Phototoxic effects of pheophorbide-a nanoconjugates on melanoma tumor spheroids

Melanoma is the deadliest form of skin cancer, with a rapidly rising incidence and a poor prognosis for patients diagnosed at advanced stages. Photodynamic therapy (PDT) represents a promising, minimally invasive treatment for melanoma. However, its clinical use is often limited by poor tumour selectivity and suboptimal intracellular accumulation of conventional photosensitizers (PSs). To address these challenges, an active targeted photosensitizer nanoparticle that uses pheophorbide-a (Ppa) conjugated with gold nanoparticles (AuNPs) and folic acid (FA) was fabricated. AuNPs serve as nanocarriers with their unique features of high biocompatibility, good photophysical properties, and high load capacity that help in the effective delivery of PSs. AuNPs are attractive nanocarriers for PDT owing to their facile synthesis, surface functionalisation flexibility, excellent biocompatibility, and high stability. These features enable efficient PS loading and facilitate both passive and active targeting, thereby improving the pharmacokinetic profile and therapeutic index of the PS. Folic acid functionalization helps in receptor-mediated targeting through the overexpressed folate receptors on the melanoma cells. The Ppa-AuNP-FA nanobioconjugate was synthesized and characterized for morphology, stability, and photodynamic performance. The phototoxic efficacy of the nanophotosensitiser was tested on three-dimensional (3D) melanoma multicellular tumour spheroids (MCTS), a physiologically relevant model that closely mimics tumour architecture and drug resistance patterns observed in vivo. Optimization studies of free Ppa yielded an IC50 concentration of 9.3 µM, which was used throughout the study. A high concentration of Ppa significantly ruptured spheroids, indicating the need to modify this PS to reduce dose dependency and prevent unwanted side effects in patients. Upon laser irradiation (10 J/cm2), Ppa- AuNP-FA caused significant disruption of the spheroid outer membrane, increased intracellular reactive oxygen species (ROS) production, and induced severe cell death compared to free Ppa and non-targeted controls. The findings from this study demonstrate that the coupling of Ppa with AuNPs and FA greatly improves PS delivery, phototoxicity, and cellular uptake. The Ppa-AuNP-FA complex may become an excellent approach in the active targeting nanotechnology platform for improving PDT in malignant melanoma.