Intracellular dynamics and photodynamic efficacy of black phosphorus quantum dots in cancer models

• Biocompatible synthesis of black phosphorus quantum dots and their functionalisation with polyethylene glycol and hyaluronic acid. • Enhanced singlet oxygen generation and improved tumor accumulation. • Intracellular tracking in 2D and 3D cancer cell models. • Live-cell detection of intracellular singlet oxygen production. • High photodynamic efficacy in 3D tumor spheroid models. Photodynamic therapy (PDT) is a promising cancer treatment that relies on the light-induced generation of singlet oxygen ( 1 O 2 ) to trigger cytotoxic effects. Black phosphorus quantum dots (BPQDs) have emerged as ideal PDT agents owing to their high 1 O 2 quantum yield, high biocompatibility, and favourable pharmcokinetics. Here we develop BPQDs functionalised with polyethylene glycol amine (PEG-NH 2 ) and hyaluronic acid (HA) to target the CD44-receptor of A549 lung cancer cells. We present a comprehensive evaluation of their intracellular dynamics, 1 O 2 generation and therapeutic efficiency in both 2D cell monolayers and 3D tumour spheroid models. Our HA-PEG-NH 2 -BPQDs were found to efficiently internalise and accumulate in both A549 cell monolayers and spheroids. Moreover, 1 O 2 generation was monitored in living cells using HyPer7, a genetically encoded fluorescent protein sensor, for the first time. Upon irradiation with red light, BPQDs exhibited significant photocytotoxicity, reducing viability to 34% in 2D cultures and inducing extensive cell death throughout 3D spheroids. These findings not only advance our understanding of BPQD behaviour in tumours but also establish their potential as powerful photosensitisers for precision photodynamic therapy.