Abstract
Background/Objectives: Microfluidic mixing has become the gold standard procedure for manufacturing nucleic acid lipid-based delivery systems, offering precise control over critical process parameters. The choice and design of microfluidic mixers are often seen as a key driving force affecting the critical quality attributes of the resulting lipid nanoparticles (LNPs). Methods: This study aimed to evaluate LNPs manufactured using two low-cost microfluidic mixers alongside manual mixing (pipette mixing (PM)), followed by characterization studies using orthogonal analytics as well as expression studies to establish whether low-cost microfluidic manufacturing methods are suitable for bench-scale and high-throughput research. Results: The results show that all manufacturing methods can produce LNPs with sizes ranging between 95 and 215 nm with high encapsulation (70-100%), and enhanced analytics showed variations between the LNPs produced using the different mixers. Despite these differences, pipette mixing production of LNPs demonstrated its application as a high-throughput screening tool for LNPs, effectively distinguishing between different formulations and predicting consistent expression patterns both in vitro and in vivo. Conclusions: Overall, these results validate the use of low-cost microfluidic mixers without compromising the efficiency and integrity of the resulting LNPs. This study supports the increased accessibility of small-scale LNP manufacturing and high-throughput screening.