Incorporation of poly(γ-glutamic acid) in lipid nanoparticles for enhanced mRNA delivery efficiency in vitro and in vivo.

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.