Using a Microfluidics System to Reproducibly Synthesize Protein Nanoparticles: Factors Contributing to Size, Homogeneity, and Stability

The synthesis of Zein nanoparticles (NPs) using conventional methods, such as emulsion solvent diffusion and emulsion solvent evaporation, is often unreliable in replicating particle size and polydispersity between batch-to-batch syntheses. We have systematically examined the parameters for reproducibly synthesizing Zein NPs using a Y-junction microfluidics chip with staggered herringbone micromixers. Our results indicate that the total flow rate of the fluidics system, relative flow rate of the aqueous and organic phase, concentration of the base material and solvent, and properties of the solvent influence the polydispersity and size of the NPs. Trends such as increasing the total flow rate and relative flow rate lead to a decrease in Zein NP size, while increasing the ethanol and Zein concentration lead to an increase in Zein NP size. The solvent property that was found to impact the size of the Zein NPs formed the most was their hydropathy. Solvents that had a hydropathy index most similar to that of Zein formed the smallest Zein NPs. Synthesis consistency was confirmed within and between sample batches. Stabilizing agents, such as sodium caseinate, Tween 80, and Pluronic F-68, were incorporated using the microfluidics system, necessary for in vitro and in vivo use, into Zein-based NPs.