Liposomal temocene (m-THPPo) photodynamic treatment induces cell death by mitochondria-independent apoptosis

Abstract

Background: The cell death pathway activated after photodynamic therapy (PDT) is controlled by a variety of parameters including the chemical structure of the photosensitizer, its subcellular localization, and the photodynamic damage induced. The present study aims to characterize a suitable m-THPPo liposomal formulation, to determine its subcellular localization in HeLa cells and to establish the cell death mechanisms that are activated after photodynamic treatments.

Methods: Liposomes containing m-THPPo were prepared from a mixture of DPPC and DMPG at a 9:1 molar ratio. In order to procure the best encapsulation efficiency, the m-THPPo/lipid molar ratio was considered. HeLa cells were incubated with liposomal m-THPPo and the subcellular localization of m-THPPo was studied. Several assays such as TUNEL, annexin V/propidium iodide and Hoechst-33258 staining were performed after photodynamic treatments. The apoptotic initiation was assessed by cytochrome c and caspase-2 immunofluorescence.

Results: m-THPPo encapsulated in liposomes showed a decrease of the fluorescence and singlet oxygen quantum yields, compared to those of m-THPPo dissolved in tetrahydrofuran. Liposomal m-THPPo showed colocalization with LysoTracker® and it induced photoinactivation of HeLa cells by an apoptotic mechanism. In apoptotic cells no relocalization of cytochrome c could be detected, but caspase-2 was positive immediately after photosensitizing treatments.

Conclusions: Photodynamic treatment with liposomal m-THPPo leads to a significant percentage of apoptotic morphology of HeLa cells. The activation of caspase-2, without the relocalization of cytochrome c, indicates a mitochondrial-independent apoptotic mechanism.

General significance: These results provide a better understanding of the cell death mechanism induced after liposomal m-THPPo photodynamic treatment.

Keywords: Apoptosis; DMPG; DMSO; DPPC; H-33258; Hoechst-33258; Liposome; MLVs; PBS; PCS; PDT; PI; PS; Photosensitizer; ROS; TB; THF; Temocene; dimethylsulfoxide; dimyristoylphosphatidylglycerol; dipalmitoylphosphatidylcholine; m-THPPo; m-tetra (hydroxyphenyl) porphycene (temocene); multilamellar vesicles; phosphate buffered saline; photodynamic therapy; photon correlation spectroscopy; photosensitizer; propidium iodide; reactive oxygen species; tetrahydrofuran; toluidine blue.