Apoptosis induction and enhanced CASP9 expression in liver cancer cells by crocin-conjugated magnetite iron oxide nanoparticles

Hepatocellular carcinoma (HCC) is among the top reasons for tumor-associated fatalities globally, with an estimated one million new diagnoses expected annually by 2025. In this study, we examined the therapeutic potential of iron oxide nanoparticles (Fe 3 O 4 NPs) conjugated with the anti-cancer compound crocin as a novel therapeutic approach for HCC. Fe 3 O 4 @Glu-crocin NPs were synthesized and extensively characterized using a range of spectroscopic and microscopic methods, including FTIR, XRD, DLS, EDS, zeta potential analysis, SEM, and TEM. The cytotoxicity of the nanoparticles was evaluated using MTT and flow cytometry assays on treated and untreated (control) HepG2 liver cancer cells. Spherical nanoparticles exhibited a dry-state size of 10–30 nm (SEM/TEM) and a hydrodynamic diameter of 171 nm (DLS). This size variation—arising from solvation effects—optimizes cellular uptake (dry-state) while maintaining colloidal stability for targeted delivery (solution-state). The synthesized nanoparticles exhibited potent anti-cancer activity, with an IC 50 value of 129 μg/mL. Further analysis through flow cytometry revealed that the nanoparticles induced early apoptosis in 35.01 % of the cancer cells and late apoptosis in 6.10 %, significantly higher than in untreated controls. Subsequent real-time PCR analysis demonstrated a 1.6-fold increase in the expression of the apoptosis-related CASP9 gene in the treated cells, confirming the activation of apoptotic pathways. These findings highlight that the specific size and morphology of the Fe 3 O 4 @Glu-crocin NPs contribute to their enhanced cellular penetration, reduced IC 50 value, and robust induction of apoptosis, underscoring their potential as a therapeutic agent for HCC. This study underscores the potential of nanotechnology-based drug delivery systems for treating liver cancer, based on the in-depth characterization and evaluation of the nanoparticles.