The ionizable lipid cores were prepared via Michael addition chemistry as previously described (34). Briefly, the polyamine cores (purchased from Enamine Inc., Monmouth Junction, NJ) were combined with excess moles of lipid epoxide needed to saturate the amines in 4-ml amber vials with a magnetic stir bar. The lipid epoxides used in this study were epoxydodecane (C12), epoxytetradecane (C14), or epoxyhexadecane (C16) (Sigma-Aldrich, St. Louis, MO). The vial was sealed and the reaction was mixed for 2 days at 80°C. The crude reaction mixture was dried using a Rotovap R-300 (Buchi, New Castle, DE), and the crude reactions were used for screening the library with luciferase mRNA. The A-3 and B-4 reaction mixtures were further characterized by LC-MS. The resultant fractions from the reaction were separated using a CombiFlash NextGen 300+ (Teledyne ISCO, Lincoln, NE) against a gradient of 100% methanol to 100% of a solution composed of 75% dichloromethane, 22% methanol, and 3% ammonium hydroxide over 55 min. Each peak was collected and dried, and the molecular weight of the fully saturated product was confirmed by LC-MS. This purified product was used to deliver human EPO mRNA.

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