Biocompatible dual drug formulation of resveratrol and hydroxychloroquine for targeted liver cancer: in vitro HepG2 model anti-hepatocellular carcinoma and in vivo safety assessments
Abstract
Hepatocellular carcinoma (HCC) is one of the most aggressive cancers with limited therapeutic efficacy and high drug resistance. This study developed a biocompatible dual-drug delivery system based on layer-by-layer liquid crystalline nanoparticles (LbL-LCNPs) co-loaded with resveratrol (RES) and hydroxychloroquine (HCQ) to enhance their anticancer activity against HepG2 liver cancer cells. The nanoparticles were coated with chitosan and hyaluronic acid to improve stability, sustained release, and targeted cellular uptake. Physicochemical characterization using Ultraviolet-Visible spectroscopy (UV-Vis), fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD),field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM),, particle size, and zeta potential analyses confirmed successful nanoparticle formation and drug encapsulation. The formulation exhibited pH-responsive drug release and enhanced cytotoxicity toward HepG2 cells compared with free drugs, i.e., RES and HCQ, while showing lower toxicity toward normal HdFn cells. RES/HCQ-LbL-LCNPs significantly inhibited colony formation, increased cellular uptake, and induced DNA damage. Flow cytometry demonstrated significant activation of caspase-3 and modulation of LC3B expression, indicating the involvement of apoptosis and autophagy-related pathways. Annexin V/PI analysis further confirmed dose-dependent apoptotic induction. In vivo toxicity studies in mice demonstrated good biocompatibility with no mortality or severe hepatotoxicity following oral administration for 30 days. Histopathological and liver enzyme analyses revealed only mild hepatic alterations compared with free drug treatments. The present formulation represents a promising and safe nanotherapeutic platform for hepatocellular carcinoma treatment through enhanced anticancer efficacy and targeted drug delivery, providing a foundation for future efficacy studies.