All-Trans Retinoic Acid Nanocrystals for Enhanced Anticancer Activity Against Neuroblastoma Cancer Stem Cells

Background/Objectives: Neuroblastoma is a high-risk pediatric malignancy in which cancer stem cells (CSCs) contribute to treatment resistance and disease recurrence. All-trans retinoic acid (ATRA) has established differentiation-inducing activity in neuroblastoma but is constrained by poor aqueous solubility and formulation challenges. Methods: ATRA nanocrystals (ATRA-NCs) stabilized with Poloxamer 407 were developed and characterized for their physicochemical properties. Their anticancer activity was evaluated in IMR-32 neuroblastoma cells and CD133+ CSCs, with unformulated ATRA used as the comparator, using multiple in vitro biological assays. Results: ATRA-NCs exhibited a mean hydrodynamic diameter of 194 ± 5.3 nm and a zeta potential of −60.1 mV. Compared with unformulated ATRA, ATRA-NCs produced greater concentration-dependent cytotoxicity and enhanced apoptotic responses in IMR-32 neuroblastoma cells. In CD133+ CSCs, ATRA-NCs reduced cell viability and suppressed the expression of stemness-associated genes, including SOX2, NANOG, LGR5, OCT4, BMI-1, and ALDH1A1. The formulation also reduced colony-forming capacity, impaired spheroid formation, and inhibited cellular migration. Conclusions: These findings demonstrate enhanced in vitro anticancer activity against neuroblastoma cancer cells and CD133+ CSCs following ATRA nanocrystallization and indicate modulation of multiple functional phenotypes associated with neuroblastoma CSCs. ATRA nanocrystallization may therefore provide a formulation approach for enhancing ATRA activity against neuroblastoma and warrants further preclinical evaluation..