Microfluidic hydrodynamic focusing (HF) has emerged as a powerful platform for the controlled synthesis of lipid nanoparticles (LNPs) and liposomes, offering superior precision, reproducibility, and scalability compared to traditional batch methods. However, the impact of HF inlet configuration and channel geometry on nanoparticle formation remains poorly understood. In this study, we present a comprehensive experimental and computational analysis comparing 2-inlet (2-way) and 4-inlet (4-way) HF designs across various sheath inlet angles (45°, 90°, 135°) and cross-sectional geometries (square vs. circular), assessing their influence on particle size, polydispersity index (PDI), and percentage encapsulation efficiency (%EE) of siRNA and FITC-Dextran. Using 3D-printed microfluidic chips, empty and loaded liposomes and LNPs were synthesized across a range of lipid concentrations (1-8 mg/mL) and total flow rates (0.12-16 mL/min). Computational fluid dynamics (CFD) simulations revealed significant differences in mixing profiles and ethanol diffusion across configurations, correlating with observed nanoparticle properties. Interestingly, 2-way focusing outperformed 4-way designs at low flow rates due to broader diffusive interfaces, while 4-way 45° configurations provided superior control over nanoparticle formation at high flow rates. Circular channels produced smaller, more uniform nanoparticles than square channels, likely due to symmetric flow patterns and reduced stagnation zones. Higher lipid concentrations decreased PDI and improved encapsulation, particularly for siRNA-loaded LNPs. Encapsulation efficiencies were similar across most configurations; however, a statistically significant increase was observed in the 4-way 135° design at 4 mL/min. This likely reflects size-related effects rather than a specific advantage of the configuration. Furthermore, LNPs produced at higher flow rates exhibited enhanced cellular uptake, attributed to their smaller particle size. Overall, our results demonstrate that optimal nanoparticle synthesis via HF is governed by an interplay of flow rate, inlet geometry, and formulation parameters (nanoparticle type and lipid concentration) rather than the number of inlets alone. This study provides a design framework for selecting HF configurations tailored to specific throughput and encapsulation requirements in therapeutic nanocarrier production.
