Lab-scale siRNA and mRNA LNP manufacturing by various microfluidic mixing techniques – an evaluation of particle properties and efficiency
David C. Jürgens,
Leonie Deßloch,
Diana Porras-Gonzalez,
Joshua Winkeljann,
Sebastian Zielinski,
Matthias Munschauer,
Andreas L. Hörner,
Gerald Burgstaller,
Benjamin Winkeljann,
Olivia M. Merkel
Affiliations
David C. Jürgens
Department of Pharmacy, Ludwig-Maximilians-University Munich, Butenandtstrasse 5-13, Haus B, Munich 81377, Germany
Leonie Deßloch
Department of Pharmacy, Ludwig-Maximilians-University Munich, Butenandtstrasse 5-13, Haus B, Munich 81377, Germany
Diana Porras-Gonzalez
Member of the German Center for Lung Research (DZL), Comprehensive Pneumology Center (CPC) with the CPC-M bioArchive/Institute of Lung Health and Immunity (LHI), Helmholtz Zentrum München, Munich, Germany
Joshua Winkeljann
Department of experimental Physics, University of Augsburg, Universitätsstraße 1, Augsburg 86159, Germany
Sebastian Zielinski
Helmholtz Institute for RNA-based Infection Research, Helmholtz Center for Infection Research, Würzburg, Germany; Josef-Schneider-Straße 2/D15, Würzburg D-97080, Germany; University of Würzburg, Medical Faculty, Josef-Schneider-Straße 2/D15, D-97080, Würzburg, Germany
Matthias Munschauer
Helmholtz Institute for RNA-based Infection Research, Helmholtz Center for Infection Research, Würzburg, Germany; Josef-Schneider-Straße 2/D15, Würzburg D-97080, Germany; University of Würzburg, Medical Faculty, Josef-Schneider-Straße 2/D15, D-97080, Würzburg, Germany
Andreas L. Hörner
Department of experimental Physics, University of Augsburg, Universitätsstraße 1, Augsburg 86159, Germany
Gerald Burgstaller
Member of the German Center for Lung Research (DZL), Comprehensive Pneumology Center (CPC) with the CPC-M bioArchive/Institute of Lung Health and Immunity (LHI), Helmholtz Zentrum München, Munich, Germany
Benjamin Winkeljann
Department of Pharmacy, Ludwig-Maximilians-University Munich, Butenandtstrasse 5-13, Haus B, Munich 81377, Germany; Center for NanoScience (CeNS), Ludwig-Maximilians-University Munich, Munich 80799, Germany
Olivia M. Merkel
Department of Pharmacy, Ludwig-Maximilians-University Munich, Butenandtstrasse 5-13, Haus B, Munich 81377, Germany; Center for NanoScience (CeNS), Ludwig-Maximilians-University Munich, Munich 80799, Germany; Member of the German Center for Lung Research (DZL), Comprehensive Pneumology Center (CPC) with the CPC-M bioArchive/Institute of Lung Health and Immunity (LHI), Helmholtz Zentrum München, Munich, Germany; Corresponding author at: Department of Pharmacy, Ludwig-Maximilians-University Munich, Butenandtstrasse 5-13, Haus B, Munich 81377, Germany.
Lipid Nanoparticles (LNPs) are promising drug delivery systems for various RNAs such as small interfering (siRNA) and messenger RNA (mRNA). Microfluidic mixing is a common technique to encapsulate RNA in LNPs. However, high flow rates and lipid concentrations are used for LNP formation to control LNP size as well as RNA encapsulation efficiency. We investigated the feasibility of downscaling siRNA and mRNA LNP manufacturing to save materials and enable a broader access to this technology. To optimize such a down-scaled procedure, we evaluated physicochemical nanoparticle characteristics including hydrodynamic diameter, zeta potential, particle concentration, encapsulation efficiency, and recovery for LNPs produced with three different microfluidic methods. We observed differences in nanoparticle characteristics and in vitro performance regarding cellular uptake, gene silencing, and mRNA expression. We determined the gene knockdown ability of the best siRNA LNPs formulation ex vivo using precision-cut lung slices to highlight the translational character of LNPs for inhalation and observed comparable efficacy as with a commercially available transfection reagent.
