Attritional evaluation of lipophilic and hydrophilic metallated phthalocyanines for oncological photodynamic therapy

• Main Authors: Cavaco, J.E.B (Author), de Haan, L.R (Author), de Keijzer, M.J (Author), de Klerk, D.J (Author), Desclos, E. (Author), Dias, L.M (Author), Ding, B. (Author), Ernst, D. (Author), Franchi, L.P (Author), Heger, M. (Author), Huang, X. (Author), Kochan, J.A (Author), Krawczyk, P.M (Author), Mesquita, B. (Author), Pan, W. (Author), Photodynamic Therapy Study Group (Author), Scutigliani, E.M (Author), Sharifi, F. (Author), Tedesco, A.C (Author), van Wijk, A.C (Author)
• Format: Article
• Language: English
• Published: Elsevier B.V. 2021
• Subjects: A-431 cell line; Aluminum phthalocyanine; antineoplastic agent; Antineoplastic Agents; apoptosis; Apoptosis; Article; cancer therapy; cell death; Cell Line, Tumor; cell membrane permeability; Cell Membrane Permeability; cell proliferation assay; Cell survival; cell viability assay; chemistry; comparative study; confocal microscopy; Dark toxicity; Dose-Response Relationship, Radiation; drug carrier; Drug Carriers; Drug Liberation; drug release; epidermal growth factor receptor; flow cytometry; fluorescence analysis; G2 phase cell cycle checkpoint; human; human cell; Humans; incubation time; indole derivative; Indoles; LC50; liposome; Liposomes; oncological photodynamic therapy; percentage of cells in G0/G1 phase; photochemotherapy; Photochemotherapy; photodynamic therapy; photosensitization; Photosensitizers, cell death; photosensitizing agent; Photosensitizing Agents; Phototoxicity; phthalocyanine; phthalocyanine aluminum; phthalocyanine zinc; protein expression; radiation response; S phase cell cycle checkpoint; signal transduction; structure activity relation; Structure-Activity Relationship; time factor; Time Factors; treatment response; tumor cell line; tumor suppressor gene; Zinc phthalocyanine
• Online Access: View Fulltext in Publisher

 Background and aim: Oncological photodynamic therapy (PDT) relies on photosensitizers (PSs) to photo-oxidatively destroy tumor cells. Currently approved PSs yield satisfactory results in superficial and easy-to-access tumors but are less suited for solid cancers in internal organs such as the biliary system and the pancreas. For these malignancies, second-generation PSs such as metallated phthalocyanines are more appropriate. Presently it is not known which of the commonly employed metallated phtahlocyanines, namely aluminum phthalocyanine (AlPC) and zinc phthalocyanine (ZnPC) as well as their tetrasulfonated derivatives AlPCS4 and ZnPCS4, is most cytotoxic to tumor cells. This study therefore employed an attritional approach to ascertain the best metallated phthalocyanine for oncological PDT in a head-to-head comparative analysis and standardized experimental design. Methods: ZnPC and AlPC were encapsulated in PEGylated liposomes. Analyses were performed in cultured A431 cells as a template for tumor cells with a dysfunctional P53 tumor suppressor gene and EGFR overexpression. First, dark toxicity was assessed as a function of PS concentration using the WST-1 and sulforhodamine B assay. Second, time-dependent uptake and intracellular distribution were determined by flow cytometry and confocal microscopy, respectively, using the intrinsic fluorescence of the PSs. Third, the LC50 values were established for each PS at 671 nm and a radiant exposure of 15 J/cm2 following 1-h PS exposure. Finally, the mode of cell death as a function of post-PDT time and cell cycle arrest at 24 h after PDT were analyzed. Results: In the absence of illumination, AlPC and ZnPC were not toxic to cells up to a 1.5-μM PS concentration and exposure for up to 72 h. Dark toxicity was noted for AlPCS4 at 5 μM and ZnPCS4 at 2.5 μM. Uptake of all PSs was observed as early as 1 min after PS addition to cells and increased in amplitude during a 2-h incubation period. After 60 min, the entire non-nuclear space of the cell was photosensitized, with PS accumulation in multiple subcellular structures, especially in case of AlPC and AlPCS4. PDT of cells photosensitized with ZnPC, AlPC, and AlPCS4 yielded LC50 values of 0.13 μM, 0.04 μM, and 0.81 μM, respectively, 24 h post-PDT (based on sulforhodamine B assay). ZnPCS4 did not induce notable phototoxicity, which was echoed in the mode of cell death and cell cycle arrest data. At 4 h post-PDT, the mode of cell death comprised mainly apoptosis for ZnPC and AlPC, the extent of which was gradually exacerbated in AlPC-photosensitized cells during 8 h. ZnPC-treated cells seemed to recover at 8 h post-PDT compared to 4 h post-PDT, which had been observed before in another cell line. AlPCS4 induced considerable necrosis in addition to apoptosis, whereby most of the cell death had already manifested at 2 h after PDT. During the course of 8 h, necrotic cell death transitioned into mainly late apoptotic cell death. Cell death signaling coincided with a reduction in cells in the G0/G1 phase (ZnPC, AlPC, AlPCS4) and cell cycle arrest in the S-phase (ZnPC, AlPC, AlPCS4) and G2 phase (ZnPC and AlPC). Cell cycle arrest was most profound in cells that had been photosensitized with AlPC and subjected to PDT. Conclusions: Liposomal AlPC is the most potent PS for oncological PDT, whereas ZnPCS4 was photodynamically inert in A431 cells. AlPC did not induce dark toxicity at PS concentrations of up to 1.5 μM, i.e., > 37 times the LC50 value, which is favorable in terms of clinical phototoxicity issues. AlPC photosensitized multiple intracellular loci, which was associated with extensive, irreversible cell death signaling that is expected to benefit treatment efficacy and possibly immunological long-term tumor control, granted that sufficient AlPC will reach the tumor in vivo. Given the differential pharmacokinetics, intracellular distribution, and cell death dynamics, liposomal AlPC may be combined with AlPCS4 in a PS cocktail to further improve PDT efficacy. © 2021 Elsevier B.V.