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3 protocols using genvoy ilm

1

mRNA Lipid Nanoparticle Formulation and Characterization

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The synthesized RBD-mRNA was encapsulated with lipid nanoparticles (LNPs) as described below (Tai et al., 2020 (link); Wang et al., 2022 (link)). Specifically, PNI Formulation Buffer with or without the synthesized mRNA was combined with GenVoy-ILM (lipid mixture) (Precision Nanosystems) at 3:1 ratio using NanoAssemblr Benchtop Instrument (Precision Nanosystems). The LNP-formulated mRNA was concentrated using 10 kDa Amicon Ultra-15 Centrifugal Filters (EMD Millipore) and stored at 4 °C until use. The endotoxin level (<1 EU/ml) of each LNP-encapsulated mRNA was measured using Chromogenic LAL Endotoxin Assay Kit (GenScript), and related particle size was measured using DynaPro NanoStar II Light Scattering Detector (WYATT Technology).
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2

Lipid Nanoparticle Encapsulation of mRNA

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The synthesized mRNAs were encapsulated with LNPs (mRNA-LNPs) as described below.14 (link) Specifically, each mRNA diluted in PNI Formulation Buffer (Precision Nanosystems) was encapsulated with lipid mixture (GenVoy-ILM) (Precision Nanosystems) (3:1 ratio) using NanoAssemblr Ignite Instrument according to the manufacturer’s instructions (Precision Nanosystems). This was followed by filtration and concentration of mRNA-LNPs using Amicon Ultra-15 Centrifugal Filters (10 kDa). The endotoxin level of mRNA-LNPs was detected by LAL Endotoxin Assay Kit (GenScript) (<1 EU/mL), with the particle size around 80–110 nm.
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3

Formulation and Characterization of mRNA-LNP

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The mRNA contained 5′ and 3′ UTR and a poly-A tail was produced from a linearized DNA template with a T7 in vitro transcription kit (Cellscript Madison, WI, USA), and pseudouridine was used in place of uridine. Then, the mRNA was enzymatically capped. According to the protocol, the mRNA was dissolved in an aqueous buffer and combined with GenVoy ILM (Precision Nanosystems, Vancouver, BC, Canada) at a flow ratio of 3:1 through a microfluidic mixer (Precision Nanosystems, Vancouver, BC, Canada). The solvent was removed by centrifugation at 2000× g using a 100 kDa ultrafiltration tube (Milipore, Billerica, MA, USA) and the size and PDI of mRNA-LNP was measured by dynamic light scattering (DLS) on a Malvern Zetasizer Nano-ZS (Malvern, Westborough, MA, UK). The concentration of mRNA-LNPs was measured by an Invitrogen’s Quant-iT Ribogreen RNA assay kit (Invitrogen, Eugene, OR, USA).
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