LNPs A-3, B-4, or DLin-MC3-DMA, PBS, or LPS/CpG (lipopolysaccharide/CG oligonucleotide) were injected into E16 fetuses via the vitelline vein. All of the fetuses in each dam (n = 3 dams) were injected. On E19, blood from the dams was collected retro-orbitally into blood collection tubes and centrifuged (10,000g, 10 min, 4°C), and plasma was harvested for cytokine analysis, liver enzymes, and complement activation. Next, fetuses were surgically removed to avoid any variability from the natural birthing process and poor parenting, and the pups were immediately evaluated for survival. Survival was assessed by observation of gross appearance, size, spontaneous movement, and a visible heartbeat. After assessing survival, pup livers were collected from a minimum of five injected fetuses for cytokine analysis and ALT/AST analysis (see below). The remaining injected fetuses were housed with foster female mice that had given birth to pups no more than 1 day before fostering.

Dam plasma and fetal livers were snap-frozen in dry ice and stored at −80°C until use. Frozen livers were cut on dry ice, and pieces were weighed using a Mettler-Toledo scale, thawed in 5 ml/g of tissue M-PER lysis buffer (Thermo Fisher Scientific) with cOmplete protease inhibitor cocktail (Roche, Basel, Switzerland), and immediately disrupted using the TissueLyzer II (Qiagen Inc., Germantown, MD) system with 5-mm steel beads (Qiagen Inc.) under 30-Hz frequency for 60 s; the homogenization process was repeated twice. Homogenized tissues were placed on ice for 30 min to allow complete lysis. Samples were centrifuged (2200g, 30 min, 4°C) and the supernatant was collected into new tubes. Total protein content in each sample was determined using the microBCA protein assay kit (Thermo Fisher Scientific) per manufacturer instructions.

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