The ionizable polyamine-lipid cores, prepared as described above, or DLin-MC3-DMA (MedChem Express, Monmouth Junction, NJ) were combined into an ethanol phase with cholesterol (Sigma-Aldrich), DOPE (Avanti, Alabaster, AL), and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (C14-PEG2000, Avanti) at molar ratios of 35:46.5:16:2.5, respectively, in a total volume of 112.5 μl. A separate aqueous phase was prepared consisting of 25 μg of luciferase (TriLink BioTechnologies, San Diego, CA) or EPO (TriLink BioTechnologies) mRNA and 10 mM citrate buffer (pH 3) in a total volume of 337.5 μl. All mRNA was N1-methyl-pseudo-U–capped with CleanCap technology offered by TriLink BioTechnologies. The ethanol and aqueous phases were combined through channels in a microfluidic device using a syringe pump as previously described (39). NPs were dialyzed against PBS for 2 hours before sterile filtration through syringe filters with 0.2-μm pores and stored at 4°C. JetPEI (Polyplus Transfection, New York, NY)–mRNA complexes were prepared according to manufacturer protocols with N/P = 7. All materials were prepared and handled ribonuclease-free throughout the synthesis, formulation, and characterization steps.

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