Zhang Hongqian, Gao Xue, Sun Qian, Dong Xiaoxue, Zhu Zongwei, Yang Chuanxu
Key Laboratory of Colloid and Interface Chemistry of the Ministry of Education, and School of Chemistry and Chemical Engineering, Shandong University, Jinan 25010, China.
Key Laboratory of Colloid and Interface Chemistry of the Ministry of Education, and School of Chemistry and Chemical Engineering, Shandong University, Jinan 25010, China.
Acta Biomater. 2024 Mar 15;177:361-376. doi: 10.1016/j.actbio.2024.02.004. Epub 2024 Feb 10.
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.
基于信使核糖核酸（mRNA）的疗法在广泛的生物医学应用中显示出巨大潜力。然而，由于递送障碍和细胞内有效载荷释放效率低下，开发安全有效的mRNA递送载体仍然具有挑战性。在此，我们提出了一种简单的策略，通过将阴离子型聚γ-谷氨酸（PGA）掺入基于可电离脂质的脂质纳米颗粒/ mRNA（LNP/mRNA）中来提高mRNA的细胞内释放。我们系统地研究了PGA掺入对体外和体内mRNA转染的影响。PGA的分子量和配方比例极大地影响了LNP/mRNA的转染效率。体外研究表明，通过掺入分子量为80 kDa或200 kDa且电荷比（N/P/C）为25/1/1的PGA，制备了优化的LNP/mRNA/PGA。与裸露的LNP/mRNA相比，优化后的配方在HeLa细胞中实现了约3倍的mRNA表达。使用特异性DNA探针进行的细胞内释放研究表明，转染效率的提高归因于mRNA向细胞质中的释放增加。此外，优化后的LNP/mRNA/PGA分别经静脉或肌肉注射到小鼠体内后，体内荧光素酶mRNA表达分别提高了5倍或3倍。此外，PGA的掺入在器官和细胞水平上均未显著改变复合物的生物分布情况。因此，我们的工作提供了一种提高mRNA递送的简单策略，有望改善mRNA疗法在各种生物医学应用中的疗效。重要性声明：设计和筛选有效的mRNA载体的过程复杂且耗时，由于递送障碍和mRNA释放效率低下，其疗效并不总是令人满意。这项工作提出了一种替代策略，通过将阴离子型天然聚合物聚γ-谷氨酸（PGA）掺入LNP/mRNA复合物中来提高mRNA递送效率。优化后的LNP/mRNA/PGA在体外和体内的转染效率分别提高了3倍和5倍。细胞内释放分析表明，转染效率的提高主要归因于mRNA细胞内释放的增加。此外，PGA的掺入并未改变复合物的生物分布或生物安全性。这些发现表明，掺入PGA是提高mRNA疗法疗效的一种有前景的策略。
