Environmental Effects on Cellular Photosensitization: Correlation of Phototoxicity Mechanism with Transient Absorption Spectroscopy Measurements

Abstract— Little is directly known about the influence of the local environment experienced by a photosensitizer in a biological system on its photophysics and photochemistry. In this paper, we have addressed this issue by correlating mechanistic studies using laser flash photolysis with cellular phototoxicity data, obtained under the same experimental conditions. In particular, we have focused on the interaction between local concentrations of photosensitizer (deuteroporphyrin) and oxygen in determining the mechanism of phototoxicity in L1210 cells. In cells, as well as in models such as liposomes and red blood cell ghosts, hypochromicity and a reduction in fluorescence and intersystem crossing yields are observed on increasing the photosensitizer concentration between 0.5 and 20 μM, which illustrates the onset of a self-association. In aerated cellular preparations, the phototoxicity is predominantly type II (singlet oxygen) for all concentrations studied but an oxygen-independent mechanism occurs at the higher concentrations in deaerated samples. These observations are readily explained by consideration of triplet state kinetics as a function of oxygen and photosensitizer concentrations in cells. The rate constant for quenching of the photosensitizer triplet state by oxygen in cells was measured as 6.6 × 10⁸ M^?1 s^?1 and by photosensitizer ground state as -10⁶M^?1s^?1 (in terms of local concentration). The latter reaction gave rise to a long-lived species that is presumably responsible for the oxygen-independent phototoxicity observed at the higher photosensitizer concentrations used. This self-quenching of the triplet state is postulated to arise from electron transfer resulting in radical ion formation. Under conditions where no self-quenching contributes, the phototoxicity measured as a function of oxygen concentration correlates well with a model based on the determined kinetic parameters, thus, unambiguously proving the intermediacy of singlet oxygen. These effects should be borne in mind when interpreting phototoxicity mechanisms from in vitro cell studies. The excellent correlation achieved between laser flash photolysis data and measured phototoxicity gives credence to the direct use of photophysical techniques to elucidate photochemical mechanisms in biological media.     

Control of photobleaching in photodynamic therapy using the photodecarbonylation reaction of ruthenium phthalocyanine complexes via stepwise two-photon excitation

In this study, we have investigated the photochemical properties and photodynamic effects of ruthenium phthalocyanine (RuPc(CO)(Py)) and naphthalocyanine (RuNc(CO)(Py)) complexes. When a nanosecond-pulsed laser is used, the photodecarbonylation of our Ru complexes efficiently proceeds via stepwise two-photon excitation, while the reaction yields are negligibly small when a continuous-wave (CW) laser is employed. The pulsed laser selective photodecarbonylation decreases the Q-band absorbance, which satisfies what the photodynamic therapy (PDT) requires of the photobleaching. For RuPc(CO)(Py), the photochemical reactions including both the photodecarbonylation and just photobleaching occur in HeLa cells in vitro. Toxicity and phototoxicity tests indicate that our RuPc(CO)(Py) and RuNc(CO)(Py) complexes in concentrations of 0.3-1 microM and 1-2 microM, respectively, are applicable as PDT agents. The phototoxicity is consistent with the photochemical properties of these complexes, namely, excited triplet lifetimes (10 and 4.8 micros for the Pc and Nc complexes, respectively) and singlet oxygen yields (0.48 and 0.35 for the Pc and Nc complexes, respectively). On the basis of these results, we propose a novel concept for achieving a greater depth of necrosis in PDT as follows: (1) PDT of upper cellular layers using CW-laser irradiation; (2) efficient photobleaching in upper cellular layers using pulsed dye-laser irradiation, which results in an increase in the therapeutic depth of red light; (3) PDT directed toward deeper tumor tissues using CW laser irradiation. In addition, these Ru complexes are promising as CO release agents for investigative biochemistry.     

The vascular response to photodynamic therapy with ATX-S10Na(II) in the normal rat colon

The mechanism of tissue damage from photodynamic therapy (PDT) may be cellular, vascular or both, depending on the photosensitising agent and the treatment conditions. Well established photosensitisers like porfimer sodium have an optimum drug light interval of two days and may cause skin photosensitivity lasting several weeks. ATX-S10Na(II) is a new photosensitiser that remains largely in the vasculature after systemic administration and clears from the body within a few hours. The present study looks at the factors controlling the extent of PDT necrosis using ATX-S10Na(II) and correlates these with changes in the circulation after PDT. Normal Wistar rats were sensitised with ATX-S10Na(II), 2 mg/kg. At laparotomy, a laser fibre was positioned just touching the colonic mucosa and 50 J light at 670 nm delivered varying the drug light interval (0.5-24 h) and light delivery regime (100 mW continuous, 20 mW continuous or 100 mW in five fractions). Some animals were killed at three days to document the area of necrosis, others received fluorescein shortly prior to death (from a few minutes to three days after PDT) to outline the zone of PDT induced vascular shutdown. Maximum necrosis was seen with the shortest drug light interval (0.5 h), with no effect by 6 h. Fractionating the light or lowering the power did not increase the necrosis. The area of fluorescein exclusion increased over the first 2 h after PDT (in contrast to the re-perfusion seen with other photosensitisers) and correlated with the area of necrosis. PDT with ATX-S10Na(II) is most effective with a drug light interval of less than one hour. It induces irreversible vascular shutdown that extends after completion of light delivery and which is largely independent of the light delivery regime.     

Comparison of the photodynamic effect of exogenous photoprotoporphyrin and protoporphyrin IX on PAM 212 murine keratinocytes

A comparative study of the cellular photosensitizing properties of protoporphyrin IX (PpIX) and photoprotoporphyrin (Ppp) was carried out in the transformed murine keratinocyte cell line, PAM 212. Time-course fluorescence studies were performed to determine the rate of uptake by cells together with fluorescence microscopy. The sensitized cells were laser irradiated with a range of light doses at 635 or 670 nm to determine the phototoxicity of the two compounds and to investigate their relative fluorescence photobleaching properties. Ppp showed enhanced phototoxicity at both its optimal activation wavelength of 670 nm (eight times more phototoxic than PpIX activated at its optimal wavelength of 635 nm for the same fluence) and at 635 nm (three times more phototoxic than PpIX at the same wavelength), using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. The photobleaching rate of Ppp in cells was found to be higher using 670 nm irradiation compared with that of PpIX at 635 nm irradiation. At 635 nm, however, the photobleaching rate of Ppp was comparable to that of PpIX. The photobleaching quantum yields of the two compounds in cells were found to be similar at approximately 5 x 10(-4), with the same value confirmed at both 670 and 635 nm irradiation for Ppp. The fluorescence lifetime of Ppp in cells was measured as 5.4 ns using time-correlated single photon counting.     

QUANTUM YIELDS AND KINETICS OF THE PHOTOBLEACHING OF HEMATOPORPHYRIN, PHOTOFRIN II, TETRA(4-SULFONATOPHENYL)-PORPHINE AND UROPORPHYRIN

Porphyrins used as sensitizers for the photodynamic therapy (PDT) of tumors are progressively destroyed (photobleached) during illumination. If the porphyrin bleaches too rapidly, tumor destruction will not be complete. However, with appropriate sensitizer dosages and bleaching rates, irreversible photodynamic injury to the normal tissues surrounding the tumor, which retain less sensitizer, may be significantly decreased. This paper surveys the quantum yields and kinetics of the photobleaching of four porphyrins: hematoporphyrin (HP), Photofrin II (PF II), tetra(4-sulfonatophenyl)porphine (TSPP) and uroporphyrin I (URO). The initial quantum yields of photobleaching, as measured in pH 7.4 phosphate buffer in air, were: 4.7 x 10(-5), 5.4 x 10(-5), 9.8 x 10(-6), and 2.8 x 10(-5) for HP, PF II, TSPP and URO respectively; thus, the rates of photobleaching are rather slow. Low oxygen concentration (2 microM) significantly reduced the photobleaching yields. However, D2O increased the yields only slightly, and the singlet oxygen quencher, azide, had no effect, even at 0.1 M. Photosensitizing porphyrins in body fluids, cells and tissues may be closely associated with various photooxidizable molecules and electron acceptors and donors. Therefore, selected model compounds in these categories were examined for their effects on porphyrin photobleaching. A number inhibited and/or accelerated photobleaching, depending on the compound, the porphyrin and the reaction conditions. For example, 1.0 mM furfuryl alcohol increased the photobleaching yields of HP and URO more than 5-fold, with little effect on PF II or TSPP. In contrast, the electron acceptor, methyl viologen, increased the photobleaching yield of TSPP more than 10-fold, with little accelerating effect on the other porphyrins. These results suggest that the mechanism(s) of the photobleaching of porphyrin photosensitizers in cells and tissues during PDT may be complex.     

Photochemical oxygen consumption, oxygen evolution and spectral changes during UVA irradiation of EMT6 spheroids.

Remarkable rates of oxygen consumption are observed via microelectrode measurements immediately upon the onset of 325 nm irradiation of multicell tumor spheroids. Consumption is irradiance dependent over the range 20-200 mW cm-2, and its magnitude is comparable to that observed previously in the same system using exogenous photosensitizers. Oscillations in the oxygen concentrations suggest that oxygen is also being evolved during irradiation. Oxygen evolution is likely the result of enzymatic dissociation of hydrogen peroxide, which is formed through UV-induced photochemistry. Irradiation of spheroids at 442 and at 514 nm produces a much more modest but detectable oxygen consumption. The dynamics of oxygen concentration changes are quite different at these wavelengths, suggesting a different photochemical mechanism. In these cases, initial oxygen depletion is followed immediately by a more gradual, monotonic increase in the oxygen concentration, consistent with irreversible photobleaching. No oscillations in the oxygen concentration are detectable. At 662 nm, no oxygen consumption was observed over the range of irradiances studied. Fluorescence spectra of cells prior to irradiation include contributions from anthranilic acid and reduced nicotinamide adenine dinucleotide (NADH). During 325 nm irradiation, anthranilic acid is rapidly and irreversibly bleached, while NADH emission undergoes only modest reduction.     

Photobleaching kinetics of optically trapped multilamellar vesicles containing verteporfin using two-photon excitation section sign

Two-photon excitation photodynamic therapy (TPE-PDT) is being developed as an improved treatment for retinal diseases. TPE-PDT has advantages over one-photon PDT, including lower collateral damage to healthy tissue and more precise delivery of PDT. As with one-photon PDT, there can be local photochemical depletion of oxygen during TPE-PDT. Here, we investigate model systems and live cells to measure local photosensitizer photobleaching and through it, infer local oxygen consumption in therapeutic volumes of the order 1 microm3. Multilamellar vesicles (MLV) and African green monkey kidney (CV-1) cells were used to study the TPE photobleaching dynamics of the photosensitizer, Verteporfin. It was found that in an oxygen-rich environment, photobleaching kinetics could not be modeled using a mono-exponential function, whereas in hypoxic conditions a mono-exponential decay was adequate to represent photobleaching. A biexponential was found to adequately model the oxygen-rich conditions and it is hypothesized that the fast part of the decay is oxygen-dependent, whereas the slower rate constant is largely oxygen-independent. Photobleaching recovery studies in the CV-1 cells support this hypothesis.     

PHOTOADDITION OF 8-METHOXYPSORALEN TO E. coli DNA POLYMERASE I. ROLE OF PSORALEN PHOTO-ADDUCTS IN THE PHOTOSENSITIZED ALTERATIONS OF POL I ENZYMATIC ACTIVITIES

Abstract— We have previously demonstrated that 8-methoxypsoralen (8-MOP)‡ plus UVA is able to inactivate the three enzymatic activities of E. coli DNA polymerase I and that oxygen is required for these reactions (M. Granger et al. , (1982) Photochem. Photobiol. , 36 , 175–180). We now show that UV-A irradiation produces a covalent incorporation of the psoralen derivative into the enzyme either in the presence or in the absence of oxygen. The excited psoralen binds directly to the protein in an oxygen-independent reaction; no complex was detected in the absence of irradiation. Fluorescence measurements reveal that at least two photoadducts are formed.
The 8-MOP-photomodified enzyme is still fully active but further irradiation leads to an inhibition of the 5'→ 3' polymerase activity whereas the 5'→ 3' exonuclease activity is not affected. A major part of the inhibition reaction is shown to be oxygen-dependent but singlet oxygen quenchers have no effect on the kinetics. This oxygen-dependent reaction is attributed to a photosensitization, due to covalently bound 8-MOP, of neighbouring amino acids through an intermediate reactive oxygen species which is not singlet oxygen. The oxygen-independent reaction is attributed to a direct photosensitization through, for example, a radical mechanism.     

QUANTUM YIELDS AND KINETICS OF THE PHOTOBLEACHING OF HEMATOPORPHYRIN, PHOTOFRIN II, TETRA(4-SULFONATOPHENYL)-PORPHINE AND UROPORPHYRIN

Abstract Porphyrins used as sensitizers for the photodynamic therapy (PDT) of tumors are progressively destroyed (photobleached) during illumination. If the porphyrin bleaches too rapidly, tumor destruction will not be complete. However, with appropriate sensitizer dosages and bleaching rates, irreversible photodynamic injury to the normal tissues surrounding the tumor, which retain less sensitizer, may be significantly decreased. This paper surveys the quantum yields and kinetics of the photobleaching of four porphyrins: hematoporphyrin (HP), Photofrin II (PF II), tetra(4-sulfonatophenyOporphine (TSPP) and uroporphyrin I (URO). The initial quantum yields of photobleaching, as measured in pH 7.4 phosphate buffer in air, were: 4.7 × 10^-5, 5.4 × 10^-5, 9.8 × 10^-5, and 2.8 × 10^-5 for HP, PF II, TSPP and URO respectively; thus, the rates of photobleaching are rather slow. Low oxygen concentration (2 μM) significantly reduced the photobleaching yields. However, D₂O increased the yields only slightly, and the singlet oxygen quencher, azide, had no effect, even at 0.1 M. Photosensitizing porphyrins in body fluids, cells and tissues may be closely associated with various photooxidizable molecules and electron acceptors and donors. Therefore, selected model compounds in these categories were examined for their effects on porphyrin photobleaching. A number inhibited and/or accelerated photobleaching, depending on the compound, the porphyrin and the reaction conditions. For example, 1.0 mM furfuryl alcohol increased the photobleaching yields of HP and URO more than 5-fold, with little effect on PF II or TSPP. In contrast, the electron acceptor, methyl viologen, increased the photobleaching yield of TSPP more than 10-fold, with little accelerating effect on the other porphyrins. These results suggest that the mechanism(s) of the photobleaching of porphyrin photosensitizers in cells and tissues during PDT may be complex.     

Polymeric Encapsulation of Novel Homoleptic Bis(dipyrrinato) Zinc(II) Complexes with Long Lifetimes for Applications as Photodynamic Therapy Photosensitisers

The use of photodynamic therapy (PDT) to treat cancer has received increasing attention over the last years. However, the clinically used photosensitisers (PSs) have some limitations that include poor aqueous solubility, hepatotoxicity, photobleaching, aggregation, and slow clearance from the body, so the design of new classes of PSs is of great interest. We present the use of bis(dipyrrinato)zinc(II) complexes with exceptionally long lifetimes as efficient PDT PSs. Based on the heavy‐atom effect, intersystem crossing of these complexes changes the excited state from singlet to a triplet state, thereby enabling singlet oxygen generation. To overcome the limitation of quenching effects in water and improve water solubility, the lead compound 3 was encapsulated in a polymer matrix. It showed impressive phototoxicity upon irradiation at 500 nm in various monolayer cancer cells as well as 3D multicellular tumour spheroids, without observed dark toxicity.     

Comparison of nonmetal and metal hydrophilic photosensitizer, ATX-S10 (Na) and ATN-2, binding with human serum proteins using spectrophotometry

Intermolecular interactions of human serum proteins with a hydrophilic nonmetalloporphyrin, 13,17-bis(1-carboxypropionyl)carbomoylethyl-8-ethenyl-2-hydroxy-3-hydroxyiminoethylidene-2,7,12,18-tetramethylporphyrin sodium salt (ATX-S10 (Na)), or a hydrophilic gallium-metalloporphyrin, diethylenetriamine pentaacetic acid ester of 2-[1-(2-hydroxy-ethoxy)ethyl]-4-vinyl-deuteroporphyrin (IX) Ga complex (ATN-2), were investigated using spectrophotometry. ATX-S10 (Na) caused a bathochromic shift with albumin, high-density lipoprotein and low-density lipoprotein, but little or no shift was observed with hemopexin, transferrin and immunoglobulin G. In contrast, ATN-2 displayed a bathochromic shift only with hemopexin. These results suggest that the association energy of ATX-S10 (Na) with albumin might be slightly greater than that with lipoproteins and that of ATN-2 with hemopexin might be greater than that with other serum proteins.     

SPECTROSCOPIC STUDIES OF CUTANEOUS PHOTOSENSITIZING AGENTS—IV. THE PHOTOLYSIS OF BENOXAPROFEN, AN ANTI-INFLAMMATORY DRUG WITH PHOTOTOXIC PROPERTIES

Abstract— Benoxaprofen [2-(4-chlorophenyl)-α-methyl-5-benzoxazole acetic acid] is an anti-inflammatory drug that causes acute phototoxicity in many patients. Photolysis studies in organic solvents (ethanol, benzene, dimethylsulfoxide) showed that benoxaprofen underwent both Type I and Type II reactions. Irradiation of an anerobic solution of benoxaprofen in ethanol resulted in hydrogen abstraction from the solvent to yield hydroxyethyl and ethoxyl radicals. In the presence of oxygen, superoxide, singlet oxygen and hydroxyethyl radicals were detected. Photolysis of benoxaprofen in air-saturated benzene or dimethylsulfoxide gave superoxide. However, under anerobic conditions the drug yielded a carbon-centered radical in benzene that could not be identified. These findings suggest that both oxygen-dependent and oxygen-independent processes may be important in the phototoxic reactions of benoxaprofen.     

In vivo mTHPC photobleaching in normal rat skin exhibits unique irradiance-dependent features

We report measurements performed on the normal skin of rats in vivo, which provide information on the photobleaching kinetics and mechanisms of the photosensitizer meso-tetrahydroxyphenyl chlorin (mTHPC). Loss of mTHPC fluorescence was monitored using in vivo fluorescence spectroscopy during photodynamic therapy (PDT) performed using 650 nm laser irradiation. The bleaching was evaluated for irradiances of 5, 20 and 50 mW cm(-2). Two distinct phases of mTHPC photobleaching were observed. In the first phase there was no obvious irradiance dependence in the loss of fluorescence vs fluence. The second phase was initiated by an irradiance-dependent discontinuity in the slope of the bleaching curve, after which the photobleaching rates showed an irradiance dependence consistent with an oxygen-dependent reaction process. To investigate the unusual shape of the in vivo bleaching curves, we measured the PDT-induced changes in O2 concentrations in mTHPC-sensitized spheroids irradiated with 2, 5 and 20 mW cm(-2) of 650 nm light. The oxygen concentration data indicated no unusual features within the range of fluences where the discontinuities in fluorescence were observed during in vivo spectroscopy. The fluorescence from the in vivo bleaching experiments thus reports a phenomenon that is not reported by measurements of the photochemical oxygen consumption in the spheroids.     

AN EFFICIENT OXYGEN INDEPENDENT TWO-PHOTON PHOTOSENSITIZATION MECHANISM

A novel oxygen-independent photosensitization mechanism from the upper triplet state (T_n) of rose bengal has been demonstrated by selectively populating T_n by sequential two-color laser excitation. Products formed from T_n inhibit red blood cell acetylcholinesterase and decrease viability of P388D₁ mouse macrophage monocyte cells as measured by trypan blue exclusion assay. Laser flash photolysis studies indicate that T_n reacts efficiently, as evidenced by permanent photobleaching of T₁ absorption, with chemical yields approaching unit efficiency. This mechanism may have application for oxygen deficient photosensitization under high intensity, pulsed laser irradiation.     

A TEST OF THE SINGLET OXYGEN MECHANISM OF CATIONIC DYE PHOTOSENSITIZATION OF MITOCHONDRIAL DAMAGE

Aromatic cationic dyes have a potential as photo-chemotherapeutic agents because they are selectively concentrated into the mitochondria of cancerous cells. The mechanism of cytophototoxicity has been proposed to be primarily due to dye sensitized photogeneration of highly toxic singlet oxygen (1O2) at the mitochondria. We tested this hypothesis by measuring the relative phototoxicity of a collection of aromatic cationic dyes towards respiring rat-liver mitochondria (RLM), upon addition of 514 nm laser light. Effectiveness of dye photosensitization towards destruction of RLM function was assayed by its effect on the RLM membrane potential. Three physical parameters of dye phototoxicity were independently measured and a relative phototoxicity calculated assuming adherence of mechanism to the 1O2 hypothesis. Quantum yields of dye sensitized 1O2 production were estimated, either from time-resolved luminescence measurements of photosensitized 1O2 formed, or by comparing rates of photobleaching of 1O2 trap; the relative partition of dye into mitochondrial lipid was determined gravimetrically; and the optical density of dye was determined in a lipid like Triton X-100 micellar environment. Under the assumption of the 1O2 hypothesis, these parameters were used to predict a relative phototoxicity which was compared with that observed. For 12 of the 14 dyes investigated, the observed and predicted phototoxicities were linearly correlated (r = 0.85) suggesting support of the 1O2 hypothesis. Carbocyanines DiOC2(3) and DiSC2(3) did not correlate and were found to be 10 and 1000 times more potent than predicted, suggesting an additional factor at play in their phototoxicity.     

A CE Method for Measuring Phototoxicity in Vitro

This paper presents an experimental setup which employs capillary electrophoresis with electrochemical and UV detection to test phototoxicity of plant extracts and components in terms of oxygen consumption and generation of reactive oxygen species upon irradiation with visible light. The experimental setup was used to test the phototoxicity of different buckwheat extracts and individual plant derived substances. The buckwheat extracts showed differences in their phototoxic behavior which might be due to different phytochemical composition. Screening of individual components revealed that rutin and quercetin alone were not phototoxic, but quercetin in combination with hypericin and chlorophyll caused considerable oxygen consumption. It has been demonstrated that the apparatus is a valuable tool to screen in vitro potential phototoxic reactions of plant extracts and individual constituents.     

Nonlinear dynamics of intracellular methylene blue during light activation of cell cultures

Methylene blue (MB+) is a well-known dye in medicine and has been discussed as an easily applicable drug for topical treatment in photodynamic therapy (PDT). Methylene blue can potentially be used as a redox indicator to detect the important redox reactions that are induced during PDT. The kinetics of this process was analyzed on a subcellular level with confocal laser scanning microscopy. BKEz-7 endothelial cells were incubated 4 h with 1 microM MB+. The fluorescence dynamics of MB+ during irradiation with 633 nm light was observed with subcellular resolution. Images were acquired at 0.5 s intervals (frame rate 1 image/0.5 s). Fluorescence was observed in the red channel of the laser scanning microscope. Synchronously, the phase-contrast image was visualized with the green channel. Morphological changes could therefore be correlated with the dynamics of MB+. In addition, the light-dose-dependent phototoxicity at 633 nm irradiation was determined by viable cell counting. After an induction period (phase I), fast fluorescent spikes could be observed in the whole cytoplasm, which decayed with a time constant of about 20 s (phase II), followed by a period of nearly constant fluorescence intensity (phase III) and exponential photobleaching (phase IV). Phase II exhibits highly nonlinear kinetics, which is hypothesized to correlate probably with a nonlinear quantal production of reactive oxygen species (ROS). Morphological cell changes were not observed during phase II. During phase III, a pycnotic cell nucleus developed. From the determination of viable cells we can conclude that a light dose applied within phase II was only sublethal in correlation with morphological observations. Overproduction of ROS leading finally to cell killing during phases III and IV is discussed.     

Reactions of NO in a Positive Streamer Corona Plasma

The reaction of NO in a streamer corona plasma is studied systematically as a function of the composition of the gas mixture, the initial concentration of NO, and the discharge repetition rate. The experimental results show that the reactions of NO depend strongly on the composition of the gas mixture. Reduction is observed in the absence of oxidants such as oxygen and water, but at very high energy cost (>200 eV/NO). In the presence of both these oxidants, more than 90% of the NO conversion is oxidation. The lowest energy costs, 24 eV/NO for He mixtures and 45 eV/NO for N ₂ mixtures, are obtained at water and oxygen concentration above 3% and at low discharge repetition rates (<10 Hz). Chemical kinetics calculations of the production of radicals in the plasma show a good agreement with the value derived from the experiments.     

Intracellular photobleaching of 5,10,15,20-tetrakis(m-hydroxyphenyl) chlorin (Foscan) exhibits a complex dependence on oxygen level and fluence rate

The understanding of photosensitizer photobleaching is important not only for mechanistic studies, but also for the development of monitoring techniques for clinical dosimetry in photodynamic therapy. In this study, we investigated the intracellular photobleaching of 5,10,15,20-tetrakis(m-hydroxyphenyl)chlorin (mTHPC, Foscan) in the murine macrophage cell line J774A.1, using quantitative fluorescence imaging microscopy, microspectrofluorometry and microspectrophotometry. Using 652 nm laser irradiation, it was found that mTHPC exhibits oxygen- and fluence rate-dependent intracellular photobleaching. The kinetics showed an inverse dose-rate behavior, i.e. a reduction of fluence rate resulted in more photobleaching at comparable fluences. The effect of deoxygenation was found to be more complex, with decreased bleaching at low fluence rates and increased bleaching at higher fluence rates. The intracellular formation of reactive oxygen species was measured using 2',7'-dichlorodihydrofluorescein diacetate. The results are analyzed in terms of competitive Type-I and Type-II mechanisms.     

Photobleaching of chemically degraded poly(vinyl chloride)

Dilute (0.23%) tetrahydrofuran solutions of chemically degraded poly(vinyl chloride) were irradiated with monochromatic light (307,320, or 354 nm) after being degassed or saturated with oxygen. The rates of the resulting photobleaching reactions of the polyene sequences present depended in a complex way on the wavelength of the light used and on the presence or absence of oxygen. During 307-nm irradiation an initial fast decrease in absorbance at 307 nm, which proceeded with a rapidly decreasing quantum yield and which was unaffected by oxygen, was followed by a slower reaction with constant quantum yield that was strongly inhibited by oxygen. The same general trend was observed for solutions irradiated with 320- or 354-nm light but in each case the rates of changes in absorbance at wavelengths other than those irradiated were complex. A mechanism that involves intra- and intermolecular reactions of the polyenes is suggested to explain the observed effects.