Photosensitization with zinc(II)phthalocyanine as a switch in the decision between apoptosis and necrosis

Photodynamic therapy (PDT) of tumors and other diseases is based on the uptake of a photosensitizing dye in target cells, which are damaged by reactive oxygen intermediates generated on irradiation with light in which the wavelengths match the dye absorption spectrum. PDT can induce cell death by necrosis and apoptosis both in vivo and in vitro, but the factors determining the contribution of either mechanism to the overall process are not completely defined. Our studies on the photosensitization of 4R transformed fibroblasts with the second-generation photosensitizer zinc (II) phthalocyanine (ZnPc) aim at determining the effect of important experimental parameters such as time of cell incubation (2 or 24 h) with ZnPc before irradiation and ZnPc concentration in the incubation medium on cell death. Furthermore, we propose possible correlations between the cell death mechanism and primary photo-damage sites; these are mainly determined by the intracellular localization of the photosensitizer. The mechanism of cell death was determined by both electron microscopy analysis of the morphological alterations induced by photosensitization and measurement of caspase 3 activation. The initial photodamage sites were determined by measuring the activities of several functions typical of mitochondria, lysosomes, Golgi apparatus, cytosol, and plasma membrane. The intracellular localization of ZnPc after 2- or 24-h incubation was determined by fluorescence microscopy. Necrosis, associated with early loss of plasma membrane integrity and complete depletion of intracellular ATP, represents the prevailing mode of death for 4R cells dark-incubated for 2 h with ZnPc and irradiated with light doses reducing viability by 99.9%. In contrast, irradiation performed 24 h after ZnPc incubation causes only partial inhibition of plasma membrane activities, and cell death occurs largely by apoptosis. ZnPc is mainly localized in the Golgi apparatus after 2- and 24-h incubation, and in all of the cases this compartment represents a primary target of photodamage. Only after prolonged incubation is mitochondrial localization of ZnPc clearly detected by fluorescence microscopy; this could be a determining factor for promotion of apoptosis. Our data demonstrate that it is possible to modulate the mechanism of cell death by appropriate protocols; this may be relevant for enhancing the therapeutic efficacy of PDT

Meso-substituted tetra-cationic porphyrins photosensitize the death of human fibrosarcoma cells via lysosomal targeting

In this paper we present a study on the intracellular localisation and the efficiency of cell photoinactivation of a series of derivatives of 5,10,15,20-tetrakis-(4-N-methylpyridyl)-porphine (C1), whose degree of lipophilicity was varied through replacement of one methyl group with an alkyl chain of various length. Human HT1080 fibrosarcoma cells exposed to the various C1 derivatives (0.25 muM) for 24 h and irradiated with increasing doses of red-light (0.45-27 J/cm(2)) were inactivated with different efficiencies. The efficiency of cell photoinactivation increased with the increasing length of the hydrocarbon tail and lipophilicity and correlated with the efficiency of the porphyrin accumulation into the cells. Despite the presence of positive charges, these porphyrins did localise rather selectively in lysosomes while mitochondrial localisation was not evident, as demonstrated by fluorescence microscopy studies. Studies on isolated mitochondria provided evidence that the porphyrin uptake and distribution in these organelles were not modulated by the transmembrane potential but were exclusively controlled by partitioning phenomena which might have prevented mitochondria localization in whole cells. Our findings demonstrated that these porphyrins entered the cells through the endocytotic pathway and were transported to lysosomes whose pH increased rapidly upon irradiation. Lysosomal damage did not cause any intracellular redistribution of the porphyrin and represented the primary event causing cell death, very likely via necrosis

A novel 10B-enriched carboranyl-containing phthalocyanine as a radio- and photo-sensitising agent for boron neutron capture therapy and photodynamic therapy of tumours: in vitro and in vivo studies

The synthesis of a Zn(II)-phthalocyanine derivative bearing four B-10-enriched o-carboranyl units (B-10-ZnB4Pc) and its natural isotopic abundance analogue (ZnB4Pc) in the peripheral positions of the tetraazaisoindole macrocycle is presented. The photophysical properties of ZnB4Pc, as tested against model biological systems, were found to be similar with those typical of other photodynamically active porphyrin-type photosensitisers, including a singlet oxygen quantum yield of 0.67. The carboranyl-carrying phthalocyanine was efficiently accumulated by B16F1 melanotic melanoma cells in vitro, appeared to be partitioned in at least some subcellular organelles and, upon red light irradiation, induced extensive cell mortality. Moreover, ZnB4Pc, once i.v.-injected to C57BL/6 mice bearing a subcutaneously transplanted pigmented melanoma, photosensitised an important tumour response, provided that the irradiation at 600-700 nm was performed 3 h after the phthalocyanine administration, when appreciable concentrations of ZnB4Pc were still present in the serum. Analogously, irradiation of the B-10-ZnB4Pc-loaded pigmented melanoma with thermal neutrons 24 h after injection led to a 4 day delay in tumour growth as compared with control untreated mice. These results open the possibility to use one chemical compound as both a photosensitising and a radiosensitising agent for the treatment of tumours by the combined application of photodynamic therapy and boron neutron capture therapy

Tumour-localizing and -photo sensitising properties of meso-tetra(4-nido-carboranylphenyl)porphyrin (H2TCP)

A water-soluble meso-substituted porphyrin (H2TCP) bearing 36 boron atoms, which appeared to be an efficient photodynamic sensitiser (singlet oxygen quantum yield=0.44), was studied for its accumulation by murine melanotic melanoma cells (B16F1). The amount of H2TCP in the cells increased with the porphyrin dose in the incubation medium up to, and at least, 100 mu M concentrations with no significant cytotoxic effect in the dark. Moreover, the H2TCP uptake increased with the incubation time reaching a plateau value corresponding with the recovery of 0.4 nmol of H2TCP per mg of cell proteins after 24 h incubation. Fluorescence microscopy observations showed that the porphyrin was largely localized intracellularly, exhibiting a discrete distribution in the cytoplasm with a pattern which was closely similar to that observed for the endosomal probe Lucifer yellow. The photosensitising efficiency of the H2TCP toward B16F1 cells was studied for different irradiation (1-15 min) and incubation (1-24 h) times. Nearly complete (> 95%) cell mortality was obtained upon incubation with 20 mu M H2TCP and 10 min irradiation with red light (600-700 nm, 20 mW/cm(2)). The porphyrin was also accumulated in appreciable amounts by the tumour tissue after intravenous injection to C57BL/6 mice bearing a subcutaneously transplanted melanotic melanoma. Maximum accumulation in the turnout was achieved by administration of H2TCP dissolved in the ternary mixture 20% dimethylsulfoxide (DMSO)-30% polyethyleneglycol (PEG 400)-50% water. Thus, this porphyrin could act as both a photodynamic therapy agent and a radiosensitising agent for boron neutron capture therapy

Tumour-localizing and -photosensitising properties of meso-tetra(4-nido-carboranylphenyl)porphyrin (H\u3csub\u3e2\u3c/sub\u3eTCP)

A water-soluble meso-substituted porphyrin (H2TCP) bearing 36 boron atoms, which appeared to be an efficient photodynamic sensitiser (singlet oxygen quantum yield = 0.44), was studied for its accumulation by murine melanotic melanoma cells (B16F1). The amount of H2TCP in the cells increased with the porphyrin dose in the incubation medium up to, and at least, 100 μM concentrations with no significant cytotoxic effect in the dark. Moreover, the H2TCP uptake increased with the incubation time reaching a plateau value corresponding with the recovery of 0.4 nmol of H2TCP per mg of cell proteins after 24 h incubation. Fluorescence microscopy observations showed that the porphyrin was largely localized intracellularly, exhibiting a discrete distribution in the cytoplasm with a pattern which was closely similar to that observed for the endosomal probe Lucifer yellow. The photosensitising efficiency of the H2TCP toward B16F1 cells was studied for different irradiation (1-15 min) and incubation (1-24 h) times. Nearly complete (\u3e95%) cell mortality was obtained upon incubation with 20 μM H2TCP and 10 min irradiation with red light (600-700 nm, 20 mW/cm2). The porphyrin was also accumulated in appreciable amounts by the tumour tissue after intravenous injection to C57BL/6 mice bearing a subcutaneously transplanted melanotic melanoma. Maximum accumulation in the tumour was achieved by administration of H2TCP dissolved in the ternary mixture 20% dimethylsulfoxide (DMSO)-30% polyethyleneglycol (PEG 400)-50% water. Thus, this porphyrin could act as both a photodynamic therapy agent and a radiosensitising agent for boron neutron capture therapy. © 2007 Elsevier B.V. All rights reserved