Process Optimization of Charge-Reversible Lipid Nanoparticles for Cytosolic Protein Delivery Using the Design-of-Experiment Approach.

This study aimed to elucidate the manufacturing process parameters with optimal quality characteristics of protein-encapsulated dioleoylglycerophosphate-diethylenediamine (DOP-DEDA)-based lipid nanoparticles (LNPs) for intracellular protein drug delivery. DOP-DEDA is a pH-responsive and charge-reversible lipid for intracellular cargo delivery. In this study, bovine serum albumin (BSA) was used as a weakly acidic protein model, and LNPs were prepared using microfluidic technology, which has many advantages for practical applications. BSA-encapsulated DOP-DEDA-based LNPs showed pH-responsive charge reversibility and excellent quality characteristics for the intracellular delivery of proteins. A process optimization study was conducted by applying the Box-Behnken design in a design-of-experiment approach. The particle size, ζ-potential, and encapsulation efficiency were evaluated in response to the total flow rate, lipid concentration, and lipid solution ratio. The lipid solution ratio and total flow rate significantly affected the particle size and encapsulation efficiency, respectively. On the contrary, none of the process parameters affected the ζ-potential. Moreover, a map of the predicted values was constructed for the particle size and encapsulation efficiency using a multiple regression equation. In the predicted particle size range of 77-215 nm and encapsulation efficiency of 14-35%, the observed values were close to the predicted values, and 100-nm LNPs were reproduced with an encapsulation efficiency of 27%. Therefore, manufacturing process parameters were established to obtain protein-encapsulated DOP-DEDA-based LNPs with optimal quality characteristics.