Optimization of lipid nanoparticle components and microfluidic parameters for intramuscular circRNA delivery with reduced hepatic off-targeting.

Circular RNA (circRNA) represents a promising class of RNAs for sustained protein expression and vaccine applications. However, current lipid nanoparticle (LNP) platforms remain poorly optimized for efficient intramuscular circRNA delivery, as they are primarily designed for the delivery of small interfering RNA and linear messenger RNA with strong off-target expression in the liver. In this study, based on the clinical benchmark SM-102 LNP, we systematically optimized lipid components and microfluidic parameters for better intramuscular circRNA delivery. Through in vitro and in vivo screening of a variety of phospholipids and sterols, we identified a 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)- and cholesterol-based formulation, which demonstrated improved storage stability and enhanced intramuscular transfection while minimizing hepatic off-target expression. Using ovalbumin as a model antigen, we proved that this formulation-based vaccine elicited more balanced Th1/Th2 immune responses with reduced liver toxicity. Together, this study successfully develops an optimized formulation with reduced hepatic off-target effects, which holds great promise for the intramuscular delivery of circRNA-based vaccines and therapeutics.