Stability and Selectivity of Indocyanine Green Towards Photodynamic Therapy of CRL-2314 Breast Cancer Cells with Minimal Toxicity to HTB-125 Cells

<b>Background:</b> Photodynamic therapy (PDT) with indocyanine green (ICG) offers a promising, minimally invasive approach for selective tumor ablation in breast cancer. This study investigates the stability, cellular uptake, and photodynamic efficacy of ICG in CRL-2314 breast cancer cells compared with HTB-125 normal mammary epithelial cells, with a focus on population density-dependent cytotoxicity. Cells were incubated with 50 µM ICG for 1-3 h and irradiated with a 780 nm laser. Viability was assessed using the Muse^® Count & Viability Kit at 1-3 h. ICG uptake kinetics were quantified by flow cytometry. Singlet oxygen (¹O₂) generation was confirmed via 1270 nm phosphorescence and Stern-Volmer quenching. ICG uptake saturated at 2 h (89 ± 4% positive cells), with lysosomal colocalization. In CRL-2314 cells, viability decreased density- and time-dependently, reaching 40 ± 5% at 1 × 10⁶ cells after 3 h (<i>p</i> < 0.0001), with IC₅₀ = 23.8 µM (95% CI: 20-27 µM) at 72 h. HTB-125 cells maintained > 80% viability even at 300 µM, yielding no IC₅₀. Two-way ANOVA confirmed cell line specificity (F = 428.7, <i>p</i> < 0.0001). ICG-PDT exhibits high selectivity and density-dependent efficacy against CRL-2314 cells with minimal toxicity to HTB-125, driven by enhanced uptake, sustained ¹O₂ production, and differential metabolic responses. These findings support ICG-PDT as a precision modality for breast cancer therapy.