Quantification of mRNA in Lipid Nanoparticles Using Mass Spectrometry. Analytical chemistry Lipid nanoparticle-encapsulated mRNA (LNP-mRNA) holds great promise as a novel modality for treating a broad range of diseases. The ability to quantify mRNA accurately in therapeutic products helps to ensure consistency and safety. Here, we consider a central aspect of accuracy, measurement traceability, which establishes trueness in quantity. In this study, LNP-mRNA is measured in situ using a novel liquid chromatography-mass spectrometry (LC-MS) approach with traceable quantification. Previous works established that oligonucleotide quantification is possible through the accounting of an oligomer's fundamental nucleobases, with traceability established through common nucleobase calibrators. This sample preparation does not require mRNA extraction, detergents, or enzymes and can be achieved through direct acid hydrolysis of an LNP-mRNA product prior to an isotope dilution strategy. This results in an accurate quantitative analysis of mRNA, independent of time or place. Acid hydrolysis LC-MS is demonstrated to be amenable to measuring mRNA as both an active substance or a formulated mRNA drug product. 10.1021/acs.analchem.3c04406

Incorporation of poly(γ-glutamic acid) in lipid nanoparticles for enhanced mRNA delivery efficiency in vitro and in vivo. Acta biomaterialia Messenger RNA (mRNA)-based therapy shows immense potential for broad biomedical applications. However, the development of safe and efficacious mRNA delivery vectors remains challenging due to delivery barriers and inefficient intracellular payload release. Herein, we presented a simple strategy to boost the mRNA intracellular release by incorporation of anionic poly(γ-glutamic acid) (PGA) into an ionizable lipid-based LNP/mRNA. We systematically investigated the impact of PGA incorporation on mRNA transfection both in vitro and in vivo. The molecular weights and formulation ratios of PGA greatly affected the transfection efficacy of LNP/mRNA. From in vitro study, the optimized LNP/mRNA/PGA was formulated by incorporation of PGA with the molecular weight of 80 kDa or 200 kDa and the charge ratio (N/P/C) of 25/1/1. The optimized formulation achieved around 3-fold mRNA expression in HeLa cells compared to the bare LNP/mRNA. The intracellular releasing study using specific DNA probe revealed that this enhancement of transfection efficacy was attributed to the elevated mRNA release into cytoplasm. Moreover, the optimized LNP/mRNA/PGA achieved up to 5-fold or 3-fold increase of luciferase mRNA expression in vivo after being injected into mice systematically or intramuscularly, respectively. In addition, the incorporation of PGA did not significantly alter the biodistribution profile of the complexes on both organ and cellular levels. Therefore, our work provides a simple strategy to boost mRNA delivery, which holds great promise to improve the efficacy of mRNA therapeutics for various biomedical applications. STATEMENT OF SIGNIFICANCE: The process of designing and screening potent mRNA carriers is complicated and time-consuming, while the efficacy is not always satisfying due to the delivery barriers and inefficient mRNA release. This work presented an alternative strategy to boost the mRNA delivery efficacy by incorporating an anionic natural polymer poly(γ-glutamic acid) (PGA) into LNP/mRNA complexes. The optimized LNP/mRNA/PGA achieved up to 3-fold and 5-fold increase in transfection efficacy in vitro and in vivo, respectively. Intracellular releasing analysis revealed that the enhancement of transfection efficacy was mainly attributed to the elevated intracellular release of mRNA. In addition, the incorporation of PGA did not alter the biodistribution or the biosafety profile of the complexes. These findings indicate that PGA incorporation is a promising strategy to improve the efficacy of mRNA therapeutics. 10.1016/j.actbio.2024.02.004