The placenta, a temporary organ that develops during pregnancy, is one of the least understood human organs. The placental microenvironment influences the ability of placental trophoblast cells to invade the maternal endometrium to anchor the placenta and dilate maternal arteries, which is crucial to a healthy pregnancy. Shallow invasion of trophoblasts can result in complications, such as preeclampsia. Previous studies have shown that epidermal growth factor (EGF), human chorionic gonadotropin (hCG), and oxygen tension stimulate trophoblast invasion, whereas lipopolysaccharide (LPS) and tumor necrosis factor alpha (TNF-α) inhibit trophoblast invasion. Therapeutic use during pregnancy necessitates studies that investigate drug delivery and interactions with the placenta under varied growth conditions. In particular, lipid nanoparticles (LNPs) have risen in popularity for therapeutic delivery. Our work seeks to evaluate how placental trophoblast growth conditions influence the delivery of lipid nanoparticles to these trophoblast cells. Conditions including EGF, TNF-α, hCG, LPS, and oxygen tension are evaluated for their role in trophoblast invasiveness and subsequent LNP uptake. LNPs were formulated by microfluidic mixing of a low pH nucleic acid solution and a lipid mixture with a ratio of ionizable lipids: distearoylphosphatidylcholine (DSPC): cholesterol: polyethylene glycol-conjugated lipids (PEG) of 50:10:38.5:1.5 mol %, respectively. The ionizable lipids used were ALC-0315, DLin-MC3-DMA (MC3), and SM-102. LNPs were loaded with luciferase-producing messenger RNA (mRNA) at an amine to phosphate ratio (N/P) of 12. The LNPs were characterized and found to be uniform particles with an average hydrodynamic diameter of 90.7 nm, a neutral zeta potential, and an average mRNA encapsulation efficiency of 83.4%. LNPs were then delivered to HTR-8/SVneo placental trophoblast cells. After a 48- hour transfection, we observed that LPS, hCG, and TNF-α significantly decreased uptake of SM-102 LNPs compared to control media without supplementation. LPS and hCG significantly decreased uptake of ALC-0315 LNPs after a 48-hour transfection. Addition of EGF to the culture media resulted in a significant increased uptake of SM-102 and ALC-0315 LNPs after a 48-hour transfection. These results demonstrate that placental trophoblast cell uptake of lipid nanoparticles and subsequent gene expression is influenced by their growth conditions, highlighting the importance of studying changes in the placental microenvironment when assessing drug delivery for pregnancy applications.
