Factorial Design-Driven Optimization of Zein-Chitosan Nanoparticles for Oral Delivery of Silibinin

Silibinin (SLB) is a poorly water-soluble flavonolignan with relevant therapeutic potential but limited oral bioavailability. In this study, SLB-loaded zein-chitosan nanoparticles (SLB-ZNP) were developed by nanoprecipitation and optimized using a full 2⁴ factorial design to investigate the effects of zein concentration, chitosan concentration, incubation time, and organic-to-water ratio on particle size, polydispersity index (PDI), zeta potential, and encapsulation efficiency. The factorial approach enabled systematic evaluation of the most influential variables and their interactions, with zein and chitosan concentrations exerting major effects on particle size and surface charge, while the organic-to-water ratio significantly affected particle size distribution. The optimized formulation produced nanoparticles with a mean diameter of approximately 145 nm, low PDI (∼0.19), high positive zeta potential (∼+40 mV), and high encapsulation efficiency (∼90%). Transmission electron microscopy revealed spherical and homogeneous nanoparticles, with enhanced structural organization upon SLB incorporation. In vitro cytotoxicity assays in HeLa and SiHa cervical cancer cell lines showed that nanoencapsulation modulates carrier-associated cytotoxicity in a cell line- and concentration-dependent manner. Overall, this study demonstrates the utility of factorial design as a formulation-centered strategy for engineering zein-chitosan nanoparticles with well-defined physicochemical and in vitro properties.