 μM for trifluperazine for the UV–Vis spectroscopic detection of the radical cation. The calibration functions for the fluorimetric sulfoxide determination ranged from two to more than three decades, starting at the limit of quantification.     

Second-derivative spectrofluorometric determination of sulphoxide impurity in phenothiazine drug substances and formulation

A direct second-derivative spectrofluorimetric procedure for determining sulphoxide impurity in phenothiazines and their formulations has been developed. The method, which has been applied to the analysis of chlorpromazine hydrochloride and prochlorperazine mesylate, as examples of typical phenothiazine substances, and to their formulations, is based on the measurement of the amplitude taken from the minimum at ca. 270 nm to the longer wavelength maximum in the second-derivative excitation spectrum of the sulphoxide in pH 8 buffer solution. The method is rapid, accurate and precise, and can be used to measure the concentration of sulphoxide in phenothiazines and their formulations at concentrations down to 0.1% m/m of that of the parent phenothiazine.     

四-（4-吡啶基）卟啉二酸聚集体的共振拉曼光谱

研究了近激子吸收带激发下四-（4-吡啶基）卟啉二酸（H8TPyP^6＋）聚集体的共振拉曼光谱。测量了H8TPyP^6＋单体和聚集体的紫外可见吸收谱和共振光散射光谱．在氘代位移的基础上结合相关体系振动光谱研究,对测得的H8TPyP^6＋单体和聚集体的拉曼谱带进行了指认．聚集体的形成导致H8TPyP^6＋的卟啉环CC／CN面内伸缩振动向低波数方向位移2-6cm^-1,而卟啉环鞍形面外振动带向高波数方向位移12cm^-1．基于拉曼谱带的强度和频率变化分析了聚集引起的H8TPyP^6＋分了内结构变化和分子间氢键作用．     

One-electron redox reactions of trifluoperazine in aqueous solutions

Absolute rate constants have been measured for the reactions of the primary and specific one-electron oxidant radicals with the protonated form of trifluoperazine (TFP). The primary radicals, e^- _aq and OH·, react with TFP at diffusion controlled rates. The transients thus produced have been characterized. Halogenated aliphatic peroxyl radicals oxidize TFP with rate constants between 10⁷ and 10⁸ dm³ mol^-1 s^-1, depending on the structure of the peroxyl radical. The reactivity of peroxyl radicals has been found to vary with Taft's inductive parameter. Oxidation of TFP at acidic pH has been studied using stopped-flow technique. The reaction between TFP radical cation and ascorbic acid has also been examined using pulse radiolysis technique. The results indicate that TFP radical cation is repaired by ascorbate. One-electron reduction potential of TFP · + /TFP at pH 3.5 has been calculated to be 0.964 V vs. NHE.     

LIGHT-INDUCED LEAKAGE OF SPIN LABEL MARKER FROM LIPOSOMES IN THE PRESENCE OF PHOTOTOXIC PHENOTHIAZINES

Abstract— Liposomes prepared from dipalmitoyl lecithin, cholesterol and dicetyl phosphate and containing a trapped spin label marker were exposed to long wavelength UV light in the presence of a series of phenothiazine tranquilizers. EPR spectroscopy was used to detect spin label marker released from liposomes, taking advantage of the disappearance of line broadening from electron spin exchange which occurred on spin label release. The minimum effective phototoxic dose in mice of these phenothiazines was also determined. Kinetic studies of light-induced spin label release from phenothiazine-sensitized liposomes showed that membrane damage was rapidly induced and that the damaging species were short-lived. The damage process was oxygen dependent and could be temporarily prevented by cysteamine or α-tocopherol added immediately before irradiation. Only those phenothiazines which mediated light-dependent liposomal membrane damage had phototoxic activity in mice and the degree of photosensitization was parallel in the two systems. In both photosensitization phenomena, the nature of the substituent at the phenothiazine 2-position was more important than the phenothiazine side chain.     

Self-assembling of phenothiazine compounds investigated by small-angle X-ray scattering and electron paramagnetic resonance spectroscopy

Small-angle X-ray scattering (SAXS) and electron paramagnetic resonance (EPR) have been carried out to investigate the structure of the self-aggregates of two phenothiazine drugs, chlorpromazine (CPZ) and trifluoperazine (TFP), in aqueous solution. In the SAXS studies, drug solutions of 20 and 60 mM, at pH 4.0 and 7.0, were investigated and the best data fittings were achieved assuming several different particle form factors with a homogeneous electron density distribution in respect to the water environment. Because of the limitation of scattering intensity in the q range above 0.15 A(-1), precise determination of the aggregate shape was not possible and all of the tested models for ellipsoids, cylinders, or parallelepipeds fitted the experimental data equally well. The SAXS data allows inferring, however, that CPZ molecules might self-assemble in a basis set of an orthorhombic cell, remaining as nanocrystallites in solution. Such nanocrystals are composed of a small number of unit cells (up to 10, in c-direction), with CPZ aggregation numbers of 60-80. EPR spectra of 5- and 16-doxyl stearic acids bound to the aggregates were analyzed through simulation, and the dynamic and magnetic parameters were obtained. The phenothiazine concentration in EPR experiments was in the range of 5-60 mM. Critical aggregation concentration of TFP is lower than that for CPZ, consistent with a higher hydrophobicity of TFP. At acidic pH 4.0 a significant residual motion of the nitroxide relative to the aggregate is observed, and the EPR spectra and corresponding parameters are similar to those reported for aqueous surfactant micelles. However, at pH 6.5 a significant motional restriction is observed, and the nitroxide rotational correlation times correlate very well with those estimated for the whole aggregated particle from SAXS data. This implies that the aggregate is densely packed at this pH and that the nitroxide is tightly bound to it producing a strongly immobilized EPR spectrum. Besides that, at pH 6.5 the differences in motional restriction observed between 5- and 16-DSA are small, which is different from that observed for aqueous surfactant micelles.     

A new photostable terrylene diimide dye for applications in single molecule studies and membrane labeling

A new terrylene diimide-based dye (WS-TDI) that is soluble in water has been synthesized, and its photophysical properties are characterized. WS-TDI forms nonfluorescing H-aggregates in water that show absorption bands being blue-shifted with respect to those of the fluorescing monomeric form. The ratio of monomeric WS-TDI to aggregated WS-TDI was determined to be 1 in 14 400 from fluorescence correlation spectroscopy (FCS) measurements, suggesting the presence of a large amount of soluble, nonfluorescent aggregates in water. The presence of a surfactant such as Pluronic P123 or CTAB leads to the disruption of the aggregates due to the formation of monomers in micelles. This is accompanied by a strong increase in fluorescence. A single molecule study of WS-TDI in polymeric films of PVA and PMMA reveals excellent photostability with respect to photobleaching, far above the photostability of other common water-soluble dyes, such as oxazine-1, sulforhodamine-B, and a water-soluble perylenediimide derivative. Furthermore, labeling of a single protein such as avidin is demonstrated by FCS and single molecule photostability measurements. The high tendency of WS-TDI to form nonfluorescent aggregates in water in connection with its high affinity to lipophilic environments is used for the fluorescence labeling of lipid membranes and membrane containing compartments such as artificial liposomes or endosomes in living HeLa cells. The superior fluorescence imaging quality of WS-TDI in such applications is demonstrated in comparison to other well-known membrane staining dyes such as Alexa647 conjugated with dextran and FM 4-64 lipophilic styryl dye.     

PHOTOCHEMICAL BINDING OF PHENOTHIAZINES ON BIOLOGICAL MEMBRANE PROTEINS

Abstract— The photobinding of phenothiazine derivatives (chlorpromazine, fluphenazine, promazine and promethazine) was studied on four different types of biological membranes (microsomes, myelin and synaptosomes from rat brain as well as human erythrocytes). The photoreaction was performed by ultraviolet irradiation of the tritiated compounds in their long wavelength absorption band (313 nm) and bound photoproducts were analysed by autoradiography of the proteins separated by polyacrylamide gel electrophoresis. The specificity of binding is low, however, a 34000 dalton band is intensely labeled on synaptic membranes with chlorpromazine and fluphenazine. All the phenothiazines bind on erythrocyte membrane proteins and specially on band 4.2 and on a peptide located before actin on the electrophoresis gel. These results show the generality of the phenothiazine photobinding on membrane proteins. These photobinding properties can be used for the identification and localization of some of these proteins.     

Effects of different light irradiations on structure and optical properties of methoxy-substituted tetraphenylporphyrins

UV lamp, filtered halogen lamp (at 425 nm) and Green laser (532 nm) experiments on a series of meso-substituted tetra phenyl porphyrin, TPP, bearing methoxy peripheral groups together with a metal derivate of 3,4 dimethoxy TPP were lead to different protonation and aggregation structures. Properties of irradiated porphyrins were investigated using their absorption and emission spectra in dichloromethane solution. The results show that the optical properties of the TPP derivates depend on light irradiation source, which shows the tuning of the absorption and emission spectra of the TPP derivates. From the dynamic light scattering measurements, the size distribution of samples was estimated about 5–15 nm in solvent after irradiation. Atomic force microscopy images of deposited porphyrins on the glass surface were shown average particle size between 10 and 30 nm. Particularly, self-assembly of the porphyrin derivates was also observed when green laser was used. We suggest that the irradiation source plays an important role in the controlling of size and morphology of products, and we propose a self-organization model to explain the formation of the porphyrin nanostructures.