The ionizable lipids used in LNP formulations A-3 and B-4 were purified as described above. LNPs were prepared with human EPO mRNA (TriLink BioTechnologies) and injected into E16 fetuses via the vitelline vein. Fetal livers were harvested at 4 and 24 hours after injection and kept on ice during processing. Livers were rinsed three times with 1× PBS to remove excess blood and were then homogenized in 5 ml of 1× PBS using 15-ml tissue grinders (Thermo Fisher Scientific). Lysates were brought through two repeated overnight freeze-thaw cycles to break up cell membranes. After the final thaw, processed liver samples were centrifuged (5 min, 5000g) and the supernatant was collected for analysis. EPO content in the supernatant was quantified by ELISA using the Human Erythropoietin Quantikine IVD ELISA Kit (R&D Systems, Minneapolis, MN) per manufacturer recommendations. Data shown are average and SEM of EPO concentrations from at least three fetuses injected in each experimental group.

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