Effects of Serum Incubation on Lipid Nanoparticle PEG Shedding, mRNA Retention, and Membrane Interactions.

Lipid nanoparticles (LNPs) are widely used for RNA delivery, but their efficiency remains limited, largely due to poor endosomal escape. Upon administration, proteins bind to the surface of the LNPs, influencing cellular uptake and potentially altering their interfacial properties. Such alterations may also affect their interaction with endosomal membranes, thus influencing the critical endosomal escape step. Using fluorescence microscopy imaging with single-LNP resolution, this study investigates how incubation in 10% fetal bovine serum alters the PEG modification and mRNA content of LNPs, as well as how serum incubation-induced alterations influence the interaction between LNPs and an anionic supported lipid bilayer (SLB), serving as a simplistic mimic for the anionic lipid membrane of late endosomes. We demonstrate that serum incubation leads to the desorption of PEG-modified lipids and a significant release of mRNA cargo from the LNPs. PEG shedding occurred consistently with a half-life time of around 10 min, while mRNA release displayed higher variability between individual LNPs. We also observed that serum preincubation enhanced attractive interactions between tethered LNPs and the anionic SLB at physiological pH 7.4, and fusion of LNPs with the anionic SLB upon pH reduction was more efficient for serum-preincubated LNPs than for their pristine counterparts, particularly during moderate acidification from pH 6.5 to 6.0. This enhanced fusion efficiency may be attributed to a reduced steric hindrance from PEG-lipids following serum preincubation. The findings highlight that serum-induced modifications enhance LNP fusion efficiency with an endosomal membrane mimic while potentially compromising mRNA retention, thus balancing the overall efficacy of LNP-assisted mRNA delivery.