Department of Plant Biosecurity and MOA Key Laboratory of Pest Monitoring and Green Management, College of Plant Protection, China Agricultural University, Beijing, 100193, People's Republic of China.
Institute of Plant Protection, Beijing Academy of Agriculture and Forestry Sciences, Beijing, 100097, People's Republic of China.
J Nanobiotechnology. 2022 Mar 9;20(1):124. doi: 10.1186/s12951-022-01336-6.
Nanoparticles have been widely applied as gene carrier for improving RNA interference (RNAi) efficiency in medical and agricultural fields. However, the mechanism and delivery process of nanoparticle-mediated RNAi is not directly visualized and elucidated. Here we synthesized a star polymer (SPc) consisted of a hydrophilic shell with positively-charged tertiary amine in the side chain, which was taken as an example to investigate the mechanism in gene delivery. The SPc could assemble with dsRNA spontaneously through electrostatic force, hydrogen bond and van der Waals force. Interestingly, the SPc could protect dsRNA from degradation by RNase A and insect hemolymph, thus remarkably increasing the stability of dsRNA. Meanwhile, the SPc could efficiently promote the cellular uptake and endosomal escape for intracellular spreading of dsRNA. Transcriptome analysis revealed that the SPc could up-regulate some key genes such as Chc, AP2S1 and Arf1 for activating clathrin-mediated endocytosis. Furthermore, the suppression of endocytosis hindered the cellular uptake of SPc-delivered dsRNA in vitro, and the subsequent RNAi effect was also disappeared in vivo. To our knowledge, our study is the first direct visualization of the detailed cellular delivery process and mechanism of nanocarrier-mediated gene delivery. Above mechanism supports the application of nanocarrier-based RNAi in gene therapy and pest management.
纳米颗粒已广泛应用于基因载体，以提高医学和农业领域的 RNA 干扰 (RNAi) 效率。然而，纳米颗粒介导的 RNAi 的机制和递药过程尚不能直接可视化和阐明。在此，我们合成了一种星形聚合物 (SPc)，它由带正电荷的叔胺侧链的亲水壳组成，以此为例来研究基因递药中的机制。SPc 可以通过静电、氢键和范德华力自发地与 dsRNA 组装。有趣的是，SPc 可以保护 dsRNA 免受 RNase A 和昆虫血淋巴的降解，从而显著提高 dsRNA 的稳定性。同时，SPc 可以有效地促进细胞摄取和内涵体逃逸，从而使 dsRNA 在细胞内扩散。转录组分析表明，SPc 可以上调一些关键基因，如 Chc、AP2S1 和 Arf1，从而激活网格蛋白介导的内吞作用。此外，抑制内吞作用会阻碍 SPc 递送的 dsRNA 在体外的细胞摄取，体内的 RNAi 效应也随之消失。据我们所知，我们的研究首次直接可视化了纳米载体介导的基因递药的详细细胞递药过程和机制。上述机制支持基于纳米载体的 RNAi 在基因治疗和害虫管理中的应用。
