The microenvironment of DNA switches the activity of singlet oxygen generation photosensitized by berberine and palmatine

The effect of the interaction between DNA and the photosensitizer on photosensitized singlet oxygen (1O2) generation was investigated using DNA-binding alkaloids, berberine and palmatine. These photosensitizers were bound to DNA by electrostatic force. Near-infrared luminescence measurement demonstrated that the photoexcited alkaloids can generate 1O2 only when the photosensitizers are bound to DNA. A fluorescence decay study showed significant enhancement of the lifetime of their photoexcited state with the DNA binding. A calculation study suggested that the electrostatic interaction with DNA inhibits the quenching of the photoexcited state of these alkaloids via intramolecular electron transfer, leading to the prolongation of the lifetime of their excited state. This effect should enhance their intersystem crossing and the yield of energy transfer to molecular oxygen. The results show that the electrostatic interaction with DNA significantly affects the 1O2 generation activity of a photosensitizer. In addition, this interaction may be applied to the control and the design of photosensitizers for medical applications such as photodynamic therapy.     

Experimental tests of the feasibility of singlet oxygen luminescence monitoring in vivo during photodynamic therapy

Singlet oxygen (1O2) is thought to be the cytotoxic agent in photodynamic therapy (PDT) with current photosensitizers. Direct monitoring of 1O2 concentration in vivo would be a valuable tool in studying biological response. Attempts were made to measure 1O2 IR luminescence during PDT of cell suspensions and two murine tumour models using the photosensitizers Photofrin II and aluminium chlorosulphonated phthalocyanine. Instrumentation was virtually identical to that devised by Parker in the one positive report of in vivo luminescence detection in the literature. Despite the fact that our treatments caused cell killing and tissue necrosis, we were unable to observe 1O2 emission under any conditions. We attribute this negative result to a reduction in 1O2 lifetime in the cellular environment. Quantitative calibration of our system allowed us to estimate that the singlet oxygen lifetime in tissue is less than 0.5 microsecond. Some technical improvements are suggested which would improve detector performance and perhaps make such measurements feasible.     

Triplet formation of 4-thiothymidine and its photosensitization to oxygen studied by time-resolved thermal lensing technique

Excited-state dynamics of 4-thiothymidine (S4-TdR) and its photosensitization to molecular oxygen in solution with UVA irradiation were investigated. Absorption and emission spectra measurements revealed that UVA photolysis of S4-TdR gives rise to a population of T1(pipi*), following S2(pipi*) --> S1(npi*) internal conversion. In transient absorption measurement, the 355 nm laser photolysis gave broad absorption (380-600 nm) bands of triplet S4-TdR. The time-resolved thermal lensing (TRTL) signal of S4-TdR containing the thermal component due to decay of triplet S4-TdR was clearly observed by the 355 nm laser excitation. The quantum yield for S1 --> T1 intersystem crossing was estimated to be unity by a triplet quenching experiment with potassium iodide. In the presence of molecular oxygen, the photosensitization from triplet S4-TdR gave rise to singlet oxygen O2 (1Deltag) with a quantum yield of 0.50. Therapeutic implications of such singlet oxygen formation are discussed.     

Time-Resolved Studies of the Excited-State Dynamics of meso-Tetra(hydroxylphenyl)chlorin in Solution

Meso-tetra(hydroxyphenyl)chlorin (m-THPC) is a new photosensitizer developed for potential use in photodynamic therapy (PDT) for cancer treatment. In PDT, the accepted mechanism of tumor destruction involves the formation of excited singlet oxygen via intermolecular energy transfer from the excited triplet-state dye to the ground triplet-state oxygen. Femtosecond transient absorption measurements are reported here for the excited singlet state dynamics of m-THPC in solution. The observed early time kinetics were best fit using a triple exponential function with time constants of 350 fs, 80 ps and > or = 3.3 ns. The fastest decay (350 fs) was attributed to either internal conversion from S2 to S1 or vibrational relaxation in S2. Multichannel time-resolved absorption and emission spectroscopies were also used to characterize the excited singlet and triplet states of the dye on nanosecond to microsecond time scales at varying concentrations of oxygen. The nanosecond time-resolved absorption data were fit with a double exponential with time constants of 14 ns and 250 ns in ambient air, corresponding to lifetimes of the S1 and T1 states, respectively. The decay of the T1 state varied linearly with oxygen concentration, from which the intrinsic decay rate constant, ki, of 1.5 x 10(6) s-1 and the biomolecular collisional quenching constant, kc, of 1.7 x 10(9) M-1 s-1 were determined. The lifetime of the S1 state of 10 ns was confirmed by fluorescence measurements. It was found to be independent of oxygen concentration and longer than lifetimes of other photosensitizers.     

Photoinduced Electron Transfer‐based Halogen‐free Photosensitizers: Covalent meso‐Aryl (Phenyl,Naphthyl, Anthryl,and Pyrenyl) as Electron Donors to Effectively Induce the Formation of the Excited Triplet State and Singlet Oxygen for BODIPY Compounds

Pristine BODIPY compounds have negligible efficiency to generate the excited triplet state and singlet oxygen. In this report, we show that attaching a good electron donor to the BODIPY core can lead to singlet oxygen formation with up to 58 % quantum efficiency. For this purpose, BODIPYs with meso ‐aryl groups (phenyl, naphthyl, anthryl, and pyrenyl) were synthesized and characterized. The fluorescence, excited triplet state, and singlet oxygen formation properties for these compounds were measured in various solvents by UV/Vis absorption, steady‐state and time‐resolved fluorescence methods, as well as laser flash photolysis technique. In particular, the presence of anthryl and pyrenyl showed substantial enhancement on the singlet oxygen formation ability of BODIPY with up to 58 % and 34 % quantum efficiency, respectively, owing to their stronger electron‐donating ability. Upon the increase in singlet oxygen formation, the fluorescence quantum yield and lifetime values of the aryl‐BODIPY showed a concomitant decrease. The increase in solvent polarity enhances the singlet oxygen generation but decreases the fluorescence quantum yield. The results are explained by the presence of intramolecular photoinduced electron transfer from the aryl moiety to BODIPY core. This method of promoting T₁ formation is very different from the traditional heavy atom effect by I, Br, or transition metal atoms. This type of novel photosensitizers may find important applications in organic oxygenation reactions and photodynamic therapy of tumors.     

Identification and reactivity of the triplet excited state of 5-hydroxytryptophan

Both the neurotransmitter serotonin and the unnatural amino acid 5-hydroxytryptophan (5HT), contain the 5-hydroxyindole chromophore. The photochemistry of 5HT is being investigated in relation to the multiphoton excitation of this chromophore to produce a characteristic photoproduct with green fluorescence ('hyperluminescence'). Laser flash photolysis (308 nm) of 5HT in aqueous solution at neutral pH produces both the neutral 5-indoloxyl radical (lambda(max) 400-420 nm) and another transient absorption with lambda(max) 480 nm and lifetime of 2 micros in deaerated solutions. Based on quenching by oxygen and beta-carotene, the species at 480 nm is identified as the triplet excited state of 5HT. In acidic solution a new oxygen-insensitive intermediate with lambda(max) 460 is assigned to the radical cation of 5HT. Time-resolved measurements of luminescence at 1270 nm have shown that the triplet state of 5HT is able to react with oxygen to form singlet excited oxygen (1O2*) with a quantum yield of approximately 0.1. However, 5HT has also been found to be an effective quencher of singlet oxygen with a second order rate constant of 1.3 x 10(8) dm3 mol(-1) s(-1). The results are discussed in the light of recent observations on the multiphoton-excited photochemistry of serotonin.     

Photophysical studies of pheophorbide a and pheophytin a. Phosphorescence and photosensitized singlet oxygen luminescence

The triplet states of pheophorbide a and pheophytin a were studied in several environments by direct measurement of the phosphorescence of the pigments and photosensitized singlet oxygen (1O2) luminescence. The spectra, lifetimes and quantum yields of phosphorescence and the quantum yields of 1O2 generation were determined. These parameters are similar for monomeric molecules of both pigments in all the environments studied. Aggregation of the pigment molecules leads to a strong decrease in the phosphorescence and 1O2 luminescence intensities, which is probably due to a large decrease in the triplet lifetime and triplet quantum yield in the aggregates. The results obtained for pheophorbide a and pheophytin a are compared with those previously reported for chlorophyll alpha. The data suggest that the photodynamic activity of the pigments in living tissues is probably determined by the monomeric pigment molecules formed in hydrophobic cellular structures. Aggregated molecules seem to have a much lower activity.     

Laser Flash Photolysis Studies of the UVA Sunscreen Mexoryl SX

Abstract The results of a nanosecond laser flash photolysis investigation of the UVA sunscreen Mexoryl* SX in various solvent environments and within a commercial sunscreen formulation are reported. To the best of our knowledge this is the first laser flash photolysis study of a commercial suncare formulation. In each of these environments kinetic UV-visible absorption measurements following nanosecond 355 nm laser excitation reveals a short-lived species with a solvent-dependent absorption maximum around 470–500 nm and a solvent-dependent lifetime of 50–120 ns. This transient absorption is attributed to the triplet state of Mexoryl* SX on the basis that it is quenched by molecular oxygen leading to the formation of singlet oxygen in acetonitrile. The singlet oxygen quantum yield (φ_Δ), determined by comparative time-resolved near-infrared luminescence measurements and extrapolated to the limit of complete triplet state quenching, is estimated as 0.09 ± 0.03 in acetonitrile. In aqueous solution the shorter triplet state lifetime combined with lower ambient oxygen concentrations precludes significant triplet state quenching. For the commercial sunscreen formulation there was no observable difference in the measured triplet lifetime between samples exposed to oxygen or argon, suggesting that the singlet oxygen quantum yield in such environments is likely to be orders of magnitude lower than that measured in acetonitrile.     

Time-resolved investigations of singlet oxygen luminescence in water, in phosphatidylcholine, and in aqueous suspensions of phosphatidylcholine or HT29 cells

Singlet oxygen was generated by energy transfer from the photoexcited sensitizer, Photofrin or 9-acetoxy-2,7,12,17-tetrakis-(beta-methoxyethyl)-porphycene (ATMPn), to molecular oxygen. Singlet oxygen was detected time-resolved by its luminescence at 1270 nm in an environment of increasing complexity, water (H2O), pure phosphatidylcholine, phosphatidylcholine in water (lipid suspensions), and aqueous suspensions of living cells. In the case of the lipid suspensions, the sensitizers accumulated in the lipids, whereas the localizations in the cells are the membranes containing phosphatidylcholine. By use of Photofrin, the measured luminescence decay times of singlet oxygen were 3.5 +/- 0.5 micros in water, 14 +/- 2 micros in lipid, 9 +/- 2 micros in aqueous suspensions of lipid droplets, and 10 +/- 3 micros in aqueous suspensions of human colonic cancer cells (HT29). The decay time in cell suspensions was much longer than in water and was comparable to the value in suspensions of phosphatidylcholine. That luminescence signal might be attributed to singlet oxygen decaying in the lipid areas of cellular membranes. The measured luminescence decay times of singlet oxygen excited by ATMPn in pure lipid and lipid suspensions were the same within the experimental error as for Photofrin. In contrast to experiments with Photofrin, the decay time in aqueous suspension of HT29 cells was 6 +/- 2 micros when using ATMPn.     

Triplet state spectroscopic studies on some 5,10,15,20-tetrakis(methoxyphenyl)porphyrins

Extensive triplet state spectroscopic investigations were carried out with a series of 5,10,15,20-tetrakis(methoxyphenyl)porphyrins. Triplet absorption spectra, triplet lifetime, triplet quantum yield and quantum yield for singlet oxygen production were determined with different absorption and emission techniques, using the frequency-doubled beam of a Nd:YAG laser. It has been found that these synthetic porphyrins are effective photosensitizers which can be used as model compounds to investigate the theoretical and instrumental aspects of PDT.     

Effect of sensitizer protonation on singlet oxygen production in aqueous and nonaqueous media

The yield of singlet molecular oxygen, O2(a(1)Delta(g)), produced in a photosensitized process can be very susceptible to environmental perturbations. In the present study, protonation of photosensitizers whose chromophores contain amine functional groups is shown to adversely affect the singlet oxygen yield. Specifically, for bis(amino) phenylene vinylenes dissolved both in water and in toluene, addition of a protic acid to the solution alters properties of the system that, in turn, result in a decrease in the efficiency of singlet oxygen production. In light of previous studies on other molecules where protonation-dependent changes in the yield of photosensitized singlet oxygen production have been ascribed to changes in the quantum yield of the sensitizer triplet state, Phi(T), and to possible changes in the triplet state energy, E(T), our results demonstrate that this photosystem can respond to protonation in other ways. Although protonation-dependent changes in the amount of charge-transfer character in the sensitizer-oxygen complex may influence the singlet oxygen yield, it is likely that other processes also play a role. These include (a) protonation-dependent changes in sensitizer aggregation and (b) nonradiative channels for sensitizer deactivation that are enhanced as a consequence of the reversible protonation/deprotonation of the chromophore. The data obtained, although complicated, are relevant for understanding and ultimately controlling the behavior of photosensitizers in systems with microheterogeneous domains that have appreciable pH gradients. These data are particularly important given the use of such bi-basic chromophores as two-photon singlet oxygen sensitizers, with applications in spatially resolved singlet oxygen experiments (e.g., imaging experiments).     

PHOTOACOUSTIC DETECTION OF TRIPLET STATE AND SINGLET OXYGEN IN HIGHLY ABSORBING SAMPLES

A photoacoustic (PA) effect theory taking into account two heat sources corresponding to the radiationless relaxation processes of two states of different lifetimes and to the heat diffusion across the sample is herewith presented. Results obtained demonstrate that the amplitude and the phase of the PA signal depend on the sample's thermal properties, on its optical absorption coefficient, on the lifetime of the long-lived excited state, and on the ratio of the two heat sources. This ratio can be expressed as a function of the product of the energy of the excited state times the quantum yield of its production. Simulations of PA amplitude and phase variations vs light modulation frequency exhibit new features of the PA signal:phase inversion and fast decrease of the amplitude. Experimental verifications were carried out on solutions and gels. Fitting of the amplitude and phase variations allow us to measure the lifetime and conversion yield of the intermediate state which can be a triplet state or singlet oxygen, O2(1 delta g). The addition of an acceptor, specific to O2(1 delta g), induces changes in the amplitude of the PA signal which can be used to study the production and deactivation of this excited form of oxygen. This work demonstrates the usefulness of PA in the detection of metastable excited states such as the triplet state and singlet oxygen and in their quantitative analysis.     

Zinc-porphyrin Solvation in folded and unfolded states of Zn-cytochrome c

After a brief review of the use of photochemical triggers and heme metal substitution to probe the folding dynamics of cytochrome c, we present new results on the photophysics and photochemistry of folded and unfolded states of the zinc-substituted protein (Zn-cyt c). Our measurements of Zn-cyt c triplet state decay kinetics reveal a systematic isotope effect on lifetimes: the decay in the folded protein (tau(H)2(O) approximately 10 ms) is only modestly affected by isotopically substituted buffers (k(H)2(O)/k(D)2(O) = 1.2), whereas a reduced triplet lifetime (approximately 1.3 ms) and greater isotope effect (1.4) were found for the chemically denatured, fully unfolded protein. The shortest lifetime (0.1-0.4 ms) and greatest isotope effect (1.5) were found for a fully exposed model compound, zinc-substituted N-acetyl-microperoxidase-8 (ZnAcMP8), implying that the unfolded protein provides some protection to the Zn-porphyrin group even under fully denaturing conditions. Further evidence for partial structure in unfolded Zn-cyt c comes from bimolecular quenching experiments using Ru(NH(3))(6)(3+) as an external Zn-porphyrin triplet state quencher. In the presence of quencher, partially unfolded protein at midpoint guanidinium chloride (GdmCl) and urea concentrations exhibits biphasic triplet decay kinetics, a fast component corresponding to an extended, solvent-exposed state (6.6 x 10(8) M(-1) s(-1) in GdmCl, 6.3 x 10(8) M(-1) s(-1) in urea) and a slow component attributable to a compact, relatively solvent-inaccessible, state (5.9 x 10(7) M(-1) s(-1) in GdmCl, 8.6 x 10(6) M(-1) s(-1) in urea). The variation in Zn-porphyrin solvation for the compact states in the two denaturants reveals that the cofactor in the partially unfolded protein is better protected in urea solutions.     

Determination of the electron transfer parameters of a covalently linked porphyrin-quinone with mesogenic substituents - optical spectroscopic studies in solution

The photoinduced electron transfer (PET) of a covalently linked porphyrin-quinone with mesogenic substituents was studied using visible and near-IR (NIR) spectroscopy. Mesogenic substituents were introduced at the porphyrin moiety in order to mimic the anisotropic membrane properties of the native reaction centre of photosynthesis. Photophysical characterization of this system in homogeneous solution is a prerequisite for a better understanding of the effects occurring in anisotropic medium. For this reason, we studied the fluorescence and phosphorescence quenching and lifetime of the charge-separated state. Time-resolved fluorescence measurements indicated an effective singlet PET. The complete set of PET parameters was calculated using Marcus theory of non-adiabatic electron transfer (ET). Steady state measurement of singlet oxygen luminescence, which allows indirect access to phosphorescence quenching, indicated that no triplet PET was involved in the decay processes. Using transient absorption spectroscopy, the lifetime of the charge-separated state was found to be 1.9 ns.     

极性敏感的BDP分子溶剂化效应的光谱性质

合成了具有分子内电荷转移(ICT)性质的三重态光敏剂分子BDP,研究了其稳态吸收光谱、荧光光谱、荧光寿命、飞秒/纳秒瞬态吸收光谱及诱导产生单线态氧的能力等性质,发现强极性溶剂对BDP分子的溶剂化效应降低了其ICT态和第一激发三重态(T1态)的能量,从而降低了BDP分子单线态氧的产量.     

Temperature-dependent energy transfer in cadmium telluride quantum dot solids

Efficiently luminescing colloidal CdTe quantum dots (QDs) were used for the preparation of monodispersed and mixed size QD solids. Luminescence spectra and decay times of the QD emission were measured as a function of temperature to study energy transfer (ET) processes in the QD solids. In the luminescence decay curves of the emission of the largest QDs (acceptors), a rise time of the luminescence signal is observed due to energy transfer from smaller QDs. Both the rise time (a measure for the energy transfer rate) and the luminescence decay time lengthen upon cooling. This is explained by the decreased dipole strength of the excitonic emission of the QDs in the solid due to the presence of a singlet and a lower lying triplet level. Studies of energy transfer in heteronuclear QD solids reveal that single-step ET dominates.     

Photophysical properties of glucoconjugated chlorins and porphyrins and their associations with cyclodextrins

Glucoconjugated analogues of the meta-hydroxyphenyl porphyrin (m-THPP) and meta-hydroxyphenyl chlorin (m-THPC) has been recently synthesized. The characteristics of their triplet states have been determined with regard to their involvement in the photodynamic (PDT) efficiency. In the case of porphyrin derivatives, triplet quantum yields (Phi(T)) were ranging from 0.42 to 0.55 and triplet life times (tau(T)) from 1 to 5 micros. High reaction rate constants (k(q)) with molecular oxygen (k(q): 1.2-1.6 x 10(9)s(-1)) have been found. The triplet lifetimes of chlorin derivatives were about four times higher than those of porphyrins whereas the Phi(T) and k(q) values remained quite similar. Singlet oxygen yields of glucosylated and non-glucosylated porphyrins and chlorins were not significantly different within experimental errors (Phi(Delta)((1)O(2)): 0.41-0.58). Furthermore, it has been shown that glucoconjugated photosensitizers could undergo associations with the methyl-beta-cyclodextrin (Me-beta-CD) which exhibit high triplet lifetimes and singlet oxygen yields ranging from 0.27 to 0.48.     

KINETIC PROPERTIES OF THE TRIPLET STATES OF METHYLENE BLUE AND OTHER PHOTOSENSITIZING DYES

Abstract— The long-lived (> 1 μsec) transients formed in the flash excitation of the representative photosensitizers methylene blue, eosin Y and pyrene have been investigated and various criteria have been used to distinguish between triplet state intermediates and chemical intermediates. Previous assignments of the triplet transients of methylene blue appeared less secure in view of the photochemical reactivity of this dye and its lack of phosphorescence. Earlier assignments of monomeric and dimeric triplet transients of methylene blue are substantiated, however, by the observations that the rate constant for quenching by oxygen is approximately 1/9^th diffusion controlled and the formation rates are commensurate with singlet decay rates and by the observation of triplet-triplet annihilation. Additional evidence in support of monomer triplet assignments for methylene blue and eosin Y is provided by the effect of heavy atom quenchers Cs⁺, Hg²⁺ and T1⁺ on decay rates. Due to chemical reactivity, quenching by I⁻appears less suitable as a diagnostic test for triplet state intermediates. The effect of N₃⁻, which is known to quench singlet oxygen molecules and to alter the course of photosensitized oxidations, on the triplet decay of methylene blue, eosin Y and pyrene is also investigated.     

Effect of aggregation on bacteriochlorin a triplet-state formation: a laser flash photolysis study

Bacteriochlorin a (BCA) is a potential photosensitizer for photodynamic therapy of cancer. It has been shown previously that the photoefficiency of the dye is mainly dependent on singlet oxygen (1O2) generation. Nanosecond laser flash photolysis was used to produce and to investigate the excited triplet state of the dye in methanol, phosphate buffer and dimiristoyl-L-alpha-phosphatidylcholine (DMPC) liposomes. The transients were characterized in terms of their absorption spectra, decay kinetics, molar absorption coefficients and formation quantum yield of singlet-triplet intercrossing. The lifetime of the BCA triplet state was measured at room temperature. The triplet-state quantum yield is quite high in methanol (0.7) and in DMPC (0.4) but only 0.095 in phosphate buffer. In the last case, BCA is in a monomer-dimer equilibrium, and the low value of the quantum yield observed was ascribed to the fact the triplet state is only formed by the monomers.