Systematic evaluation and statistical modeling of dual centrifugation and high-pressure homogenization as promising methods for scalable preparation of polymeric nanoparticles.

Nanobiomaterials have emerged as critical platforms for modern therapeutics, offering targeted delivery, controlled release, and improved performance. Among these, polymeric PLGA nanoparticles (PLGA-NPs) are widely studied due to their tailorability, biocompatibility, biodegradability, and regulatory acceptance. However, their clinical translation depends on robust manufacturing approaches that support scalability, reproducibility, and continuous manufacturing. Conventional methods, such as high-shear homogenization, suffer from limitations, including high energy input, process sensitivity, and contamination risk. This study presents a comprehensive experimental and statistical approach to systematically compare three PLGA-NPs preparation methods: high-shear homogenization (HSH), dual centrifugation (DC), and high-pressure homogenization (HPH). A full factorial Design of Experiments (DoE) combined with Response Surface Methodology (RSM) evaluated the effects of formulation and process parameters, including PVA and PLGA concentrations, homogenization speed, bead diameter, and pressure on Critical Quality Attributes (CQAs; particle size, polydispersity index, and zeta potential). HSH produced uniform nanoparticles with narrow size distribution but was highly process-sensitive. DC showed more variability in results, while HPH achieved consistent results. PVA concentration was the dominant factor across all methods. In conclusion, DC and HPH emerged as viable candidates for scalable nanoparticles manufacturing.