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. 2021 Jul 14;21(13):5671-5680.
doi: 10.1021/acs.nanolett.1c01353. Epub 2021 Jun 30.

Scalable mRNA and siRNA Lipid Nanoparticle Production Using a Parallelized Microfluidic Device

Sarah J Shepherd  1 , Claude C Warzecha  2 , Sagar Yadavali  1 , Rakan El-Mayta  1 , Mohamad-Gabriel Alameh  3 , Lili Wang  2 , Drew Weissman  3 , James M Wilson  2 , David Issadore  1   4   5 , Michael J Mitchell  1   6   7   8   9
Affiliations

Scalable mRNA and siRNA Lipid Nanoparticle Production Using a Parallelized Microfluidic Device

Sarah J Shepherd et al. Nano Lett. .

Abstract

A major challenge to advance lipid nanoparticles (LNPs) for RNA therapeutics is the development of formulations that can be produced reliably across the various scales of drug development. Microfluidics can generate LNPs with precisely defined properties, but have been limited by challenges in scaling throughput. To address this challenge, we present a scalable, parallelized microfluidic device (PMD) that incorporates an array of 128 mixing channels that operate simultaneously. The PMD achieves a >100× production rate compared to single microfluidic channels, without sacrificing desirable LNP physical properties and potency typical of microfluidic-generated LNPs. In mice, we show superior delivery of LNPs encapsulating either Factor VII siRNA or luciferase-encoding mRNA generated using a PMD compared to conventional mixing, with a 4-fold increase in hepatic gene silencing and 5-fold increase in luciferase expression, respectively. These results suggest that this PMD can generate scalable and reproducible LNP formulations needed for emerging clinical applications, including RNA therapeutics and vaccines.

Keywords: gene therapy; lipid nanoparticles; mRNA; siRNA.

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Conflict of interest statement

Complete contact information is available at: https://pubs.acs.org/10.1021/acs.nanolett.1c01353

The authors declare the following competing financial interest(s): J.M.W. is a paid advisor to and holds equity in Scout Bio and Passage Bio; he holds equity in Surmount Bio; he also has sponsored research agreements with Amicus Therapeutics, Biogen, Elaaj Bio, Janssen, Moderna, Passage Bio, Scout Bio, Surmount Bio, and Ultragenyx, which are licensees of Penn technology. J.M.W. and L.W. are inventors on patents that have been licensed to various biopharmaceutical companies and for which they may receive payments. S.J.S., S.Y., D.I., and M.J.M. are inventors on a patent related to this work filed by the Trustees of the University of Pennsylvania (63/131,008). D.W. is an inventor on several patents related to this work filed by the Trustees of the University of Pennsylvania (11/990,646; 13/585,517; 13/839,023; 13/839,155; 14/456,302; 15/339,363; and 16/299,202). The remaining authors declare that they have no conflicts of interest.

Figures

Figure 1.
Figure 1.
Fabrication of a scalable PDMS-based microfluidic platform for precise and large-scale RNA lipid nanoparticle (LNP) formulations. (A) Microfluidic formulation produces smaller and more homogeneous LNPs for potent RNA delivery, while larger and more heterogeneous LNPs produced by bulk methods are more variable in terms of RNA delivery. Microfluidic formulation can be scaled up using the same design (staggered herringbone micromixers) for rapid mixing to produce comparable LNPs for RNA delivery. (B) Photograph of the PDMS device, which incorporates 128 (4 × 32) mixing channels with only two inlets and one outlet. Scale bar: 5 mm. (C) Production rate comparison of different continuous LNP formulation methods (T-junction;,,– hydrodynamic focusing;, SHM;, NanoAssemblr Ignite, NxGen Blaze, GMP system), highlighting the total volumetric production rate and respective LNP size.
Figure 2.
Figure 2.
Incorporation of ladder design architecture, flow resistors, and herringbone micromixers out of the parallelized microfluidic device (PMD) enable large-scale LNP production. (A–C) Schematic diagram for the design of this device containing 4 rows of 32 mixing channels (A), highlighting the individual mixing unit design with a top view and a side view (B) and the individual mixing cycle design with a top, angled, and side view (C). Direction of flow is indicated by white arrows. Schematics are not to scale. (D) Circuit model of the delivery channels (Rdelivery), resistors (Rresistor), mixing channels (Rmixing), and individual mixing channel unit (Rdevice). (E) Resistance of an individual mixing channel unit versus length of the mixing channel, where the Rdevice is dominated by the resistance of the fluidic resistors, not the mixing channels. Rdevice is normalized to the resistance of an individual mixing channel unit with resistors and a mixing channel length of 20 cycles. (F) Device features. Scale bar: 200 μm. (G) Cross section of the PMD. Scale bar: 200 μm.
Figure 3.
Figure 3.
Validation of uniform mixing across all scales of microfluidic devices. Mixing characterization for microfluidic devices: single channel (A, B), single row PMD (C–F), and PMD (G–J). (A) Schematic for the mixing experiment, where FITC and rhodamine were flowed through a single channel device to quantify mixing at various channel lengths, showing the red and green plot profiles versus channel distance at the outlet (inset). Scale bar: 200 μm. (B) Quantification of mixing versus channel length for five different flow rates, where the channel length for 90% mixing (±standard error mean) is directly correlated with the natural log of the Peclet number (inset). (C) Schematic of the mixing experiment with a single row PMD consisting of 10 parallel channels. (D–E) Fluorescent images of mixing in a channel, showing the red and green plot profiles versus channel distance at the outlet (inset). Scale bars: 200 μm. (F) Comparison of the channel length required for 90% mixing (±standard error mean) for all 10 channels and the single channel device. Samples were compared by one-way ANOVA. ns: p > 0.05. (G) Schematic for the mixing experiment with the PMD. (H, I) Fluorescent images of mixing in a channel, showing the red and green plot profiles versus channel distance at the outlet (inset). Scale bars: 200 μm. (J) Comparison of the channel length required for 90% mixing (±standard error mean) for three channels across the device and the single channel device. Samples were compared by one-way ANOVA. ns: p > 0.05.
Figure 4.
Figure 4.
Validation of mixing, uniform LNP physical properties, and potent RNA delivery in vitro for the single row PMD. (A) Ten mixing channels are organized in a single row, with the two inlets connected by a ladder geometry to each channel, showing the beginning (left), center (middle), and end (right) of each channel. Scale bars: 200 μm. (B) Quantification of the ratio of red to green inlets (±standard deviation) for each channel. (C) DLS curves for firefly luciferase siRNA LNPs produced from each channel. (D) Luciferase expression (±standard deviation) in HeLa cells after treatment with 5 nM of luciferase siRNA LNPs produced from each channel. Data is normalized to cells without treatment with background subtracted. n = 10. (E) Photograph of the single row PMD with two inputs (blue, orange dye) and 10 outputs, one for each mixing channel, used to collect LNPs from each channel. Scale bar: 5 mm. (F) Calculated IC50 (±standard deviation) indicates the luciferase siRNA dose needed to silence 50% of the firefly luciferase gene for each formulation method. Samples were compared by one-way ANOVA. ns: p > 0.05.
Figure 5.
Figure 5.
Scalable PMD formulates LNPs with uniform physical properties for potent in vivo therapeutic RNA delivery compared to bulk mixing methods. (A) Schematic for comparison between microfluidic single channel device, single row PMD, and bulk mixing. (B) Microfluidic-formulated siRNA LNPs targeting Factor VII were delivered to C57BL/6 mice to determine the optimal dose and collection time point. LNPs were formulated with Factor VII siRNA by a microfluidic single channel device, and Factor VII activity is reported as a percentage of activity 1 day prior to LNP administration. n = 3. (C) Mice were dosed with 0.2 mg/kg of Factor VII siRNA LNPs, and Factor VII activity was quantified 48 h later. Factor VII activity is reported as a percentage of activity 1 day prior to LNP administration. *p < 0.05 (p = 0.0116); ****p < 0.0001 in unpaired t test to siControl LNP. n = 4. (D) LNPs were formulated with mRNA encoding firefly luciferase and administered to mice via tail vein injection at a dose of 0.2 mg/kg. Luminescent flux of a region of interest was quantified 4 h after LNP administration. ***p < 0.001 in unpaired t test to bulk mixed LNPs. n = 3–4.
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