Highly Pifithrin-μ Loaded Melanin Nanosphere: A Safe and Efficient Approach to Hepatocellular Carcinoma Therapy via Thermo-Controlled Drug Release

Pifithrin-[Formula: see text], a potent dual-pathway inhibitor of p53 and HSP70, suffers from poor aqueous solubility and dose-limiting systemic toxicity, which severely hinder its anticancer efficacy. To overcome these limitations, we developed melanin-based nanospheres (MNSs) through alkaline-driven self-polymerization of dopamine. Capitalizing on [Formula: see text]–[Formula: see text] stacking interactions, we achieved efficient surface loading of pifithrin-[Formula: see text] (forming MNS-[Formula: see text] with a high drug-loading capacity of 40%. The resulting MNS-[Formula: see text] platform exhibited excellent colloidal stability under simulated physiological conditions and displayed sustained, thermoresponsive drug release kinetics, retaining [Formula: see text] of the payload at physiological temperature while triggering rapid release at [Formula: see text]C. Flow cytometry and confocal microscopy analyzes confirmed enhanced cellular internalization of MNS-[Formula: see text] in HepG2 cells. Upon laser irradiation, MNS-[Formula: see text] mediated localized photothermal effects, enabling spatiotemporally controlled drug release and significant cytotoxicity, reducing HepG2 cell viability to 35% at 200[Formula: see text][Formula: see text]g/mL. In contrast, PEGylated MNS (MNS-PEG) controls showed negligible photothermal toxicity. In a HepG2 xenograft mouse model, the combination of MNS-[Formula: see text] and near-infrared (NIR) irradiation achieved superior tumor growth inhibition ([Formula: see text] reduction in tumor volume) compared to free pifithrin-[Formula: see text] monotherapy, attributable to enhanced tumor accumulation and on-demand drug release. Histopathological examination revealed extensive tumor necrosis in the combination group without observed systemic toxicity. This study highlights MNSs as a versatile and biocompatible nanoplatform for enhancing the therapeutic index of hydrophobic agents via photothermally augmented targeting and stimuli-responsive delivery.