Development and In Vitro Proof-of-Concept of Interstitially Targeted Zinc- Phthalocyanine Liposomes for Photodynamic Therapy

Abstract

Photodynamic therapy (PDT) has been successfully used to treat various solid tumors. However, some cancer types respond poorly to PDT, including urothelial carcinomas, nasopharyngeal carcinomas, and extrahepatic cholangiocarcinomas. The therapeutic recalcitrance is in part due to the use of photosensitizers with suboptimal optical/ photochemical properties and unfavorable pharmacokinetics. To circumvent these drawbacks, a second-generation photosensitizer with improved optical/photochemical properties, zinc phthalocyanine (ZnPC), was encapsulated in interstitially targeted, polyethylene glycol-coated liposomes (ITLs) intended for systemic administration. The ZnPC-ITLs were examined for reactive oxygen species (ROS) generation and oxidation capacity and validated for tumoricidal efficacy in human extrahepatic cholangiocarcinoma (Sk-Cha1) cells. ZnPC-ITL uptake and the mechanism and mode of PDT-induced cell death were studied. The ITL formulation was optimized on the basis of fluorescence spectroscopy and photon correlation spectroscopy. The extent of ROS generation, protein oxidation, and membrane oxidation were determined by the 2',7'-dichlorodihydrofluorescein, tryptophan oxidation, and calcein leakage assays, respectively. PDT efficacy was evaluated by measuring mitochondrial activity and apoptosis-/necrosis-specific staining in combination with flow cytometry. The uptake of fluorescently labeled ITLs was assayed by confocal microscopy, flow cytometry, and fluorescence spectroscopy. ZnPC-ITLs exhibited maximum ROS-generating and oxidation potential at a ZnPC:lipid molar ratio of 0.003. PDT of Sk-Cha1 cells incubated with ZnPC-ITLs induced cell death in a lipid concentration- dependent manner. The mode of PDT-induced cell death comprised both apoptosis and necrosis, with necrotic cell death predominating. Post-PDT cell death was attributable to pre-PDT ZnPC-ITL uptake by cancer cells, which was more efficient at smaller ITL diameters and a more positive surface charge. ZnPC-ITLs constitute a nanoparticulate photosensitizer delivery system capable of inducing apoptosis and necrosis in cultured extrahepatic cholangiocarcinoma cells by PDT-mediated oxidative processes. PDT-induced cell death is dependent on the extent of ITL uptake, which in turn relies on ITL size and zeta potential.