For liver toxicity studies, plasma samples from dams or fetal liver lysates were assessed for AST and ALT liver enzyme levels using AST or ALT colorimetric activity assay kits, respectively (Cayman Chemical, Ann Arbor, MI, USA) according to manufacturer recommendations at 3 days after LNP or PBS injection. Of note, because of the small fetal size and limited fetal serum availability, fetal cytokine production and fetal liver enzymes were assessed in fetal liver lysates rather than serum. Thus, fetal AST/ALT data were normalized to the protein concentration in the sample as determined by the microBCA assay. Complement system activation from plasma collected from the dams was assessed using Immunotag Mouse C3 (complement C3) and Immunotag Mouse C4 (complement C4) ELISA kits (G-Biosciences, St. Louis, MO, USA) per manufacturer instructions. We were unable to assess complement system activation directly from fetal plasma due to the low volume of blood present in fetuses at this gestational age.

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