Department of Chemistry and Biochemistry , University of Bern , Freiestrasse 3 , 3012 Bern , Switzerland.
Bioconjug Chem. 2019 Aug 21;30(8):2165-2182. doi: 10.1021/acs.bioconjchem.9b00403. Epub 2019 Aug 9.
Transfecting nucleic acids into cells is an essential procedure in biological research usually performed using nonviral transfection reagents. Unfortunately, most transfection reagents have polymeric or undisclosed structures and require nonstandard synthetic procedures. Herein we report peptide dendrimers accessible as pure products from standard building blocks by solid-phase peptide synthesis and acting as nontoxic single component siRNA transfection reagents for a variety of cell lines with equal or better performance than the gold standard lipofectamine L2000. Structure-activity relationships and mechanistic studies illuminate their transfection mechanism in unprecedented detail. Stereoselective dendrimer aggregation via intermolecular β-sheets at neutral pH enables siRNA complexation to form nanoparticles which enter cells by endocytosis. Endosome acidification triggers protonation of amino termini and rearrangement to an α-helical conformation forming smaller dendrimer/siRNA nanoparticles, which escape the endosome and release their siRNA cargo in the cytosol. Two particularly efficient d-enantiomeric dendrimers are proposed as new reference reagents for siRNA transfection.
将核酸转染到细胞中是生物学研究中的一个基本步骤，通常使用非病毒转染试剂来完成。然而，大多数转染试剂具有聚合或未公开的结构，并且需要非标准的合成程序。在此，我们报告了肽树突状聚合物，它们可以通过固相肽合成得到，作为纯产物，并且可以作为无毒的单一组分 siRNA 转染试剂，用于多种细胞系，其性能与金标准脂质体 L2000 相当，甚至更好。结构-活性关系和机制研究以前所未有的细节阐明了它们的转染机制。在中性 pH 下通过分子间β-折叠进行立体选择性的树突状聚合物聚集，使 siRNA 能够形成纳米颗粒，这些纳米颗粒通过内吞作用进入细胞。内涵体酸化引发氨基末端质子化和重排形成α-螺旋构象，形成更小的树突状聚合物/siRNA 纳米颗粒，这些纳米颗粒从内涵体逃逸，并在细胞质中释放其 siRNA 货物。两种特别有效的 d-对映体树突状聚合物被提议作为 siRNA 转染的新参考试剂。
