Indocyanine Green as a Theragnostic Agent in MCF-7 Breast Cancer Cells

Background/Objectives: Indocyanine green (ICG) is an FDA-approved, near-infrared fluorescent dye widely used for tumor imaging. This study aimed to evaluate the photodynamic efficacy and selectivity of ICG as a photosensitizer in photodynamic therapy (PDT) against MCF-7 breast cancer cells in 2D monolayers and 3D collagen-embedded cell cultures that simulate ECM diffusion, and to confirm direct generation of singlet oxygen (¹O₂) as the primary cytotoxic species. Methods: MCF-7 breast adenocarcinoma cells and HMEC normal mammary epithelial cells were cultured in 2D monolayers, with MCF-7 cells additionally grown in 3D collagen type I matrices to mimic tumor environments. Cells were incubated with 50 µM ICG for 30 min, washed, and irradiated with a 780 nm diode laser at 39.8 mW/cm². Cell viability was quantified using the Muse^® Count & Viability assay at multiple time points, while ICG uptake and penetration were assessed via flow cytometry, fluorescence microscopy, and confocal imaging. Direct ¹O₂ production was measured through its characteristic 1270 nm phosphorescence using time-resolved near-infrared spectrometry. Results: ICG-PDT reduced MCF-7 viability to 58.3 ± 7.4% in 2D cultures (41.7% cell kill, p < 0.0001) and 70.2 ± 10.7% in 3D cultures (29.8% cell kill, p = 0.0002). In contrast, normal HMECs maintained 91.0 ± 1.3% viability (only 9% reduction, p = 0.08), resulting in a therapeutic index of approximately 4.6. IC₅₀ values in 2D MCF-7 cultures decreased over time from 51.4 ± 3.0 µM at 24 h to 27.3 ± 3.0 µM at 72 h. ICG uptake was higher in 2D (78%) than in 3D (65%) MCF-7 cultures, with diffusion in 3D collagen exhibiting linear depth-dependent penetration. Notably, the singlet-oxygen phosphorescence signal, though weak and requiring highly sensitive detectors, provided direct evidence of efficient ¹O₂ generation. Conclusions: ICG as a photosensitizer in photodynamic therapy using clinically compatible parameters is highly cytotoxic to MCF-7 breast cancer cells while largely sparing HMECs in 2D cell culture. Direct spectroscopic evidence confirms efficient ¹O₂ generation, which contributes significantly to the cytotoxicity. The reduced efficacy in 3D versus 2D models highlights the importance of penetration barriers also present in solid tumors. These results support further preclinical and clinical investigation of ICG as a dual imaging-and-therapy (theragnostic) agent for selective photodynamic treatment of breast cancer.