Abstract

Inhalable messenger RNA (mRNA) has demonstrated great potential in therapy and vaccine development to confront various lung diseases. However, few gene vectors could overcome the airway mucus and intracellular barriers for successful pulmonary mRNA delivery. Apart from the low pulmonary gene delivery efficiency, nonnegligible toxicity is another common problem that impedes the clinical application of many non-viral vectors. PEGylated cationic peptide-based mRNA delivery vector is a prospective approach to enhance the pulmonary delivery efficacy and safety of aerosolized mRNA by oral inhalation administration. In this study, different lengths of hydrophilic PEG chains were covalently linked to an amphiphilic, water-soluble pH-responsive peptide, and the peptide/mRNA nano self-assemblies were characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM). The in vitro mRNA binding and release, cellular uptake, transfection, and cytotoxicity were studied, and finally, a proper PEGylated peptide with enhanced pulmonary mRNA delivery efficiency and improved safety in mice was identified. These results showed that a proper N-terminus PEGylation strategy using 12-monomer linear monodisperse PEG could significantly improve the mRNA transfection efficiency and biocompatibility of the non-PEGylated cationic peptide carrier, while a longer PEG chain modification adversely decreased the cellular uptake and transfection on A549 and HepG2 cells, emphasizing the importance of a proper PEG chain length selection. Moreover, the optimized PEGylated peptide showed a significantly enhanced mRNA pulmonary delivery efficiency and ameliorated safety profiles over the non-PEGylated peptide and Lipofectamine^TM 2000 in mice. Our results reveal that the PEGylated peptide could be a promising mRNA delivery vector candidate for inhaled mRNA vaccines and therapeutic applications for the prevention and treatment of different respiratory diseases in the future.

摘要

可吸入的信使 RNA (mRNA) 在对抗各种肺部疾病的治疗和疫苗开发方面已显示出巨大的潜力。然而，很少有基因载体能够克服气道粘液和细胞内障碍，成功地将 mRNA 递送到肺部。除了肺部基因传递效率低之外，不可忽视的毒性是阻碍许多非病毒载体临床应用的另一个常见问题。基于聚乙二醇化阳离子肽的 mRNA 递送载体是一种通过口服吸入给药增强雾化 mRNA 肺部递送功效和安全性的前瞻性方法。在这项研究中，不同长度的亲水性 PEG 链与两亲性、水溶性 pH 响应肽共价连接，肽/mRNA纳米自组装体通过动态光散射（DLS）和透射电子显微镜（TEM）进行表征。这体外研究了 mRNA 结合和释放、细胞摄取、转染和细胞毒性，最终确定了一种合适的聚乙二醇化肽，该肽可增强肺部 mRNA 递送效率并提高小鼠的安全性。这些结果表明，使用12单体线性单分散PEG的适当的N端聚乙二醇化策略可以显着提高非聚乙二醇化阳离子肽载体的mRNA转染效率和生物相容性，而较长的PEG链修饰会不利地降低细胞对mRNA的摄取和转染。 A549 和 HepG2 细胞，强调选择适当 PEG 链长度的重要性。此外，与非聚乙二醇化肽和 Lipofectamine 相比，优化的聚乙二醇化肽显示出显着增强的 mRNA 肺部递送效率和改善的安全性^TM 2000 在小鼠中。我们的结果表明，聚乙二醇化肽可能是一种有前途的 mRNA 递送载体候选物，可用于吸入 mRNA 疫苗和未来预防和治疗不同呼吸道疾病的治疗应用。