Christine Vever-Bizet

Singlet molecular oxygen quenching by saturated and unsaturated fatty-acids and by cholesterol.

The rate constants of molecular singlet oxygen quenching by saturated and unsaturated fatty‐acids and by cholesterol‐membrane critical components ‐ membrane critical components ‐ have been measured by time resolved detection of the 1270 nm phosphorescence of singlet molecular oxygen [O2(1δg)]. We have determined (i) an increment of 5.7 times 102M‐1 s‐1 per ‐CH2‐ in C6D6 and CD3OD for saturated fatty acids between C4 and C20, (ii) an increment of 3 times 104M‐1s‐1 per non‐conjugated cu‐double bond for C18 unsaturated fatty acids, identical in C6D6 and CD3OD, (iii) a lower quenching rate constant by a factor of 2.7 for the trans‐C16 and trans‐C18 as compared to the corresponding cis‐monounsaturated fatty acids, (iv) a rate constant of O2(1δg) quenching by cholesterol of 5.7 times 104M‐l s‐l in benzene. These rate constants are compared to those obtained for other membrane cellular components.

Interactions of dicarboxylic porphyrins with membranes in relation to their ionization state

The interactions of dicarboxylic porphyrins with membrane systems are discussed with particular emphasis on the effect of the charge of the porphyrin and the nature of the side-chains. The incorporation of hematoporphyrin or related dicarboxylic porphyrins within small unilamellar vesicles as membrane models is favored by a decrease of the pH in the range of physiological pH values. This effect might play an important role in the retention of porphyrins by tumors, which are more acidic than normal tissues. Kinetics studies also show that the partition of the porphyrin between the lipidic bilayer and the aqueous phase is governed by its release rate rather than by its incorporation rate.

Anoxic photodamage in the presence of porphyrins: evidence for the lack of effects on mitochondrial membranes.

Abstract When isolated mitochondria are irradiated in the presence of 50 μg/ml hematoporphyrin or hematoporphyrin derivative in the respiratory medium, the irradiation is at least twenty‐fold less effective in impairing the Ca2+ pump in the absence of oxygen than in its presence. This result suggests that photosensitization by non oxygen‐depending pathways has few or no effects on cell membranes, a result somewhat at variance with those observed with liposomes.

Kinetics of incorporation of porphyrins into small unilamellar vesicles

The kinetics of hematoporphyrin or deuteroporphyrin incorporation in egg phosphatidylcholine small unilamellar vesicles have been investigated by fluorescence stopped-flow measurements. The processes can be described by a fast equilibrium. The on-rate constant is nearly diffusion controlled regardless of the compound used and the pH. The affinity of these porphyrins for the vesicles is merely governed by the exit rate which depends on the structure of the porphyrin and on its charge determined by pH.

Photofrin II uptake by atheroma in atherosclerotic rabbits. Fluorescence and high performance liquid chromatographic analysis on post-mortem aorta.

Abstract— Atherosclerotic lesions were induced in normal and Watanabe rabbits by atherogenic diet and stripping of aorta endothelium. The rabbits were injected with Photofrin II and sacrificed two days later. Atheromatous aorta as well as normal aorta from control animals were characterized by their fluorescence spectra using front face excitation. Characteristic emission peaks at 631 and 694 nm were displayed at atheromatous plaques. The excitation spectrum shows a strong band at 394 nm and weaker bands at 446, 504, 536 and 574 nm. Although no fluorescence of normal aorta can be seen by visual inspection, emission with a maximum at 626 nm was detected by spectrofluorimetry. Normal phase high performance liquid chromatography analysis of extracts from atheroma and control aorta were also carried out. The specific labelling of atheroma involves mainly protoporphyrin, hematopor‐phyrin and also minor components of Photofrin II which are accumulated. Some other components are accumulated but do not appear to be specifically retained by atheroma.

Photodynamic inactivation of cell-free HIV strains by a red-absorbing chlorin-type photosensitizer

We have investigated the photodynamic activity of a new chlorin-type photosensitizer on a reference human immunodeficiency virus type 1 (HIV-1) strain, two wild-type HIV-1 isolates and two drug-resistant HIV-1 isolates. This chlorin was highly effective for the inactivation of free viruses, as assessed by two different quantitative cell culture assays. In the absence of blood components, all the HIV strains, including wild-type and drug-resistant mutant isolates, were totally inactivated using 30 micrograms ml-1 of chlorin and 0.75 J cm-2 of 661 nm light. Successful killing of HIV-1 strains in either plasma or whole blood was also obtained by increasing the chlorin concentration moderately. Our results demonstrate the antiviral efficiency of this chlorin, suggesting the potential application of dye-sensitized photoirradiation to decontaminate blood products.

Photophysical properties of a chlorin, a potent sensitizer for photochemotherapy

Abstract— Photophysical and photodynamic properties of a ehlorin type molecule derived from hydroxyethylvinyldeuteroporphyrin are presented. It photosensitizes singlet oxygen production as efficiently as mesotetraphenylporphin. The high absorptions of both its ground and triplet states in the red (660 nm) make it a potent photosensitizer which might act not only by photo‐oxidation via singlet oxygen but also by radicals produced via sequential biphotonic absorption.

UPTAKE and PHOTODYNAMIC EFFICIENCY OF HEM ATOPORPH YRIN, HYDROXYETHYLVINYLDEUTEROPORPHYRIN and HEMATOPORPHYRIN DERIVATIVE (PHOTOFRIN II®): A STUDY WITH ISOLATED MITOCHONDRIA

Abstract— The uptake of Photofrin II® (PFII), hematoporphyrin (Hp) and hydroxyethylvinyldeuter‐oporphyrin (HVD) by isolated mitochondria was studied using the high performance liquid chromatography (HPLC) technique. The various PFII components show a high affinity for mitochondria. At 5.75 μg/ml PFII, their ratio of incorporation was found to be very similar, except for Hp which is about two times less incorporated. These results were reproduced with pure Hp and pure HVD. The uptake of Hp and HVD increases with concentration but, while that of Hp reaches a plateau, the uptake of HVD continues to increase. At a high porphyrin concentration (˜ 10−5M), the loss of respiratory control is obtained with the same light dose for Hp and PFII. Taking into account the uptake and the known photophysical parameters of the various porphyrins, the photodynamic efficiency of HVD seems equivalent to that of Hp. The present results and known data on cell photoinactivation suggest that the activity of these porphyrins is mainly dependent on their incorporation.

Self-association of Disulfonated Deuteroporphyrin and its Esters in Aqueous Solution and Photosensitized Production of Singlet Oxygen by the Dimers¶

Dimerization of free acid and ester forms of disulfonated deuteroporphyrin is investigated in aqueous solution by absorbance and fluorescence spectroscopies. The dimerization equilibrium constant increases with the extent of esterification. In phosphate buffer saline (pH 7.4, 20°C), it ranges from 1.4 × 106M−1 to 7.8 × 107M−1 for the free acid and the diethyl ester forms, respectively. The dimer formation is favored by an increase of ionic strength, as predicted by the Debye–Hückel law. The dimers display a marked shift to the blue of their Soret band. In agreement with the exciton model, a cofacial stacking of the molecules with some offset is postulated. The sulfonate groups on each molecule are likely to stand on opposite directions to reduce repulsion. Both the analysis of porphyrin self‐association and careful examination of the fluorescence excitation spectra show that the dimers of disulfonated deuteroporphyrins do not fluoresce at all. The quantum yield of formation of singlet oxygen by the disulfonated deuteroporphyrins in deuterated methanol is 0.71, a value typical of monomers. In deuterated water, the yield is 0.44 for all the compounds studied though they are dimerized. The fact that nonfluorescent dimers of porphyrins can be efficient photosensitizers is emphasized.

In vitro uptake of dicarboxylic porphyrins by human atheroma. Kinetic and analytical studies.

Human atheromatous aorta segments as well as presumably disease‐free control aorta were obtained at autopsy. They were incubated with solutions of various purified dicarboxylic porphyrins including hematoporphyrin (HP) and hydroxyethylvinyldeuteroporphyrin (HVD), and with solutions of Photofrin®. Selective labelling of the atheroma was shown by macroscopic and microscopic observations of the characteristic porphyrin fluorescence associated with the atheromatous plaques. The time dependence of the uptake, monitored by absorption spectrophotometry or by high performance liquid chromatography, was inferred from the disappearance of the porphyrins in the incubation medium. Significant binding was observed in the absence of albumin or serum proteins. The uptake of HP was less than that of the more hydrophobic compounds HVD or Photofrin when these porphyrins were used alone. The presence of albumin or serum drastically reduces atheroma labelling. Some competition between HP and HVD for binding sites is also seen. The present results do indicate that hydrophobic porphyrins have an intrinsic affinity for atheroma and that they can be taken up through passive processes. Taking into account previous data on animal models (Photochem. Photobiol. (1989) 49,731–737), it appears however that, in vivo, interactions with proteins and pharmacokinetics will primarily determine plaque labelling.