Michel Julliard

Photodynamic activities of silicon phthalocyanines against achromic M6 melanoma cells and healthy human melanocytes and keratinocytes

Dichlorosilicon phthalocyanine (Cl2SiPc) and bis(tri-n-hexylsiloxy) silicon phthalocyanine (HexSiPc) have been evaluated in vitro as potential photosensitizers for photodynamic therapy (PDT) against the human amelanotic melanoma cell line M6. Each photosensitizer is dissolved in a solvent-PBS mixture, or entrapped in egg-yolk lecithin liposomes or in Cremophor EL micelles. The cells are incubated for 1 h with the sensitizer and then irradiated for 20 min, 1 h or 2 h (lambda > 480 nm, 10 mW cm-2). The photocytotoxic effect is dependent on the photosensitizer concentration and the light dose. Higher phototoxicity is observed after an irradiation of 2 h: treatment with a solution of photosensitizer (2 x 10(-9) M) leads to 10% (HexSiPc in egg-yolk lecithin liposomes) or 20% (Cl2SiPc in DMF-PBS solution) cell viability. After 1 h incubation and 20 min of light exposure, the photodynamic effect is connected with the type of delivery system used. For HexSiPc, lower cell viability is found when this photosensitizer is entrapped in egg-yolk lecithin instead of solvent-PBS or for Cremophor EL micelles with Cl2SiPc. Liposome-delivered HexSiPc leads to lipid damage in M6 cells, illustrated by an increase of thiobarbituric acid-reacting substances (TBARs), but the change is not significant with Cremophor EL. The same is observed for the antioxidative defences after photodynamic stress. The cells irradiated with HexSiPc entrapped in liposomes display an increase of superoxide dismutase (SOD) activity and a decrease of glutathione (GSH) level, glutathione peroxidase (GSHPx) and catalase (Cat) activities.

Electron paramagnetic resonance evidence of the generation of superoxide (O2.− and hydroxyl (.OH) radicals by irradiation of a new photodynamic therapy photosensitizer, Victoria Blue BO

Electron paramagnetic resonance (EPR) experiments were performed on Victoria Blue BO, a cationic dye whose photocytotoxicity has been studied against the human leukaemic cell lines K-562 and TF-1. EPR experiments with 2,2,6,6-tetramethyl-4-piperidone and spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide showed that, on illumination in aerated aqueous solution or DL-alpha-dipalmitoylphosphatidylcholine liposomes, photoexcited Victoria Blue BO is unable to generate 1O2, whereas O2.- and .OH are trapped by 5,5-dimethyl-1-pyrroline-N-oxide in the presence or absence of electron donors. The O2.- formed probably leads to the .OH radical, with an efficiency which is increased by electron donors such as FE2+.

Photophysical and Redox Properties of a Series of Phthalocyanines: Relation with Their Photodynamic Activities on TF‐1 and Daudi Leukemic Cells

Abstract— The photodynamic therapy (PDT) efficiency of five phthalocyanines, chloroaluminum phthalocyanine (AlPc), dichlorosilicon phthalocyanine (SiPc), bis(tri‐n‐hexylsi‐loxy)silicon phthalocyanine (PcHEX), bis(triphenyl‐siloxy)silicon phthalocyanine (PcPHE) and nickel phthalocyanine (NiPc), was assessed on two leukemic cell lines TF‐1 and erythroieukemic and B lymphoblastic cell lines, Daudi, respectively. AlPc showed the best photocytotox‐icity leading to 0.008 surviving fraction at 2 × 10−9M for TF‐1 and 4 × 10−9M for Daudi. At 5 × 10−7M, SiPc and PcHEX induced a significant photokilling, whereas NiPc and PcPHE were inactive. Laser flash photolysis and photoredox properties of the phthalocyanines were investigated to try to relate these parameters with the biological effects. AlPc showed the longest triplet lifetime: 484 fis in dimethyl sulfoxide/H2O. This value was increased up to 820 u.s when AlPc was complexed with human serum albumin used as a membrane model. Such an enhancement was not observed with the silicon phthalocyanines. Upon irradiation, all the phthalocyanines generated singlet oxygen with 0.29–0.37 quantum yield values. The reduction potentials of the excited states obtained from measurement in the ground state and energy of the excited triplets show that AlPc is the best electron acceptor. The in vitro photocytotoxicity observed and the measured parameters are in agreement with a key role of electron transfer in PDT assays involving these phthalocyanines.

SYNTHESIS AND CHARACTERIZATION OF A SERIES OF HEXADECACHLORINATED PHTHALOCYANINES

Abstract Several methods known for the preparation of metallated (Al, Co, Li, Si, Zn) or unmetallated phthalocyanines were explored to synthesize the corresponding hexadecachlorophthalocyanines. The one pot condensation of tetrachlorophthalic anhydride or tetrachlorophthalonitrile in molten urea in the presence or not of a metal salt, led to chlorinated phthalocyanines in 5–36% yield depending on the metal. Al and Zn atoms can be inserted in free hexadecachlorophthalocyanine in 46–80% yield. The substitution of the two lithium atoms of the dilithium phthalocyanine by Al salt get to 68% aluminum phthalocyanine. All phthalocyanines synthesized were purified and fully characterized. 1 H, 13 C and 27 Al NMR data were obtained. As a rule, the Q band of the UV–Vis spectrum displayed a 30 nm bathochromic shift relative to the band of unchlorinated compounds.

Preferential photoinactivation of leukemia cells by aluminum phthalocyanine

The efficacy of chloroaluminum phthalocyanine (AlPc) for photodynamic therapy (PDT) has been evaluated in vitro on acute myeloid leukemia (AML) cells, normal peripheral blood leukocytes (PBL) and mobilized peripheral blood stem cells (mPBSC). The selectivity of the treatment has been evaluated by mixing PBL and TF-1, an erythroleukemic cell line. Upon photoradiation, this photosensitizer leads to a significant and preferential photokilling of leukemia cells in comparison to normal cells. The use of stimulated lymphocytes in PBL/TF-1 mixtures instead of resting cells also leads to a preferential killing towards TF-1 although activated PBL are more affected than resting PBL. The analysis of AlPc intracellular emission by flow cytometry shows that the uptake of the dye by leukemia cells is faster. This good efficacy towards AML and the observed lower phototoxicity towards normal cells (PBL, normal progenitors) suggest that this phthalocyanine is a potential bone marrow purging agent.

EFFICIENT PHOTODYNAMIC ACTION OF VICTORIA BLUE BO AGAINST THE HUMAN LEUKEMIC CELL LINES K‐562 AND TF‐1

Abstract— Photodynamic induced cytotoxicity by Victoria blue BO (VB‐BO), merocyanine 540 (MC540), Nile blue A (NB) and 4‐tetrasulfonatophenyl‐porphyrin (4‐TSPP) has been studied on two human leukemic cell lines: K‐562 and TF‐1. Cells were incubated with dyes and irradiated with different doses of white light. Cell survival was assessed by propidium iodide (PI) staining using flow cytometry analysis. Concentrations of 5 x 10 8M VB‐BO were found to kill 75% of cells, and a concentration of 1 × 10−7M induced more than 99% of cell killing. To obtain the same cytotoxic level, the presence of 2.6 × 10−5M of MCS40 during irradiation was needed. Under the conditions used, NB was ineflective as a photosensitizer, although uptake studies showed that this dye was taken by the cells in much greater amounts than any other studied dye. Cell cycle distribution of TF‐1 cells, surviving MC540 or VB‐BO photoscnsitization has bccn studied by flow cytometry analysis after staining with Hoechst 33342 and PI. It was found that cells in G1 phase were slightly more resistant toward MCS40– and VB‐BO‐mediated photosensitization than cells in other phascs of the ccll cycle

Killing efficacy of a new silicon phthalocyanine in human melanoma cells treated with photodynamic therapy by early activation of mitochondrion-mediated apoptosis.

Abstract:  Photodynamic therapy (PDT) is a promising therapeutic modality that utilizes a combination of a photosensitizer and visible light for the destruction of diseased tissues. Using human‐pigmented melanoma cells, we examined the photokilling efficacy of new silicon‐phthalocyanines (SiPc) that bore bulky axial substituents. The bis(cholesteryloxy) derivate (Chol‐O‐SiPc) displayed the best in vitro photokilling efficacy (LD50 = 6–8 × 10−9 M) and was seven to nine times more potent than chloro‐aluminium Pc (ClAlPc), a known photosensitizer used as a reference. Although Chol‐O‐SiPc was half as potent as ClAlPc for promoting photo‐oxidative membrane damage in a cell‐free assay, early events of mitochondrion‐mediated apoptosis upon PDT were triggered much faster, as demonstrated by kinetics studies examining cells with permeabilized mitochondrial membranes, cytochrome c release and caspase‐9 activation. Inhibition of caspase‐9 activity by a substrate analogue argued for its central role in the proapoptotic events leading to cell death by Chol‐O‐SiPc PDT. In addition, immunoblots showed that Bcl‐2 antiapoptotic oncoprotein was not a primary target of Chol‐O‐SiPc in M3Dau cells treated with PDT. Conclusively, Chol‐O‐SiPc is a useful new photosensitizer with the property of triggering cell apoptosis mediated by mitochondria.

Spectroscopic properties of thiophene linked [2H]-chromenes

Abstract The influence of an oligothiophenic substituent upon the spectroscopic behaviour of chromenes is discussed. Fluorescence emission is observed for several compounds. The photochromism/fluorescence ratio depends on the polythiophenic chain length and on the substitution site on the chromene.

Supported redox photosensitizers for the activation of alkylaromatics towards oxygen

Abstract Several 2,4,6-trinitrobenzene derivatives and 2,4,7-trinitrofluorenone were grafted on silica and used as supported electron-acceptor photosensitizers. The grafting was effected by two methods: reaction of a halogenated derivative with aminopropyl silica or condensation on silica of the electron acceptor previously transformed into monochlorosilane. The oxidation of 4-methyl anisole by redox photosensitization was used as a test reaction to compare the efficiencies of these heterogeneous photosensitizers. Silica grafted 2,4,6-trinitrobenzene was then used to oxidize 4-substituted methyl aromatic compounds into the corresponding aromatic aldehydes with 76%–90% yields and 100% selectivity.

Photodynamic Therapy: In Vitro Photosensitizing Efficacy of Bis(tri-n-hexylsiloxy)silicon Phthalocyanine against Achromic M6 Melanocytes. ESR Study of Active Oxygen Intermediates

The in vitro photodynamic effect of bis(tri-n-hexylsiloxy)silicon phthalocyanine has been evaluated against the melanotic M6 cell line. The results showed that at 10-5 M dose, LD50 is obtained for a 150 J cm-2 light dose and LD90 for 540 J cm-2. Electron spin resonance spectroscopy was used with spin traps to study the type I and type II photochemical pathways involved and to detect active oxygen intermediates such as singlet oxygen, oxygen superoxide and hydroxyl radical. The two mechanisms occurred simultaneously and no change was observed when the phthalocyanine was entrapped in liposomes.