title: Comparative kinetics of damage to the plasma and mitochondrial membranes by intra-cellularly synthesized and externally-provided photosensitizers using multi-color FACS.
abstract: Photodynamic therapy (PDT) of cancer involves inflicting lethal damage to the cells of malignant tumors, primarily by singlet oxygen that is generated following light-absorption in a photosensitizer molecule. Dysfunction of cells is manifested in many ways, including peroxidation of cellular components, membrane rupture, depolarization of electric potentials, termination of mitochondrial activity, onset of apoptosis and necrosis and eventually cell lysis. These events do not necessarily occur in linear fashion and different types of damage to cell components occur, most probably, in parallel. In this report we measured the relative rates of damage to two cellular membranes: the plasma membrane and the mitochondrial membrane. We employed photosensitizers of diverse hydrophobicities and used different incubation procedures, which lead to their different intra-cellular localizations. We monitored the damage that was inflicted on these membranes, by employing optical probes of membrane integrity, in a multi-color FACS experiment. The potentiometric indicator JC-1 monitored the electric cross-membrane potential of the mitochondria and the fluorometric indicator Draq7 monitored the rupture of the plasma membrane. We show that the electric depolarization of the mitochondrial membrane and the damage to the enveloping plasma membrane proceed with different kinetics that reflect the molecular character and intracellular location of the sensitizer: PpIX that is synthesized in the cells from ALA causes rapid mitochondrial damage and very slow damage to the plasma membrane, while externally added PpIX has an opposite effect. The hydrophilic sensitizer HypS4 can be taken up by the cells by different incubation conditions, and these affect its intracellular location, and as a consequence either the plasma membrane or the mitochondria is damaged first. A similar correlation was found for additional extracellularly-provided photosensitizers HP and PpIX.
authors.affiliation: Department of Physics and Institute of Nanotechnology and Advanced Materials, Bar Ilan University, Ramat Gan, Israel.
authors.forename: Sara
authors.initials: S
authors.lastname: Haupt
authors.forename: Zvi
authors.initials: Z
authors.lastname: Malik
authors.forename: Benjamin
authors.initials: B
authors.lastname: Ehrenberg
mesh_terms: D000622:Aminolevulinic Acid / Q000494:pharmacology*; D018943:Anthracyclines; D001562:Benzimidazoles; D002232:Carbocyanines; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008027:Light; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects*; D051336:Mitochondrial Membranes / Q000187:drug effects*; D009474:Neurons / Q000187:drug effects / Q000473:pathology; D017319:Photosensitizing Agents / Q000494:pharmacology*; D011524:Protoporphyrins / Q000494:pharmacology*
chemical_list: D018943:Anthracyclines; D001562:Benzimidazoles; D002232:Carbocyanines; C582241:DRAQ7 fluorescent probe; D005456:Fluorescent Dyes; D017319:Photosensitizing Agents; D011524:Protoporphyrins; C068624:5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine; D000622:Aminolevulinic Acid; C028025:protoporphyrin IX