Title:Development and In Vitro Proof-of-Concept of Interstitially Targeted Zinc- Phthalocyanine Liposomes for Photodynamic Therapy
Volume: 21
Issue: 3
Author(s): M. Broekgaarden, A. I.P.M. de Kroon, T. M. van Gulik and M. Heger
Affiliation:
Keywords:
Sk-Cha1 human cancer cell line, extrahepatic cholangiocarcinoma, liposomal drug delivery system, oxidation reactions,
molecular probes and model proteins, fluorescence spectroscopy and flow cytometry.
Abstract: Background: 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. Objective: To circumvent these drawbacks, a secondgeneration
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. Methods: 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. Results: 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. Conclusions: 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.
