LASER PHOTOSENSITIZATION OF CELLS BY HYPERICIN

Abstract— Administering a light dose of 90 J/cm² at 599 nm during incubation with hypericin to a highly differentiated normal epithelial cell line(FRTL–5), derived from Fisher rat thyroid, and to a neoplastic cell line(MPTK–6), derived from the lung metastases of a thyroid carcinoma induced in Fisher rats, produces cell kill at drug doses 1000 times lower than those necessary to cause the same mortality in the dark. The photocytocidal activity of this polycyclic quinone drug on neoplastic cells is superior to that of antitumor anthraquinone drugs, such as daunomycin and mitoxanthrone, and to the photosensitized antiviral activity previously reported for hypericin.     

HETEROEXCIMER SYSTEMS IN AQUEOUS MICELLAR SOLUTIONS

Fluorescence quenching due to charge transfer interactions in the excited state has been studied in aqueous micellar solutions. Fluorescers used were pyrene, various pyrene derivatives and several other conjugated π-electronic systems, and quenchers were N, N-dimethylaniline, N. N-dimethylaniline sulfonate, dicyanobenzenes and cyanopyridine. Strong quenching of the aromatic hydrocarbon fluorescence with dimethylanilines as well as dicyanobenzenes was observed, while no heteroexcimer emission was detected. The dependences of relative fluorescence yield and fluorescence decay curve upon the quencher concentration have been explained with equations derived on the basis of a simple model. Based on the obtained results, some discussions were given on the nature of micelle 'interior' and micelle 'surface'.     

PHOTOSENSITIZATION TISSUE MODEL

Photosensitization in a light scattering matrix was investigated with a tissue model consisting of polystyrene microsphere scattering particles, in the presence of a Photofrin II as a photosensitizing agent, and subtilisin Carlsberg as an enzyme target. The photodynamic rate constant for irradiation at 435 nm, 545 nm, and red light was measured at different microsphere concentrations. The reaction rate was almost independent of the microspheres, ruling out a significant effect of light scattering on the integrated photosensitization efficiency. Modeling with the one-dimensional diffusion approximation showed that increasing scatterer concentrations led to lower transmission and higher diffuse reflection, such that the fractional absorption was almost constant. The quantum efficiency of enzyme inactivation in the light-scattering systems was 0.0014 +/- 0.0003. In terms of incident dose, red and green light had approximately the same effectiveness and blue light was two-fold more efficient.     

PHASE EQUILIBRIA IN AQUEOUS LOW MOLECULAR CATANIONIC SURFACTANT SYSTEMS

Phase equilibria in systems alkanoic acid-alkylamine-water have been determined at 298.2 K. In the system hexanoic acid (A) - hexylamine (B) only one isotropic solution phase is formed. This solution phase is in eqilibrium with almost pure water. The strong A₁B₁ complex shows catanionic surfactant properties as concluded from phase equilibria studies in the system A₁B₁-heptane-water. This system behaves like ordinary microemulsions. The dominant feature of the phase diagram with ethanoic acid -dodecylamine is the large D phase. In addition there exist a large solution phase, a hexagonal phase and a small cubic I phase.     

PHOTOSENSITIZATION AND COMPLEX FORMATION IN A MODEL SYSTEM: 2-AMINO-PYRIMIDINE IN ETHANOL

Abstract— In the presence of 2-amino-pyrimidine, ethanol free radicals are produced in rigid glasses at 77°K by light not absorbed by the alcohol. ESR measurements indicate that a photo-sensitization occurs following a step-wise biphotonic process via triplet-triplet absorption. Absorption, fluorescence and phosphorescence measurements show that the photosensitization quenching effect at high sensitizer concentration is correlated with formation of a molecular complex, probably generated from an excited state of 2-amino-pyrimidine.     

ON THE NATURE OF CHLOROPHYLL a IN AQUEOUS MICELLAR SYSTEMS

Abstract— Absorption and fluorescence spectra of chlorophyll a in aqueous micellar solutions were studied. Solutions in anionic micelles gave an absorption band at 740 nm, attributable to microcrystal- line chlorophyll a. Solutions in cationic and nonionic micelles do not show this band. The positions of the spectral bands in anionic micelles and the possible structure of microcrystalline form are discussed. A comparison is made of the behaviour of microcrystalline form in hydrocarbon solvents and in micellar solutions.     

SPECTROSCOPIC AND PHOTOACOUSTIC STUDIES OF HYPERICIN EMBEDDED IN LIPOSOMES AS A PHOTORECEPTOR MODEL*

Abstract— In photoresponsive ciliates, like Blepharisma japonicum and Stentor coeruleus, the photoreceptor pigments responsible for photomotile reactions are hypericin-type chromophores packed in highly osmiophilic subpellicular granules. Liposomes loaded with hypericin can constitute a simple model system, appropriate for understanding the primary light-induced molecular events triggering the sensory chain in these microorganisms. Optical absorption, steady-state and time-resolved fluorescence and pulsed photoacoustic calorimetry have been used to measure spectral distributions, fluorescence lifetimes, radiative and radiationless transition quantum yields of hypericin when assembled into egg L-a-phosphatidylcholine liposomes. With respect to hypericin ethanol solutions, both absorption and fluorescence maxima are 5 nm red shifted when the pigment is inserted into the lipidic microenvironment, regardless of the hypericin local concentration. Increasing by 100 times the hypericin local concentration decreases the relative fluorescence quantum yield by a factor of around 150 and the fraction of thermally released energy, conversely, increases from 0.6 to 0.9. From the analysis of fluorescence lifetimes and their relative amplitudes it appears that a subnanosecond living component is predominant at the highest hypericin local concentrations.     

MEASUREMENT OF FORMAL POTENTIALS FOR HYPERICIN IN DIMETHYLSULFOXIDE

The formal potentials for hypericin are measured in dimethylsulfoxide using cyclic voltammetry. Two reversible one-electron reductions and a more poorly defined multielectron oxidation are observed. The formal potentials for these processes are consistent with the visible spectrum of hypericin and they indicated that hypericin is both a good oxidizing agent and a good reducing agent in the excited state.     

SUBCELLULAR DISTRIBUTION OF HYPERICIN IN HUMAN CANCER CELLS

Confocal laser microspectrofluorometric measurements on human T47D mammary tumor cells have been performed to assess the intracellular distribution of hypericin within the various cell compartments: cytoplasmic membrane, cytoplasm and nucleus. Confocal fluorescence measurements obtained from microvolumes (? 1 μm³) located within the three sites of interest show that, while being primarily located in the cell membrane and cytoplasm after a short-term incubation in a 10^?6M hypericin-containing culture medium, hypericin actually reaches the inside of the cell nucleus after a long-term incubation (210 min). Moreover, owing to the relative fluorescence quantum yields of hypericin determined in vitro when the molecule interacts with DNA, membrane and protein model systems, it is assumed that there is a significant accumulation of the drug into the cell nucleus. Consequently, the nucleus has to be considered as a possible target for the toxic action of hypericin.     

LONG RANGE ELECTRON TUNNELING IN BIOLOGICAL AND MODEL SYSTEMS

Abstract— This review paper reports the data on the role of long range electron tunneling in photosynthetic and model systems. The main papers concerned with elucidation mechanisms and kinetical peculiarities of electron transfer in reaction centers of bacteria and green plants are considered. The paper reviews the articles on long range electron transfer in reactions of metalloporphyrins—synthetic analogs of the main natural pigment of photosynthesis, chlorophyll. Liquid and solid phase redox reactions of electronically excited porphyrin molecules, intramolecular and photosensitized electron transfer processes are considered.     

PHOTOSENSITIZATION SYSTEMS OF COVALENTLY LINKED PHTHALOCYANINE COMPLEXES IN BOTH BILAYER LIPID MEMBRANES AND TIN OXIDE PHOTOVOLTAIC CELL

Abstract Zinc phthalolocyanine photosensitized donor-acceptor systems for light energy conversion and for the design of photoelectrochemical molecular devices are presented. Covalently linked phthalocyanine complexes were incorporated in bilayer lipid membranes (BLM) and deposited on SnO₂ transparent electrodes. Their photovoltages were measured and compared. It has been found that a more favorable orientation and closer proximity are attained in the diad compounds between the donor (phthalocyanine)-acceptor (anthraquinone) pair than in the reference compound for efficient light-induced charge separation and transfer. The triad compound is the best among all tested compounds. The decrease in the fluorescence yield and lifetime induced by quinones was examined and the apparent electron-transfer rate constants were calculated.     

PHOTOSENSITIZATION BY DRUGS IN SURFACTANT SOLUTIONS

Abstract— Chlorpromazine, promazine, anthracene and furosemide were tested as photosensitizers using 365 nm UV light in micellar solutions of cationic, anionic and nonionic surfactants. In all cases, micelles enhanced the ability of these compounds to photosensitize the oxidation of 2,5-dimethylfuran and the free radical polymerization of acrylamide. pH variation showed that the base form of chlorpromazine and the acid form of furosemide are the principal photosensitizing forms of these compounds. Rate differences between cationic and anionic surfactant media indicate the cation radical to be the major photochemical species formed from chlorpromazine and promazine in micellar media. Photodechlorination of chlorpromazine accounted for a significantly higher reactivity of chlorpromazine over promazine. Anthracene was found to be a very active photosensitizer by the singlet oxygen mechanism but also yielded a small concentration of cation radicals in micellar solution. In its neutral form, furosemide reacted strongly in both photooxidation and photopolymerization systems.
The implications of this study to drug-induced photosensitivity are that (i) free radical reactions may play a major role, and (ii) these sensitizers are more reactive in a hydrophobic environment, suggesting that the cellular membrane or the hydrophobic surfaces of proteins or DNA are more important sites of action in photosensitivity.     

AQUEOUS AGGREGATES OF BACTERIOCHLOROPHYLL c AS A MODEL FOR PIGMENT ORGANIZATION IN CHLOROSOMES

Chlorosomes isolated from Chloroflexus aurantiacus were extracted with chloroform/methanol. The extract contained bacteriochlorophylls c and a and lipids, but was devoid of proteins. This crude extract spontaneously formed aggregates when a methanol solution was dispersed in aqueous buffer. The aggregates could be sedimented by ultracentrifugation and appeared in electron micrographs as stain-excluding bodies with diameters between 70 and 170 nm. The absorption spectrum is remarkably similar to that of intact chlorosomes with an absorption maximum of bacteriochlorophyll c at around 740 nm. The circular dichroism spectrum of the aggregate is also very similar to that of intact chlorosomes. A conservative (±) band centered at 740 nm confirms the highly aggregated state of bacteriochlorophyll c in both systems. Steady-state fluorescence studies showed that in the aggregate energy-transfer from bacteriochlorophyll c to a component emitting at 830 nm took place. When the aggregate was suspended in buffer saturated with 1-hexanol the 740 nm form of bacteriochlorophyll c was reversibly converted to a form with spectral properties resembling the monomer absorbing at 670 nm but still in an aggregated state. This form of bacteriochlorophyll c showed no circular dichroism signal.     

HYPERICIN DERIVED TRIPLET STATES and TRANSIENTS IN ALCOHOLS and WATER

Abstract— Direct pulse photoexcitation of an antivirally active compound, hypericin sodium salt in ethanol, results in a short-lived transient, attributed to a triplet state. In the presence of reducing agents, a long-lived transient is observed, which indicates a radical anion species. In isopropanol solution, an identical triplet state is formed, accompanied by a long-lived intermediate that consists of a semiquinone-type radical. Laser excitation of hypericin sodium salt aggregates dispersed in water produces a very short-lived transient, also assigned to a triplet state, which decays, leaving an absorption spectrum, indicating a radical anion species. The latter reacts with oxygen with a rate constant of k ∼ 6 × 10⁷ M^-1 s^-1, suggesting the formation of superoxide.     

PHOTOSENSITIZATION OF CONDUCTION IN MOLECULAR SOLIDS*

Abstract— The mechanism of spectral sensitization of conduction in molecular photoconductors has been investigated in systems in which sensitizer and photoconductor form separate layers. The efficiency of sensitization is strongly influenced by external fields, approaching 100 per cent at 10⁶ V/cm in certain cases. The spectral response, efficiency, and field dependence are characteristic primarily of the sensitizer, while the transport characteristics are largely determined by the photoconductor. These results are considered in the light of available information concerning the molecular energy levels of the materials studied. They support the view that light absorption produces mobile electronic charge carriers in the sensitizer, which may be separated, injected, and transported across the photoconductor under an applied electric field. This mechanism is consistent with the interpretation of work on electrolyte-photoconductor contacts, and appears to account for all that is presently known about optical sensitization of molecularly bonded photoconductors.     

PHOTOPOLYMERIZATION IN THE MICELLAR SYSTEMS Ⅳ THE PHOTOSENSITIZATION EFFECTS OF THE BENZOPHENONES WITH DIFFERENT CHARGE IN IONIC MICELLAR SYSTEMS*

The photosensitization effects of the benzophenone derivatives having different charges, such as cationic BP(BP—CH_2—N~+ (C_2H_5)_3Br~-) (PKT-1), anionic BP(BP—COONa) and the neutral BP in the various ionic micellar systems were studied. From the photopolymerization of MMA initiated by the various BP/TEA in different micellar systems, the results obtained show that the polymerization rate is in the order of PKT-1>BP>BP—COONa in the SDS anionic micelle. On the contrary, the order changes to BP—COONa>BP>PKT-1 in the CTAB cationic mieelle, and to BP—COONa≈PKT-1>BP in the PKT-3 functional micelle. It is noteworthy that the PKT-1/SDS system is more efficient with its quantum yield more than 50 times compared with that of simple BP/H_2O system.     

PHOTOOXYGENATION OF METHYL LINOLEATE SENSITIZED BY PORPHYRINS AND DYES IN ACETONITRILE SOLUTION AND AQUEOUS EMULSION SYSTEMS

Photooxygenation reaction of an unsaturated fatty acid ester, methyl linoleate (methyl 9- cis. 12- cis -octadecadienoate, ML-H), sensitized by porphyrins and several types of dyes has been studied in aqueous emulsion and acetonitrile solution under air at 40°C. The oxygen (O₂) uptake proceeded slowly in the absence of sensitizers upon irradiation of an aqueous emulsion and an acetonitrile solution of ML-H (20 m M ) at ℷ_ex > 290 nm (11.4 and 6.1 μmol h^-1, respectively). The rate of O₂ uptake was enhanced by a catalytic amount (0.1 m M ) of porphyrins and dyes; hematoporphyrin (HP), zinc tetrakis(N-methyl-4-pyridiniumyi)porphyrin (ZnTMPyP), methylene blue (MB), rose bengal (RB), acridine orange (AO), and acriflavine (AF). In both systems, the sensitized photooxidation of ML-H by O₂ proceeded equimolarly to produce isomeric mixture of C9 and C13 hydroperoxides having the trans,cis and trans,trans conjugated diene configurations, independent of the types of the sensitizers used. The yield ratio of trans,trans/ trans,cis products in the MB-sensitized photooxygenation in acetonitrile and aqueous emulsion were almost equal (0.32 and 0.35. respectively). The sensitizing activity of the sensitizers, as measured by the quantum yield of O₂ uptake, increased in the order: MB (≃ 0) < ZnTMPyP < RB < HP < AF < AO in the aqueous emulsion and AO < AF < HP < RB = MB in the acetonitrile solution. The order in homogeneous acetonitrile solution was interpreted by the sensitizing ability of the dyes to produce singlet oxygen, while that in heterogeneous aqueous emulsion was correlated to the lipophilicity of dyes as well as the singlet-oxygen-producing ability.     

TEMPERATURE AND VISCOSITY DEPENDENCE OF FLUORESCENCE QUENCHING BY OXYGEN IN MODEL SYSTEMS

Abstract— The bimolecular rate constant, k _q, for the quenching of the fluorescence of pyrenebutyric acid and naphthaleneacetic acid by molecular oxygen has been studied as a function of temperature and viscosity, η. Fluorescence lifetime measurements were used to monitor the degree of quenching. Glycerol-water and sucrose-water mixtures were used to increase the bulk viscosity. Plots of log k _q vs log η show deviations from Stokes-Einstein behavior, but the data are consistent with the patterns that have been observed for other diffusion limited reactions. This work provides background information, regarding the viscosity dependence of oxygen quenching reactions, which is essential for the correct interpretation of oxygen quenching/viscosity dependence studies with more complex systems (i.e. quenching of tryptophan residues in proteins).