(175af) Messenger RNA Loaded Lipid Nanoparticle Delivery and Gene Expression in Trophoblast Cells Is Impacted By Ionizable Lipid Content

Lipid nanoparticles (LNPs) are a rising non-viral drug and gene delivery vehicle most notably used for the delivery of nucleic acids in the COVID-19 vaccines from Pfizer-BioNTech and Moderna, as well as in a medication for polyneuropathy from Alnylam [1]. These three therapeutics each use a different ionizable lipid – one of five main components of a nucleic acid-loaded LNP. Ionizable lipids play key roles in nucleic acid encapsulation and endosomal escape due to their ability to become protonated in low pH environments [2]. The unique and promising nature of LNP delivery sparked interest in their use for women’s health conditions, specifically delivering therapeutics to treat pregnancy-related complications and pre-existing maternal conditions during pregnancy. Defined as “the death of a woman while pregnant or within 42 days of termination of pregnancy” [3], maternal deaths have risen over the last five years, nearly doubling from 17.4 to 32.9 deaths per 100,000 live births in 2018 and 2021, respectively [4]. The limited information on pharmaceutical interactions during pregnancy drives the need for research on the interactions of drug and gene delivery vehicles with the placental barrier for the development of therapies for pregnancy-related complications and the ease of treating pre-existing conditions during pregnancy. This study aimed to compare ionizable lipids that have been used in FDA-approved formulations for nucleic acid delivery to placental trophoblast cells. LNPs were formulated through microfluidic mixing to contain ionizable lipids, helper lipids, cholesterol, and polyethylene glycol-conjugated lipids (Figure 1A). These LNP formulations contained either SM-102, ALC-0315, or Dlin-MC3-DMA (MC3) ionizable lipids. All LNPs were loaded with luciferase-producing messenger RNA (mRNA). HTR-8/SVneo trophoblast cells were seeded in a 96-well plate at 10,000-15,000 cells per well and dosed with mRNA loaded LNPs, and gene expression was assessed at various timepoints. Luminescence was elicited by using a Pierce™ Firefly Luciferase Glow Assay kit and measured on a microplate reader. These results demonstrated all LNPs had successful uptake by trophoblast cells and subsequent gene expression. The SM-102 formulation resulted in a ~10-fold increased gene expression compared to the formulations containing ALC-0315 and MC3 (Figure 1B). GFP-tagged mRNA loaded LNPs were formulated and characterized to confirm luciferase results. Based on this data, SM-102 will be further investigated for mRNA delivery for placental cell applications.

Figure 1: Experimental workflow and gene expression results. (A) Schematic of LNP formulation and characterization, delivery to placental trophoblast cells, and evaluation of uptake and gene expression. Created with BioRender.com. (B) Luminescence expression from trophoblast cells transfected with LNPs formulated with different ionizable lipids ALC-0315, MC3, and SM-102 at 12-, 24-, and 48-hour timepoints. Statistical analysis performed using Two-way ANOVA with α=0.05, ***p<0.001, ****p<0.0001.

[1] Tenchov et. al., ACS Nano, 2021. [2] Tang et. al., Adv. NanoBiomed Research, 2023. [3] WHO, International statistical classification of diseases and related health problems, 2009. [4] Hoyert et. al., NCHS Health E-Stats, 2023.