Optimizing mRNA delivery: A microfluidic exploration of DOTMA vs. DOTAP lipid nanoparticles for GFP expression on human PBMCs and THP-1 cell line

Abstract

This study highlights lipid nanoparticle (LNP) formulations incorporating DOTMA or DOTAP as cationic lipids for the delivery of mRNA encoding Enhanced Green Fluorescent Protein (eGFP-mRNA). The performance of these tailored formulations was benchmarked against a commercial formulation (LipidFlex®, Precigenome), which can also be combined with DOTMA or DOTAP but contains helper lipids of undisclosed composition. LNPs were synthesized using a microfluidic device equipped with a passive Y-shaped microchip, operating at an optimized total flow rate of 6 mL/min and a flow rate ratio of 1:3, with a total lipid concentration ranging from 0.7 to 30 mM. This method produced Single Unilamellar Vesicles (SUVs) with an average size of 150 ± 53 nm and a surface charge of 18 mV. The nitrogen-to-phosphate (N/P) ratio was varied between 250 and 6, modulating the surface charge (from 48 to 18 mV) and the mRNA-eGFP encapsulation efficiency (from 80 % to 70 %, respectively). Cytotoxicity assays and IC₅₀ evaluations on a Hamster Ovarian cell line confirmed that the c-DOTMA formulation achieved an optimal balance of low toxicity and high transfection efficiency. In THP-1 cells, c-DOTMA delivered the highest eGFP expression, reaching up to 25 % transfection efficiency, extremely higher if compared to those observed in the total PBMC population under similar conditions. This selective behavior highlights its potential for precise mRNA delivery to specific immune cell subsets, though further research is required to assess in vivo performance, biodistribution, and immunogenicity.