Nanoscopy for Nanomedicine, Institute for Bioengineering of Catalonia, Barcelona, Spain.
Nanoscale. 2019 Oct 3;11(38):17869-17877. doi: 10.1039/c9nr02858g.
The future of gene therapy relies on the development of efficient and safe delivery vectors. Poly(β-amino ester)s are promising cationic polymers capable of condensing oligonucleotides into nanoparticles - polyplexes - and deliver them into the cell nucleus, where the gene material would be expressed. The complexation state during the crossing of biological barriers is crucial: polymers should tightly complex DNA before internalization and then release to allow free DNA to reach the nucleus. However, measuring the complexation state in cells is challenging due to the nanometric size of polyplexes and the difficulties to study the two components (polymer and DNA) independently. Here we propose a method to visualize and quantify the two components of a polyplex inside cells, with nanometre scale resolution, using two-colour direct stochastic reconstruction super-resolution microscopy (dSTORM). With our approach, we tracked the complexation state of pBAE polyplexes from cell binding to DNA release and nuclear entry revealing time evolution and the final fate of DNA and pBAE polymers in mammalian cells.
基因治疗的未来依赖于高效和安全的输送载体的发展。聚（β-氨基酯）是一种很有前途的阳离子聚合物，能够将寡核苷酸浓缩成纳米颗粒 - 聚合物复合物，并将其递送到细胞核，在那里基因物质将被表达。在穿越生物屏障的过程中，复合物的状态至关重要：聚合物在被内化之前应该紧密地将 DNA 复合，然后释放，以使游离的 DNA 能够到达细胞核。然而，由于聚合物复合物的纳米级尺寸以及独立研究两个组分（聚合物和 DNA）的困难，测量复合物在细胞中的状态具有挑战性。在这里，我们提出了一种使用双色直接随机重建超分辨率显微镜（dSTORM）在纳米级分辨率下可视化和定量细胞内聚合物复合物的两种组分的方法。通过我们的方法，我们跟踪了 pBAE 聚合物复合物从细胞结合到 DNA 释放和核进入的复合物状态，揭示了 DNA 和 pBAE 聚合物在哺乳动物细胞中的时间演变和最终命运。
