ComparativeInvestigation of the Impact of SodiumCitrate Buffers on Lipid Nanoparticles of circRNA or Linear mRNA

Circular RNAs (circRNAs) are an emerging modality of RNA medicine. By bypassing terminal degradation, circRNA is highly stable with prolonged protein/peptide expression duration relative to its linear mRNA counterpart, which may benefit its disease therapeutic or prophylactic efficacy. Currently, mRNA-delivering ionizable lipid nanoparticles (LNPs) and their formulation conditions, such as buffer, are often adopted for the delivery of circRNA delivery. However, it remains elusive whether the LNPs and their formulation methods optimized for mRNA are still optimal for the delivery of circRNA. Here, by comparing circRNA versus mRNA payloads, we investigated the impact of sodium citrate buffer concentrations on the physicochemical properties and RNA delivery efficiency of the resulting RNA-LNPs of three current FDA-approved LNPs, DLin-MC3-DMA (MC3), SM-102, and ALC-0315, respectively. First, for each LNP formulated using the same sodium citrate buffer concentration, mRNA and circRNA showed comparable physicochemical characteristics and in vitro transfection efficiency; by contrast, high-concentration sodium citrate compromised the in vivo expression of circRNA LNPs but not mRNA LNPs. Second, relative to SM-102 LNPs, MC3-LNPs showed a lower RNA expression efficiency in mice, especially for circRNA. Third, LNPs formulated using high-concentration sodium citrate buffer showed comparable integrity of mRNA and circRNA after multiday storage at room temperature. Overall, our findings underscore the differential impact of citrate buffer concentrations on different LNPs loaded with circRNA and mRNA, calling for the customized optimization of different LNPs for the delivery of mRNA and circRNA.