Self‐assembled verteporfin nanoparticles decrease drug efflux by P‐glycoprotein without light activation in drug‐resistant cancer cells

P‐glycoprotein (P‐gp)‐mediated multidrug resistance (MDR) remains one of the major obstacles to successful chemotherapy for cancer. Previous studies have shown that photodynamic therapy and priming using verteporfin (VP) decrease the drug efflux by P‐gp and improve chemotherapy efficacy. Despite these promising results, the inhibitory effects on P‐gp are reversible, as continuous protein synthesis restores P‐gp expression. The effects of VP under non‐illuminated conditions remain underexplored in the context of P‐gp‐mediated MDR. In this study, we investigate the capacity of non‐illuminated self‐assembled nanoaggregates of VP (NanoVP) to modulate P‐gp efflux function by targeting mitochondrial metabolism in vitro. We found that high concentrations of NanoVP, when exposed to drug‐resistant cells for longer time periods, decrease oxygen consumption rate and adenosine triphosphate (ATP production in mitochondria. We determined a concentration (5 μM) and time point (72 h) for NanoVP treatment that significantly decreases mitochondrial ATP levels with minimal cytotoxicity. This metabolic perturbation results in enhanced accumulation of P‐gp substrates and decreased binding of P‐gp‐specific antibodies, indicating the sustained inhibition of P‐gp function. Notably, NanoVP‐mediated inhibition of P‐gp without light activation enhances the efficacy of chemotherapeutic agents that are P‐gp substrates in drug‐resistant cells. Thus, our findings introduce a novel, light‐independent application for VP as a metabolic priming agent to overcome P‐gp‐mediated MDR and improve chemotherapeutic efficacy in drug‐resistant cancers.