In vitro transcribed messenger RNA (IVT-mRNA) holds great promise for the development of novel therapeutics, such as immunotherapy and vaccination. However, the main obstacle towards clinical translation is the lack of effective delivery systems. Herein, we have synthesized a series of ionizable lipids by the addition of an alkyl-acrylate to amine-containing molecules (amine-head groups) as a key component of ionizable lipid nanoparticles (iLNPs) and thoroughly investigated the impact of the amine-head group on the transfection efficiency of iLNPs/mRNA lipoplexes both in vitro and in vivo. The top-performing iLNP (114-iLNP), composed of a lipid with spermine as the amine-head, demonstrated the strongest cellular uptake, membrane disruption and endosomal escape, and further achieved the highest protein expression in HeLa cells with more than 95% transfection efficiency. More importantly, intravenous injection of luciferase mRNA loaded 114-iLNP enables the most efficacious in vivo protein expression, predominantly in the liver. Biodistribution and biosafety evaluation of 114-iLNP/mRNA further demonstrated the liver-selective delivery capability and high biocompatibility. In addition, 114-iLNP facilitated efficient in vivo delivery of a therapeutic gene, human erythropoietin (hEPO) mRNA, and induced hEPO expression in a dose-dependent manner. Therefore, these results demonstrate that the amine-head group in the ionizable lipid significantly affects mRNA delivery efficacy and the leading candidate 114-iLNP composed of a lipid with spermine as the amine-head has great potential for mRNA therapeutics development.

中文翻译：

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通过优化脂质胺头基团促进可电离脂质纳米颗粒介导的体内 mRNA 递送

体外转录的信使 RNA (IVT-mRNA) 为开发新疗法（如免疫疗法和疫苗接种）带来了巨大希望。然而，临床转化的主要障碍是缺乏有效的传递系统。在此，我们通过将丙烯酸烷基酯添加到含胺分子（胺头基团）作为可电离脂质纳米粒子 (iLNPs) 的关键组分合成了一系列可电离脂质，并彻底研究了胺头的影响iLNPs/mRNA lipoplexes体外和体内转染效率组. 性能最好的iLNP（114-iLNP），由以精胺为胺头的脂质组成，表现出最强的细胞摄取、膜破坏和内体逃逸，并进一步在HeLa细胞中实现了最高的蛋白质表达，超过95%转染效率。更重要的是，静脉注射载有荧光素酶 mRNA 的 114-iLNP 能够实现最有效的体内蛋白质表达，主要是在肝脏中。114-iLNP/mRNA 的生物分布和生物安全性评估进一步证明了肝脏选择性递送能力和高生物相容性。此外，114-iLNP 促进了体内有效递送治疗基因、人促红细胞生成素 (hEPO) mRNA，并以剂量​​依赖性方式诱导 hEPO 表达。因此，这些结果表明可电离脂质中的胺头基团显着影响 mRNA 传递效率，主要候选 114-iLNP 由以精胺为胺头的脂质组成，具有巨大的 mRNA 治疗开发潜力。