Cellular Imaging and Time-Domain FLIM Studies of Meso-Tetraphenylporphine Disulfonate as a Photosensitising Agent in 2D and 3D Models

• Published in: International journal of molecular sciences Vol. 25; no. 8; p. 4222
• Main Authors: Balukova, Andrea, Bokea, Kalliopi, Barber, Paul R, Ameer-Beg, Simon M, MacRobert, Alexander J, Yaghini, Elnaz
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 11.04.2024
• Subjects: Cancer; Cancer therapies; Cell death; Cell Line, Tumor; Chemotherapy; Collagen; Female; Fluorescence; fluorescence lifetime imaging microscopy; Humans; Hypoxia; Light; Localization; Lysosomes - drug effects; Lysosomes - metabolism; Microscopy; Optical Imaging - methods; Ovarian cancer; Ovarian Neoplasms - drug therapy; Ovarian Neoplasms - metabolism; Ovarian Neoplasms - pathology; Photochemotherapy; Photochemotherapy - methods; photodynamic therapy; Photosensitizing Agents - chemistry; Photosensitizing Agents - pharmacology; Porphyrins - chemistry; Porphyrins - pharmacology; reactive oxygen species; Reactive Oxygen Species - metabolism; United Kingdom; Germany; photodynamic therapy; reactive oxygen species; fluorescence lifetime imaging microscopy
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms25084222

Fluorescence lifetime imaging (FLIM) and confocal fluorescence studies of a porphyrin-based photosensitiser (meso-tetraphenylporphine disulfonate: TPPS ) were evaluated in 2D monolayer cultures and 3D compressed collagen constructs of a human ovarian cancer cell line (HEY). TPPS is known to be an effective model photosensitiser for both Photodynamic Therapy (PDT) and Photochemical Internalisation (PCI). This microspectrofluorimetric study aimed firstly to investigate the uptake and subcellular localisation of TPPS , and evaluate the photo-oxidative mechanism using reactive oxygen species (ROS) and lipid peroxidation probes combined with appropriate ROS scavengers. Light-induced intracellular redistribution of TPPS was observed, consistent with rupture of endolysosomes where the porphyrin localises. Using the same range of light doses, time-lapse confocal imaging permitted observation of PDT-induced generation of ROS in both 2D and 3D cancer models using fluorescence-based ROS together with specific ROS inhibitors. In addition, the use of red light excitation of the photosensitiser to minimise auto-oxidation of the probes was investigated. In the second part of the study, the photophysical properties of TPPS in cells were studied using a time-domain FLIM system with time-correlated single photon counting detection. Owing to the high sensitivity and spatial resolution of this system, we acquired FLIM images that enabled the fluorescence lifetime determination of the porphyrin within the endolysosomal vesicles. Changes in the lifetime dynamics upon prolonged illumination were revealed as the vesicles degraded within the cells.