Single pot organic solvent-free thermocycling technology for siRNA-ionizable LNPs: a proof-of-concept approach for alternative to microfluidics.

Ionizable LNPs are the latest trend in nucleic acid delivery. Microfluidics technology has recently gained interest owing to its rapid mixing, production of nucleic acid-ionizable LNPs, and stability of nucleic acid inside the body. Industrial scale-up, nucleic acid-lipid long-term storage instability, and high production costs prompted scientists to seek alternate solutions to replace microfluidic technology. We proposed a single-pot, organic solvent-free thermocycling technology to efficiently and economically overcome most of the limitations of microfluidic technology. New thermocycling technology needs optimization of process parameters such as sonication duration, cooling-heating cycle, number of thermal cycles, and lipid:aqueous phase ratio to formulate precisely sized particles, effective nucleic acid encapsulation, and better shelf-life stability. Our research led to the formulation of siRNA-ionizable LNPs with particle sizes of 104.2 ± 34.7 nm and PDI 0.111 ± 0.109, with 83.3 ± 4.1% siRNA encapsulation. Thermocycling siRNA-ionizable LNPs had comparable morphological structures with commercialized microfluidics ionizable LNPs imaged by TEM and cryo-TEM. When compared to microfluidics ionizable LNPs, thermocycling siRNA-ionizable LNPs had a longer shelf life at 4°C. Our thermocycling technology showed an effective alternative to microfluidics technology in the production of nucleic acid-ionizable LNPs to meet global demand.

Thermocycling technology is a low-energy, low-temperature, self-assembling cooling­heating process in which lipid droplets spontaneously break apart into much smaller droplets to form siRNA-ionizable LNPs.The new technology is an alternative to multistep, costly, and complex microfluidics technology for the formulation and bulk up of siRNA-ionizable LNPs economically.Thermocycling siRNA-ionizable LNPs formulation focused on optimizing process parameters such as thermal cycle rate, number of thermal cycles, and lipid:aqueous phase ratio.The thermocycling technology is able to overcome the limitations of the storage stability limitations of commercialized ionizable LNPs.