Dual centrifugation – a novel perspective on lipid nanoparticle formulation development

Abstract

The application of therapeutic RNAs, such as miRNAs, siRNAs, and mRNAs, has emerged as a promising therapeutic approach for treating diseases like cancer. Lipid nanoparticles (LNPs) are widely used for RNA delivery, typically produced using microfluidic mixing systems (MMS). However, MMS manufacturing is rather complex, time-consuming, and cost intensive. Dual centrifugation (DC), an in-vial homogenization technique, offers a straightforward, fast, and efficient alternative for preparing nanoscale lipid formulations, including LNPs. This study focuses on downscaling a DC-based LNP production method to enable 1 mg batch sizes, greatly reducing reagent consumption and allowing high-throughput research. Using a Design-of-Experiment (DoE) approach, key process parameters (lipid concentration, bead amount, and homogenization time) were optimized for small batches of LNPs formulated with cationic or ionizable lipids (DODMA, SM-102, ALC-0315). The optimized process was then applied to produce mRNA- and miRNA-loaded LNPs, which were evaluated for size, size distribution, encapsulation efficiency, and in vitro transfection performance. This work introduces DC as a novel and cost-efficient method for producing small-batch size LNPs, with satisfactory size characteristics and in vitro performance.