Microfluidic Production and Application of Lipid Nanoparticles for Nucleic Acid Transfection

Abstract

Lipid nanoparticles (LNPs) are established in the biopharmaceutical industry for efficient encapsulation and cytosolic delivery of nucleic acids for potential therapeutics, with several formulations in clinical trials. The advantages of LNPs can also be applied in basic research and discovery with a microfluidic method of preparation now commercially available that allows preparations to be scaled down to quantities appropriate for cell culture. These preparations conserve expensive nucleic acids while maintaining the particle characteristics that have made LNPs successful in later stages of genetic medicine development. Additionally, this method and the resulting LNPs are seamlessly scalable to quantities appropriate for in vivo models and development of nucleic acid therapeutics.The present work describes the methodology for preparing LNPs loaded with siRNA, mRNA or plasmids using a commercially available microfluidic instrument and an accompanying transfection kit. Guidelines for application to cultured cells in a well-plate format are also provided.

Development of siRNA-Loaded Lipid Nanoparticles Targeting Long Non-Coding RNA LINC01257 as a Novel and Safe Therapeutic Approach for t(8;21) Pediatric Acute Myeloid Leukemia

“…In this study, we made use of NxGen microfluidic technology to precisely generate LNPs with high batch-to-batch reproducibility and ensure the correct manufacturing of larger volumes needed to progress into in vivo assays [18]. This technology also has the potential for use in the delivery of other nucleic acids such as plasmid DNA and antisense oligonucleotides (ASOs) [44], thereby broadening the applications of LNP-based therapies in AML. Finally, the accumulation of LNP-siRNA particles in the liver following a systemic delivery presents a major challenge with siRNA-based therapies.…”

Advances in Lipid Nanoparticles for mRNA-Based Cancer Immunotherapy

“…However, nanoparticles generated with early fluidic devices based on macroscopic mixing techniques have often shown high polydispersity and poor reproducibility. For this reason, microfluidic chip devices have been recently developed for the synthesis of mRNA lipid-based nanoformulations (Cullis and Hope, 2017;Thomas et al, 2018). The use of microfluidic mixing devices can ensure a rapid mixing of the aqueous and organic phases, with a consequent fast increase of the polarity of the solution.…”

Current Status and Future Perspectives on MRNA Drug Manufacturing

“…For nearly a decade, microfluidic mixing methods have been reported in the literature to formulate RNA–LNPs for cancer therapeutics, ,, protein replacement therapies, − gene editing, − and RNA vaccines. − Microfluidics enables nonturbulent mixing of the aqueous and organic phases to control self-assembly. The technology has been demonstrated to be scalable from microliters, enabling bench-scale formulation development, to liters where integration with in-line analytical methods enables an important step toward an integrated manufacturing suite . Historically, the staggered herringbone mixer (SHM) has been widely used, utilizing typical flow rates in tens of mL/min, which is an order of magnitude faster than typical hydrodynamic flow focusing devices .…”

“…Historically, the staggered herringbone mixer (SHM) has been widely used, utilizing typical flow rates in tens of mL/min, which is an order of magnitude faster than typical hydrodynamic flow focusing devices . However, the structure of the staggered herringbone chevrons adds multidimensional dependencies and practical limitations making it hard to achieve throughput speeds required to GMP specifications, which results in multiple SHM mixers needing to be arrayed in parallel to achieve higher throughput while retaining the same critical quality attributes such as size, PDI, and encapsulation efficiency. ,, Innovations in microfluidic mixers have introduced next-generation toroidal mixers (TrM) that retain nonturbulent advective mixing of SHM but enable single-mixer flow rates an order of magnitude greater (200 mL/min vs 12 mL/min) by increasing mixer dimensions while maintaining critical quality attributes of RNA–LNP. , This retains compatibility with discovery and preclinical-scale production and eases process scale-up. ,,, The details of how mRNA-1273 and BNT162b2 were produced for preclinical and clinical development have not been clarified; however; Moderna has published numerous studies utilizing microfluidic SHM technology ,, and ethanol-drop nanoprecipitation, , and publications describing the nonhuman primate studies of BNT162b2 indicate LNPs were formed by transfer of an ethanolic solution of lipids into an aqueous buffer by diafiltration. , …”