Poly(beta-amino ester) (PBAE)-based nanoparticles have emerged as promising carriers for RNA delivery, yet clear design rules linking formulation parameters to performance are still lacking. In this study, a Quality by Design (QbD)-guided and Design of Experiments (DoE)-driven approach was combined with high-throughput microfluidics to rapidly identify formulations with favorable physicochemical properties and consistent critical quality attributes (CQAs). Response Surface Modeling revealed that high total flow rates (TFR >/= 10), nitrogen to phosphorus (N/P) ratios >/=10, and a Flow Rate Ratio (FRR) of 1 : 3 (buffer : ethanol) led to the formation of smaller, more stable particles. Among the polymers tested, a polymer candidate with a balanced composition of hydrophobic and hydrophilic side chains demonstrated optimal intraparticle stability and gene silencing performance. Notably, transfection efficiency depended strongly on formulation parameters beyond polymer type and N/P ratio, with flow rate ratio emerging as a key driver of gene knockdown kinetics. The lead formulation achieved approximately 95% gene knockdown even after two weeks of storage at 4 degrees C. Scale-up production of the lead candidate confirmed the transferability of optimized Critical Process Parameters (CPPs) and preserved CQA profiles, validating the robustness of the design space. This study establishes a robust and scalable QbD-guided workflow for the development of microfluidically manufactured siRNA nanoparticles, enabling rapid optimization, reliable scale-up, and clinically relevant performance.
