<scp>A549</scp> Alveolar Carcinoma Spheroids as a Cytotoxicity Platform for Carboxyl‐ and Amine‐Polyethylene Glycol Gold Nanoparticles

Gold nanoparticles (AuNPs) present with unique physicochemical features and potential for functionalization as anticancer agents. Three-dimensional spheroid models can be used to afford greater tissue representation due to their heterogeneous phenotype and complex molecular architecture. This study developed an A549 alveolar carcinoma spheroid model for cytotoxicity assessment and mechanistic evaluation of functionalized AuNPs. A549 spheroids were generated using an agarose micro-mold and were characterized (morphology, acid phosphatase activity, protein content) over 21 culturing days. The 72-h cytotoxicity of carboxyl-polyethylene glycol- (PCOOH-) and amine-polyethylene glycol- (PNH₂-) functionalized AuNPs against Day 7 spheroids was assessed by determining spheroid morphology, acid phosphatase activity, protein content, caspase-3/7 activity, and cell cycle kinetics. Spheroids remained stable over the experimental period. Although the A549 spheroids' volume increased while remaining viable over the culturing period, structural integrity decreased from Day 14 onwards. The PCOOH-AuNPs lacked cytotoxicity at a maximum concentration of 1.2 × 10¹² nanoparticles/mL with no prominent alteration to the cellular processes investigated, while the PNH₂-AuNPs (at a maximum of 4.5 × 10¹² nanoparticles/mL) displayed dose- and time-dependent cytotoxicity with associated loss of spheroid compactness, debris formation, DNA fragmentation, and a 75% reduction in acid phosphatase activity. Differentiation between cytotoxic and non-cytotoxic AuNPs was achieved, with preliminary elucidation of cytotoxicity endpoints. The PNH₂-AuNPs promote cytotoxicity by modulating cellular kinetics while destabilizing the spheroid ultrastructure. The model serves as a proficient platform for more in-depth elucidation of NP cytotoxicity at the preclinical investigation phase.