Editing Cleancap mRNA Invitro Transcription (section)

== <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">LNP</span> ==

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">LNP were prepared by mixing ethanol and aqueous phase at a 1:3 volumetric ratio in a microfluidic device, using syringe pumps as previously described (Chen, Love et al., 2012). The ethanol phase was prepared by solubilizing a mixture of ionizable lipidoid cKK-E12, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE, Avanti), cholesterol (Avanti), and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy-(polyethyleneglycol)-2000] (ammonium salt) (C14-PEG 2000, Avanti) at a molar ratio of 35:16:46.5:2.5 The aqueous phase was prepared in 10 mM citrate buffer (pH 3) with either trastuzumab m 164 RNA or luciferase mRNA (Firefly luciferase mRNA, TranslateBio). LNPs were dialyzed against PBS in a Slide-A-Lyzer™ G2 Dialysis Cassettes, 20,000 MWCO (Thermo Fisher) for 2h at RT. The concentration of mRNA encapsulated into LNPs nanoparticles was analyzed using Quant-iT RiboGreen assay (Thermo Fisher), according to manufacturer’s protocol. The efficacy of mRNA encapsulation into LNPs was calculated by comparing measurements in the absence and presence of 1% (v/v) Triton X- 100. Nanoparticle size, polydispersity (PDI), and ζ-potential were analyzed by dynamic light scattering (DLS) using Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK). LNP hydrodynamic diameters are reported in the volume weighting mode and are an average of three independent measurements (Supplemental Table 5).</span>