Comprehensive Evaluation of Gastrointestinal Factors Influencing the Stability and Function of mRNA‐Encapsulated Lipid Nanoparticles

Abstract

ABSTRACT Owing to the intrinsic instability of mRNA, its routes of administration are limited, and to date, only injectable delivery has been clinically applied. In contrast, oral administration exposes mRNA to a variety of substances in the gastrointestinal (GI) tract. Despite these challenges, oral delivery remains the most familiar and convenient route for patients, making the development of orally administrable mRNA‐encapsulated lipid nanoparticles (mRNA–LNPs) highly desirable. However, no studies have directly examined how mRNA–LNPs are affected within the GI environment. In the present study, we investigated the effects of representative GI components on mRNA–LNPs. Among several digestive enzymes, pepsin markedly reduced the protein‐expression efficiency of the particles, whereas amylase had no effect. Pepsin‐treated particles exhibited increased size, suggesting that structural alterations contributed to the observed functional loss. Lipase at high concentrations impaired nanoparticle function. Because lipase hydrolyzes ester bonds in lipids, we examined LNPs incorporating C12‐200, an ionizable lipid lacking ester bonds, instead of SM‐102. The C12‐200 formulation showed reduced susceptibility to lipase‐induced functional loss, indicating that enzymatic lipid degradation underlay the observed effects. Simulated gastric fluid also significantly diminished particle functionality. A detailed analysis of the relationship between functionality and pH revealed that impairment primarily occurred at approximately pH 1 but not at pH ≥ 3. Collectively, these findings identify the key GI factors that compromise mRNA–LNP integrity and provide important insights into the rational design of orally administrable mRNA–LNP formulations.