Inherited retinal diseases (IRDs) are a group of genetically heterogeneous neurodegenerative disorders that cause progressive vision loss. Gene therapies based on adeno-associated virus vectors have achieved notable progress in IRDs, but their limited packaging capacity and potential long-term toxicity constrain broader applications. Lipid nanoparticles (LNPs) have been clinically validated as nonviral delivery vehicles for nucleic acid therapeutics. Previous ocular studies have primarily focused on strategies such as surface modification and polyethylene glycol lipid engineering, which have provided valuable insights. However, systematic exploration of core formulation parameters, such as lipid component ratios, has received comparatively limited attention. In this study, we applied a design of experiments strategy to construct and evaluate LNP libraries for ocular delivery. Systematic screening identified formulation B11, which produced 2.6-fold and 3.0-fold reporter gene expressionfollowing intravitreal administration compared with benchmark SM-102 and MC3 formulations. Mechanistic analyses revealed that LNP B11 is internalized predominantly through caveolae-mediated endocytosis and macropinocytosis., LNP B11 mediated mRNA expression localized to the retinal pigment epithelium, as demonstrated by mCherry expression. Furthermore, Cre mRNA delivery using B11 formulation induced tdTomato activation in Ai14 reporter mice more effectively than standard SM-102 and MC3 formulations, indicating that B11 supports functional protein expression in retinal tissue. Together, these findings identify B11 as a promising optimized LNP formulation for retinal mRNA delivery, highlighting the critical role of systematic formulation optimization in advancing nucleic acid therapeutics for retinal diseases. in vivo In vivo
