Characterization and preliminary cytotoxic effects of pomegranate peel extract-loaded nanoparticles on HepG2 cells

Abstract Punica granatum (pomegranate) peel is recognized for its rich phytochemical profile and traditional use in medicinal applications, making it a promising source of anticancer agents. In this study, an aqueous pomegranate peel extract (PPE) was prepared and encapsulated into chitosan nanoparticles (CSPPE) using the ionic gelation method. The resulting CSPPE nanoparticles were characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM), revealing nanoscale size distribution (Z-average 408.8 ± 98.8 nm by DLS, TEM mean 51.5 ± 12.6 nm) and good colloidal stability, with positive zeta potential (+ 15–35 mV) supporting electrostatic stabilization. Fourier-transform infrared spectroscopy (FTIR) indicated successful physical entrapment of PPE within the chitosan matrix, with characteristic functional groups preserved. The cytotoxic activity of both PPE and CSPPE was assessed against human hepatocellular carcinoma (HepG2) cells using the MTT assay. PPE showed moderate cytotoxicity only at high concentrations (IC₅₀ = 921.8 ± 18.6 µg/mL), while CSPPE induced markedly stronger, concentration-dependent cytotoxic effects across all tested doses (IC₅₀ = 12.38 ± 0.28 µg/mL PPE-equivalent), representing approximately a 75-fold increase in potency. Blank chitosan nanoparticles without PPE exhibited minimal cytotoxicity (> 90% cell viability), confirming that the observed enhancement is attributable to PPE encapsulation. Microscopic observations corroborated the dose-dependent cytotoxic effects, showing reduced cell density, loss of adhesion, and morphological changes consistent with cell death. These findings demonstrate that nanoencapsulation significantly enhances the bioactivity of pomegranate peel extract against hepatocellular carcinoma cells. While this study provides strong preliminary evidence, further in vitro mechanistic assays and in vivo investigations are warranted to elucidate the mode of cell death, assess selectivity towards cancer versus normal hepatocytes, and confirm therapeutic potential and safety.