Nanoscopy for Nanomedicine Group, Institute for Bioengineering of Catalonia (IBEC) , The Barcelona Institute of Science and Technology (BIST) , Carrer Baldiri Reixac 15-21, 08024 Barcelona , Spain.
Department of Biomedical Engineering, Institute for Complex Molecular Systems (ICMS) , Eindhoven University of Technology , 5612AZ Eindhoven , The Netherlands.
Nano Lett. 2019 May 8;19(5):2784-2792. doi: 10.1021/acs.nanolett.8b04407. Epub 2019 Apr 26.
The successful application of gene therapy relies on the development of safe and efficient delivery vectors. Cationic polymers such as cell-penetrating peptides (CPPs) can condense genetic material into nanoscale particles, called polyplexes, and induce cellular uptake. With respect to this point, several aspects of the nanoscale structure of polyplexes have remained elusive because of the difficulty in visualizing the molecular arrangement of the two components with nanometer resolution. This limitation has hampered the rational design of polyplexes based on direct structural information. Here, we used super-resolution imaging to study the structure and molecular composition of individual CPP-mRNA polyplexes with nanometer accuracy. We use two-color direct stochastic optical reconstruction microscopy (dSTORM) to unveil the impact of peptide stoichiometry on polyplex structure and composition and to assess their destabilization in blood serum. Our method provides information about the size and composition of individual polyplexes, allowing the study of such properties on a single polyplex basis. Furthermore, the differences in stoichiometry readily explain the differences in cellular uptake behavior. Thus, quantitative dSTORM of polyplexes is complementary to the currently used characterization techniques for understanding the determinants of polyplex activity in vitro and inside cells.
基因治疗的成功应用依赖于安全高效的输送载体的发展。阳离子聚合物，如细胞穿透肽（CPPs），可以将遗传物质浓缩成纳米级颗粒，称为聚阳离子，从而诱导细胞摄取。在这方面，由于难以以纳米分辨率可视化两个组件的分子排列，聚阳离子的纳米级结构的几个方面仍然难以捉摸。这种局限性阻碍了基于直接结构信息的聚阳离子的合理设计。在这里，我们使用超分辨率成像技术以纳米精度研究了单个 CPP-mRNA 聚阳离子的结构和分子组成。我们使用双色直接随机光学重建显微镜（dSTORM）揭示了肽化学计量比对聚阳离子结构和组成的影响，并评估了它们在血清中的不稳定性。我们的方法提供了关于单个聚阳离子大小和组成的信息，允许在单个聚阳离子基础上研究这些性质。此外，化学计量比的差异很容易解释细胞摄取行为的差异。因此，聚阳离子的定量 dSTORM 补充了目前用于理解聚阳离子在体外和细胞内活性的决定因素的表征技术。
