The production of singlet oxygen ( 1 O 2 ) by bacteriochlorin a (BCA) was studied in phosphate buffer and in dimyristoyl--α-phosphatidylcholine (DMPC) unilamellar liposomes. The comparative method used to measure 1 O 2 production was a quantitative analysis of photooxidation reactions leading to the loss of absorbance of the water-soluble specific probe: anthracene-9,10-dipropionic acid. Rose Bengal, whose 1 O 2 quantum yield (Φ RB ) is well known in alcohols and phosphate buffer, was used as the standard for the quantification of the BCA singlet oxygen production. Our results confirm quantitatively that solubilization of BCA in liposomes leads to an increase in 1 O 2 production. Indeed, the quantum yield of 1 O 2 production by BCA (Φ BCA ) is 0.05 in phosphate buffer and 0.33 in DMPC liposomes.Furthermore, the diffusion characteristics of 1 O 2 produced by BCA bound to liposome were also examined using the isotopic lifetime enhancement effect of D 2 O. It was shown that 1 O 2 spent at least 70% of its lifetime in the vesicular environment.
To describe the action mechanisms of Bacteriochlorin a (BCA), a second generation photosensitizer, in phosphate buffer (PB) and in dimyristoyl phosphatidylcholine (DMPC) liposomes we carried out oxygen consumption and ESR measurements. In PB, where BCA was in a monomer-dimer equilibrium, our results suggested that the oxygen consumption was related to the BCA monomers concentration in solution. Incorporation of BCA in DMPC liposomes, by promoting the monomerization of BCA, increased 9-fold the oxygen consumption in comparison to the value in PB. The use of specific singlet oxygen quenchers (Azide and 9, 10-Anthracenedipropionic acid) in ESR and oxygen consumption experiments allowed us to assert that BCA was mainly a type II sensitizer when it was incorporated in DMPC. Finally, the cell survival of WiDr cells after a PDT treatment was measured for cells incubated with BCA in cell culture medium and cells incubated with BCA in DMPC. Irrespective of the dye concentration, the cell survival was lower when liposomes were used. This effect could be the result of a better BCA monomerization and / or a different BCA uptake in cells.
Analysis of the bacteriochlorin a absorption spectra suggests the existence of a monomer-dimer equilibrium, particularly intense in phosphate buffer and favored by a decrease of the pH. The dye in methanolic solution is predominantly in monomeric form. Fluorescence and electron spin resonance nitroxide spin labeling measurements indicate that incorporation into the lipid phase of dimyristoyl-L-alpha-phosphatidylcholine liposomes induces dye monomerization. Moreover, the molecules are bound in the external surface of the vesicles and a complete incorporation is ensured by a lipid-to-dye ratio greater than 125.
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.
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‐α‐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.
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