Preparation of placenta-tropic mRNA lipid nanoparticles for pregnancy disorders

Lipid nanoparticles (LNPs) have garnered tremendous enthusiasm in preclinical and clinical settings for the delivery of nucleic acids such as mRNA. With applications in protein replacement therapies, vaccines and gene editing, mRNA LNPs have only recently been explored in the context of pregnancy disorders. There is a significant need for the design of novel therapeutic technologies such as mRNA LNPs to treat obstetric disorders like pre-eclampsia that are associated with placental pathology and detrimental effects on maternal and fetal health. Here, we present a step-by-step procedure for the preparation and evaluation of placenta-tropic mRNA LNPs for researchers from varied disciplines to explore their application in treating pregnancy disorders. In this Protocol, we describe steps for synthesizing and purifying the key ionizable lipid excipient of the placenta-tropic LNP formulation (4 d) before preparing mRNA LNPs using microfluidic mixing (1 d). Then, we detail in vitro mechanistic evaluations of the effect of protein adsorption on LNP-mediated mRNA transfection to placental trophoblasts (3 d). Finally, we outline methods for isolating reproductive tissues from time-dated pregnant mice to assess in vivo LNP biodistribution and mRNA transfection to the murine placenta (16 d). Compared to alternative LNP formulation procedures, this Protocol focuses on delivering mRNA LNPs to the placenta with a workflow that can be applied for a range of obstetric disorders. This Protocol seeks to increase interdisciplinary work at the interface of nanomedicine, gene modulation and reproductive health.