Mechanistic insight of the photodynamic inactivation of Escherichia coli by a tetracationic zinc(II) phthalocyanine derivative

Abstract

Photodynamic inactivation (PDI) of Escherichia coli has been studied in cultures treated with zinc(II) 2,9,16,23-tetrakis[4-(N-methylpyridyloxy)]phthalocyanine (ZnPPc+4) to obtain insight about the mechanism of damage. This phthalocyanine is rapidly bound to cells, reaching a value of ∼0.8 nmol/106 cells when the cultures were incubated with 2 μM sensitizer. After 30 min of irradiation, a 4 log decrease of E. coli survival was observed. The photocytotoxic action was investigated in plasmid and genomic DNA by electrophoretic analysis. Absorption spectroscopic studies showed that this cationic phthalocyanine interacts strongly with DNA (KDNA = 4.7 × 106 M-1). Photocleavage of calf thymus DNA sensitized by ZnPPc+4 was not found even after long irradiation periods. Similar results were also observed in genomic DNA extracted from E. coli cells after PDI treatment. Modifications of plasmid DNA isolated from bacteria were only observed after long irradiation periods. However, under these conditions transmission electron microscopy of the PDI bacteria revealed an aggregation of cytoplasmic macromolecules and irregularities in cell barriers. Also, scanning electron microscopy showed a shrunken appearance in cells after PDI. Even so, release of intracellular biopolymers was not detected by absorption. On the other hand, outer and inner membranes permeabilization assays showed an increase in the permeability. Consequently, alterations in the cell membrane functionality induced by ZnPPc+4 appear to be the major cause of E. coli inactivation upon PDI.