Formulation-dependent serum effects on functional mRNA delivery: a head-to-head comparison of lipid and polymer-lipid hybrid nanoparticles

Abstract

mRNA therapeutics offer a promising platform for gene therapy; however, clinical translation remains constrained by increased extracellular instability and intracellular transport barriers, particularly under serum-containing physiological conditions. Therefore, the success of mRNA transport in the presence of serum largely depends on the formulation used. In this study, lipid-based nanoparticles (LNP) and polymer-lipid hybrid nanoparticles (HNP) were compared for GFP mRNA transport in HEK293 cells under serum-free and 10% FBS conditions. Both formulations formed stable mRNA complexes with high complexation efficiency (∼96%) and maintained transcript integrity after incubation at 37 °C. After mRNA loading, both platforms remained monodisperse and below 200 nm, with mean diameters of 153.67 ± 2.89 nm for LNP-mRNA and 144.53 ± 1.29 nm for HNP-mRNA, and positive zeta potentials of 40.4 ± 1.04 mV and 30.3 ± 0.06 mV, respectively. Scanning transmission electron microscopy (STEM) analyses confirmed the spherical morphology. Transfection efficiency was evaluated by flow cytometry. LNP-mRNA showed 78.90 ± 4.89% GFP positivity in serum-free conditions, while it decreased to 30.7 ± 1.4% in the presence of 10% FBS (∼60% reduction). In contrast, HNP-mRNA showed 53.80 ± 6.39% GFP positivity in serum-free medium and largely maintained its expression in serum at 51.5 ± 4.4%. MTT and LDH analyses showed acceptable biocompatibility in HEK293, BEAS-2B, and PMA-differentiated THP-1 cells. These results demonstrate that serum significantly influences mRNA transport depending on the formulation, and that HNPs can better protect gene expression under more biologically realistic conditions.