Fluorescence labeling of mitochondria in living cells by the cationic photosensitizer ZnTM2,3PyPz, and the possible roles of redox processes and pseudobase formation in facilitating dye uptake.

The study of labeling selectivity and mechanisms of fluorescent organelle probes in living cells is of continuing interest in biomedical sciences. The tetracationic phthalocyanine-like ZnTM2,3PyPz photosensitizing dye induces a selective violet fluorescence in mitochondria of living HeLa cells under UV excitation that is due to co-localization of the red signal of the dye with NAD(P)H blue autofluorescence. Both red and blue signals co-localize with the green emission of the mitochondria probe, rhodamine 123. Microscopic observation of mitochondria was improved using image processing and analysis methods. High dye concentration and prolonged incubation time were required to achieve optimal mitochondrial labeling. ZnTM2,3PyPz is a highly cationic, hydrophilic dye, which makes ready entry into living cells unlikely. Redox color changes in solutions of the dye indicate that colorless products are formed by reduction. Spectroscopic studies of dye solutions showed that cycles of alkaline titration from pH 7 to 8.5 followed by acidification to pH 7 first lower, then restore the 640 nm absorption peak by approximately 90%, which can be explained by formation of pseudobases. Both reduction and pseudobase formation result in formation of less highly charged and more lipophilic (cell permeant) derivatives in equilibrium with the parent dye. Some of these are predicted to be lipophilic and therefore membrane-permeant; consequently, low concentrations of such species could be responsible for slow uptake and accumulation in mitochondria of living cells. We discuss the wider implications of such phenomena for uptake of hydrophilic fluorescent probes into living cells.

Endogenous and exogenous porphyrins as photosensitizers in the Hep-2 human carcinoma cell line.

The photodynamic activity of three photosensitizers (PS): AL-induced PPIX, the porphyrin derivative 5-(4-trimethylammoniumphenyl)-10, 5, 20-tris (2,4,6- trimethoxyphenyl) porphyrin (CP) and the molecular dyad porphyrin-C(60) (P-C(60)), the last two incorporated into liposomal vesicles, was evaluated on Hep-2 human larynx carcinoma cell line. ALA-induced accumulation of the endogenous PS PPIX, reached saturation values between 5 and 24 h incubation time; the maximal PPIX content was 5.7 nmol/106 cells. The same intracellular level was accumulated when the cationic porphyrin CP was used, while the amount of P-C(60) attained was 1.5 nmol/106 cells. Under violet-blue exciting light, the fluorescence of PPIX and P-C(60) was found in the cytoplasm showing a granular appearance indicating lysosomal localization. CP was mainly detected as a filamentous pattern characteristic of mitochondrial localization. No dark cytotoxicity was observed using 1mM ALA, 5 microM CP and 1 microM P-C(60) after 24 h incubation. Cell morphology was analyzed using Hoechst-33258, toluidine blue staining, TUNEL assay and DNA fragmentation, 24 h after irradiation with 54 J/cm2. When photosensitized with ALA and P-C(60), chromatine condensation characteristic of apoptotic cell death was found; instead, 58 % of necrotic cells were observed with CP. The results show that in the Hep-2 cells, of the three PS analyzed, the molecular dyad P-C(60) was more efficient than CP and PPIX, and confirm that PDT can induce different mechanisms of cell death depending on the PS and the irradiation dose.

Physiological parameters and biodistribution of 5,10,15,20-tetra (4-methoxyphenyl) porphyrin in rats.

Physiological parameters on hepatic and renal functionality and biodistribution, accumulation and elimination, in different organs of the 5,10,15,20-tetra (4-methoxyphenyl) porphyrin (TMP) were determined in Wistar rats. The transport of TMP by low-density (LDL) and high-density lipoproteins (HDL) was also investigated. The photosensitizer is accumulated in the spleen, where its concentration is significantly increased 21 d post-injection; it also accumulates in the liver and in a lower proportion, in the duodenum, and poorly in brain and muscle. The urine and serum biochemical parameters reached normal values both in control and treated groups. The glomerular filtrate rate was not affected by the TMP treatment in any of the studied times. These results would indicate that the sensitizer does not modify the renal glomerular function. TMP is mainly eliminated from the organism via the bile-gut pathway. Considering the total amount of porphyrin bound to both lipoproteins (LDL and HDL) in comparison with the total value of the TMP in serum, it can be inferred that a large amount of the agent is transported by lipoproteins in the plasma. This study proves information about the behavior of TMP in vivo under dark conditions. The results can be used to design photodynamic treatments using this porphyrin model as the sensitizer.

Synthesis and biological evaluation of methoxyphenyl porphyrin derivatives as potential photodynamic agents.

A new meso-2,4,6-trimethoxyphenyl porphyrin covalently linked to a 2',6'-dinitro-4'-trifluoromethylphenyl group by an amine bond 5 and its metal complex with Cd(II) 6 was prepared. The photodynamic activities of 5 and 6 were evaluated in vitro on Hep-2 cells. A considerable increase in the photocytotoxic effect was found for 6, which has higher singlet molecular oxygen, O(2)((1)Delta(g)), production.

Photodynamic studies of metallo 5,10,15,20-tetrakis(4-methoxyphenyl) porphyrin: photochemical characterization and biological consequences in a human carcinoma cell line.

The photodynamic activities of the free-base 5,10,15,20-tetrakis(4-methoxyphenyl)porphyrin (TMP) and their metal complexes with zinc(II) (ZnTMP), copper(II) (CuTMP) and cadmium(II) (CdTMP) have been compared in two systems: reverse micelle of n-heptane/sodium bis(2-ethylhexyl)sulfosuccinate/water bearing photooxidizable substrates and Hep-2 human larynx carcinoma cell line. The quantum yields of singlet molecular oxygen, O2(1 delta g), production (phi delta) of TMP, ZnTMP and CdTMP in tetrahydrofuran, were determined yielding values of 0.65, 0.73 and 0.73, respectively, while O2(1 delta g) formation was not detected for CuTMP. In the reverse micellar system, the amino acid L-tryptophan (Trp) was used as biological substrate to analyze the O2(1 delta g)-mediated photooxidation. The observed rate constants for Trp photooxidation (kobsTrp) were proportional to the sensitizer quantum yield of O2(1 delta g). A value of approximately 2 x 10(7) s-1 M-1 was found for the second-order rate constant of Trp (krTry) in this system. The response of Hep-2 cells to cytotoxicity photoinduced by these agents in a biological medium was studied. The Hep-2 cultures were treated with 1 microM of porphyrin for 24 h at 37 degrees C and the cells exposed to visible light. The cell survival at different light exposure levels was dependent on phi delta. Under these conditions, the cytotoxic effect increases in the order: Cu-TMP << TMP < ZnTMP approximately CdTMP, correlating with the production of O2(1 delta g). A similar behavior was observed in both the chemical and biological media indicating that the O2(1 delta g) mediation appears to be mainly responsible for the cell inactivation.

Photodynamic activity of 5,10,15,20-tetrakis(4-methoxyphenyl)porphyrin on the Hep-2 human carcinoma cell line: effect of light dose and wavelength range.

The photodynamic activity of 5,10,15,20-tetrakis(4-methoxyphenyl)porphyrin (TMP) has been investigated in two systems: reverse micelles of n-heptane/sodium bis(2-ethylhexyl)sulfosuccinate (AOT)/water-bearing photooxidizable substrates and on a Hep-2 human carcinoma cell line. The effect of variation in the light dose and wavelength range (360-800, 455-800, and 590-800 nm) was compared in both media. The aerobic singlet oxygen-mediated photooxidation of L-tryptophan (Trp) was used as a model of biological substrate in a micellar system. A considerable increase of the observed rate constants of Trp (k(Trp)(obs)) was noted, increasing the irradiated area of the TMP spectrum. In vitro, the survival curves of Hep-2 cells, treated with TMP, were markedly dependent on the light wavelength ranges used for irradiation. A linear behavior between k(Trp)(obs) and the photoinactivation rate of Hep-2 cells was found, indicating that the singlet oxygen (1O2 ) is the main species responsible for cell inactivation. These results contributed to an understanding of the photodynamic process yielded by this porphyrin in vitro and the sensitivity of Hep-2 cells to photodamage.

Photokilling of cultured tumour cells by the porphyrin derivative CF3.

We have analysed the photosensitizing properties of the new porphyrin 5-(4-N-(N-2',6'-dinitro-4'-trifluoromethylphenyl)aminophenyl)-10,15,20-tris(2,4,6-trimethoxyphenyl) porphyrin (CF3) on HeLa cells. The fluorescence and singlet oxygen quantum yield for CF3 were, respectively, phiF = 0.032 and phidelta = 0.25. Cell treatments were done with 5 x 10(-6) M CF3 incorporated into liposome vesicles. Under violet-blue exciting light, the red fluorescence of CF3 was mainly detected in lysosome-like granules. No dark cytotoxicity was observed using high concentration (5 x 10(-6) M) and long incubation time (18 h). Cell cultures treated for 18 h with CF3 and exposed to light (360 < lambda < 460 nm; 8 mW/cm2) for 7 min revealed a great amount of apoptotic (75.8%) and detached cells (62%) 8 h later, leading to a cell lethality of 85% (LD85). Apoptosis was identified by chromatin fragmentation and DNA ladder in gel electrophoresis. Necrotic cells were found using 15 min irradiation (LD96) and showed first small and then giant bubbles at the cell surface, with homogeneous nuclear condensation. Incubation with CF3 for 3 h followed by 7 min irradiation (LD38) produced a mitotic arrest 18 h later (mitotic index: 25.1%). Forty-eight hours after this metaphase blockage, cultures showed a great number of apoptotic cells. Taking into account these results, CF3 could be a valuable photosensitizer for the photodynamic therapy of cancer.

Expression of Fas antigen and apoptosis caused by 5,10,15, 20-tetra(4-methoxyphenyl)porphyrin (TMP) on carcinoma cells: implication for photodynamic therapy.

The photodynamic effects of 5,10,15, 20-tetra(4-methoxyphenyl)porphyrin (TMP) on a Hep-2 cell line were investigated. TMP toxicity in the dark and in relation to illumination with visible light was examined. Hep-2 cells were treated with different TMP concentrations (1, 5 and 10 microM). The uptake of TMP by Hep-2 cells increased with TMP concentration and an increase of the initial uptake rate was observed with increasing TMP concentrations. However, after 24 h of incubation, a similar value of intracellular TMP concentration was reached at all three concentrations of TMP added. Cell toxicity induced by TMP was analyzed in the dark at different concentrations of the photosensitizer and at several incubation periods. The cell mortality obtained after exposure of the cell cultures to visible light was exclusively due to the photosensitization effect of TMP produced by light irradiation. Staining with the hematoxylin-eosin method demonstrated that treatment with TMP, followed by exposure to visible light, notably increased the apoptotic figures. Fas antigen was only expressed in these conditions. The results contribute to the understanding of the photodynamic therapy (PDT) mechanism produced by TMP on Hep-2 carcinoma cell line.

Light-driven production of ATP catalysed by F0F1-ATP synthase in an artificial photosynthetic membrane.

Energy-transducing membranes of living organisms couple spontaneous to non-spontaneous processes through the intermediacy of protonmotive force (p.m.f.)--an imbalance in electrochemical potential of protons across the membrane. In most organisms, p.m.f. is generated by redox reactions that are either photochemically driven, such as those in photosynthetic reaction centres, or intrinsically spontaneous, such as those of oxidative phosphorylation in mitochondria. Transmembrane proteins (such as the cytochromes and complexes I, III and IV in the electron-transport chain in the inner mitochondrial membrane) couple the redox reactions to proton translocation, thereby conserving a fraction of the redox chemical potential as p.m.f. Many transducer proteins couple p.m.f. to the performance of biochemical work, such as biochemical synthesis and mechanical and transport processes. Recently, an artificial photosynthetic membrane was reported in which a photocyclic process was used to transport protons across a liposomal membrane, resulting in acidification of the liposome's internal volume. If significant p.m.f. is generated in this system, then incorporating an appropriate transducer into the liposomal bilayer should make it possible to drive a non-spontaneous chemical process. Here we report the incorporation of F0F1-ATP synthase into liposomes containing the components of the proton-pumping photocycle. Irradiation of this artificial membrane with visible light results in the uncoupler- and inhibitor-sensitive synthesis of adenosine triphosphate (ATP) against an ATP chemical potential of approximately 12 kcal mol(-1), with a quantum yield of more than 7%. This system mimics the process by which photosynthetic bacteria convert light energy into ATP chemical potential.