Rigorous control of the properties of lipid nanoparticles (LNPs) such as the particle size, polydispersity index (PdI), ζ-potential, and encapsulation efficiency of small interfering RNA (siRNA) with good reproducibility is important for LNP-based siRNA delivery. In this study, polyethylene glycol (PEG)-free LNPs containing a dioleoylglycerophosphate-diethylenediamine conjugate (DOP-DEDA), which is a pH-responsive lipid derivative, were prepared by using a microfluidic technology to construct a predictive model for particle size control using the design of experiment (DoE). PEG-free DOP-DEDA-based LNPs (DEDA LNPs) encapsulating siRNA, which were prepared by using a microfluidic technology, formed uniform particles and triggered effective gene silencing. The effects of the preparation conditions such as the total flow rate, lipid concentration, and lipid solution ratio on the LNP properties were investigated, and results indicated that the lipid solution ratio is the most important parameter to control the particle size and PdI of DEDA LNPs. Conversely, the ζ-potential and siRNA encapsulation efficiency of DEDA LNPs were not affected by the preparation conditions. The predictive model constructed using the DoE data showed that the particle size of the prepared DEDA LNPs was consistent with that predicted. Therefore, our approach using the DoE will contribute to the rigorous prediction and control of the particle size of PEG-free LNPs for nucleic acid delivery.
