Optimization of Zein-Casein-Hyaluronic Acid NanoparticlesObtained by Nanoprecipitation Using Design of Experiments (DoE)

Zein-based nanoparticles offer significant potential as carriers for drug delivery due to their biocompatibility. However, optimizing their formulation is essential to achieving efficient encapsulation and stability. This study aimed to optimize the formulation of zein-casein-hyaluronic acid-based nanoparticles for the encapsulation of a hydrophilic drug, focusing on achieving favorable physicochemical properties for oral drug delivery applications. A factorial experimental design was employed to evaluate the influence of key formulation parameters, including zein concentration, hyaluronic acid concentration, sodium caseinate concentration, and the organic-to-aqueous phase (O/W) ratio. Particle size (PS), polydispersity index (PDI), zeta potential, and encapsulation efficiency (EE) were analyzed as response variables. Multivariate analyses, such as hierarchical cluster analysis and principal component analysis, were performed to explore the relationships between formulation parameters and nanoparticle properties. Model validity was confirmed by using ANOVA and residual analysis. Optimized nanoparticles exhibited a PS of 217 ± 5 nm, PDI of 0.077 ± 0.022, zeta potential of −24.7 ± 1.9 mV, and EE of 31% ± 4. The nanoparticles displayed a monomodal size distribution and a spherical morphology. Multivariate analyses revealed that the O/W ratio and zein concentration were the most influential factors, while sodium caseinate played a crucial stabilizing role. The desirability function yielded a high score (<i>D</i> = 0.9338), confirming the robustness of the optimization process. Stability studies demonstrated that refrigeration at 8 °C preserved the nanoparticles’ physicochemical properties over 180 days. This study underscores the power of experimental design as a tool to refine nanoparticle formulations, paving the way for more efficient drug delivery systems and unlocking new possibilities for the oral administration of hydrophilic compounds.