Exploring the challenges of lipid nanoparticle development : the in vitro–in vivo correlation gap

Background/Objectives: The development of lipid nanoparticles (LNPs) as delivery platforms for nucleic acids has revolutionised possibilities for both therapeutic and vaccine applications. However, emerging studies highlight challenges in achieving reliable in vitro–in vivo correlation (IVIVC), which delays the translation of experimental findings into clinical applications. This study investigates these potential discrepancies by evaluating the physicochemical properties, in vitro efficacy (across three commonly used cell lines), and in vivo performance (mRNA expression and vaccine efficacy) of four LNP formulations. Methods: LNPs composed of DSPC, cholesterol, a PEGylated lipid, and one of four ionizable lipids (SM-102, ALC-0315, MC3, or C12-200) were manufactured using microfluidics. Results: All formulations exhibited comparable physicochemical properties, as expected (size 70–100 nm, low PDI, near-neutral zeta potential, and high mRNA encapsulation). In vitro studies demonstrated variable LNP-mediated mRNA expression in both immortalised and immune cells, with SM-102 inducing significantly higher protein expression (p < 0.05) than the other formulations in immortalised and immune cells. However, in vivo results revealed that ALC-0315 and SM-102-based LNPs achieved significantly (p < 0.05) higher protein expression without a significant difference between them, while MC3- and C12-200-based LNPs exhibited lower expression levels. As vaccine formulations, all LNPs elicited strong immune responses with no significant differences among them. Conclusions: These findings highlight the complexities of correlating in vitro and in vivo outcomes in LNP development and demonstrate the importance of holistic evaluation strategies to optimise their clinical translation.