In vitro anticancer activity and biodistribution analysis of optimized hybrid nanoparticulate system co-loaded with apigenin and oleanolic acid

This study addresses the limitations of traditional cancer therapies-such as high toxicity and non-selectivity-by developing a novel nanoparticulate system co-loaded with plant-derived compounds, apigenin (APG) and oleanolic acid (OA). These naturally occurring phytochemicals are known for their anticancer properties but face clinical challenges due to poor solubility and limited efficacy. The Apigenin and Oleanolic Acid Nanoparticulate System (AONS) was refined and optimized using the Box-Behnken statistical design approach, resulting in nanoparticles with a mean diameter of 169 nm and size homogeneity. A validated HPLC technique was employed to simultaneously quantify both apigenin and oleanolic acid in the developed formulation as well as in various biological matrices. In vitro drug release was enhanced at acidic pH (5.5), mimicking the tumor microenvironment. AONS showed strong physicochemical stability under refrigerated storage. Cytotoxicity tests on HepG2, MDA-MB-231, and U87 glioma cells demonstrated significantly improved anticancer activity compared to control, free drugs, or individual nanoformulations. Cellular uptake studies confirmed efficient internalization in U87 cells, and ELISA results indicated apoptosis via activation of the p53 pathway. Biodistribution analysis revealed prolonged systemic drug retention with AONS (up to 72 hours), surpassing the short circulation time of free drugs. Notably, LC-MS data confirmed that the nanoparticle system could cross the blood-brain barrier, highlighting its therapeutic potential for treating brain cancers.