(393e) Award Submission: Development of a Model Placental Lipid Bilayer

The placenta has one of the most important roles in controlling fetal development as it is a semi-permeable barrier controlling the exchange of nutrients and wastes. However, it remains one of the least understood organs in the human body. There is a lack of placenta representative in vitro and in vivo models, making studies of this organ difficult. Meanwhile, during pregnancy a number of molecules may come in contact with the placenta (e.g. environmental toxins, pharmaceuticals, etc.). For example, only approximately 2% of medications approved by the FDA have sufficient data on their risk when used during pregnancy. However, 90% of women take at least one medication throughout the course of pregnancy. Additionally, environmental toxins, such as phthalates are of concern during pregnancy. Previous studies have suggested that women who are exposed to high concentrations of di-(2-ethylhexyl) phthalate (DEHP), were 60% more likely to lose a pregnancy compared to those with lower concentrations. Lipid bilayers have been used to model biological interfaces, such as the liver, red blood cell, and myelin. Here, we sought to develop a placental model. The goal of this work was to develop an in vitro placenta model using the placental lipid composition.

First, we investigated the lipid composition of placental (trophoblast) cell lines. We extracted the lipid composition from HTR-8, TCL-1, and primary trophoblast cells using the Bligh-Dyer method. HTR-8 cells are representative of first-trimester trophoblast cells while TCL-1 cells are representative of third-trimester trophoblast cells. After extraction, liquid chromatography with tandem mass spectrometry (LC-MS/MS) was used to quantify the composition of the most abundant lipid classes including phosphatidylcholine (PC), phosphoethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), and sphingomyelin (SPH). We observed statistically significant differences between the lipid composition of HTR-8, TCL-1, and primary trophoblasts. With the known compositions, we then developed synthetic lipid vesicles using the dry lipid thin film technique and extrusion through a 100 nm polycarbonate membrane with the same lipid composition as the HTR-8, TCL-1, and primary cells. Using dynamic light scattering we confirmed the hydrodynamic diameter of our trophoblast lipid vesicles to be 109 ±1.3 nm. In order to develop a supported lipid bilayer, we used quartz crystal microbalance with dissipation monitoring (QCM-D) to measure frequency (ΔF) and dissipation (ΔD) changes occurring on a silica surface upon introduction of these synthetic vesicles. With this complex vesicle composition, the vesicles adsorb on the surface but do not spontaneously rupture (ΔF = -45.4 ± 13.0 Hz, ΔD = 5.3 x 10^-6 ± 1.6 x 10^-6). We have synthesized an α-helical peptide that is derived from the N-terminal amphipathic helix of the hepatitis C virus NS5A protein, previously used to rupture complex vesicles into supported lipid bilayers. Using this peptide, we are able to rupture our trophoblast representative vesicles into lipid bilayers on QCM-D (ΔF = -30.0 ± 4.4 Hz, ΔD = 1.3 x 10^-6 ± 0.6 x 10^-6) and have confirmed the removal of this peptide after rinsing the formed bilayer. Thus, we have successfully fabricated lipid bilayers mimicking the lipid composition of placental studies. Future studies will investigate how environmental toxins (di(2-ethylhexyl) phthalate, mono(2-ethylhexyl) phthalate) and small molecules (folic acid, caffeine, etc.) interact with these bilayers. This work will improve placental understanding so that risk to a developing fetus can be minimized.