Krieg Bettina, Hirsch Markus, Scholz Erik, Nuhn Lutz, Tabujew Ilja, Bauer Heiko, Decker Sandra, Khobta Andriy, Schmidt Manfred, Tremel Wolfgang, Zentel Rudolf, Peneva Kalina, Koynov Kaloian, Mason A James, Helm Mark
Institute of Pharmacy and Biochemistry, Johannes Gutenberg-University Mainz, Staudinger Weg 5, 55128, Mainz, Germany.
Pharm Res. 2015 Jun;32(6):1957-74. doi: 10.1007/s11095-014-1589-7. Epub 2014 Dec 9.
Release of siRNA from nanoscale polyplexes is a crucial yet little investigated process, important during all stages of therapeutic research. Here we develop new methods to characterize polyplex stability early on in the development of new materials.
We used double fluorescent labeled siRNA to compare binding and stability of a panel of chemically highly diverse nanoscale polyplexes, including peptides, lipids, nanohydrogels, poly-L-lysine brushes, HPMA block copolymers and manganese oxide particles. Conventional EMSA and heparin competition methods were contrasted with a newly developed microscale thermophoresis (MST) assay, a near-equilibrium method that allows free choice of buffer conditions. Integrity of FRET-labeled siRNA was monitored in the presence of nucleases, in cell culture medium and inside living cells. This approach characterizes all relevant steps from polyplex stability, over uptake to in vitro knockdown capability.
Diverging polyplex binding properties revealed drawbacks of conventional EMSA and heparin competition assays, where MST and FRET-based siRNA integrity measurements offered a better discrimination of differential binding strength. Since cell culture medium left siRNA in all polyplexes essentially intact, the relevant degradation events could be pinpointed to occur inside cells. Differential binding strength of the variegated polyplexes correlated only partially with intracellular degradation. The most successful compounds in RNAi showed intermediate binding strength in our assays.
We introduce new methods for the efficient and informative characterization of siRNA polyplexes with special attention to stability. Comparing FRET-labeled siRNA in different polyplexes associates successful knockdown with intermediate siRNA stability in various steps from formulation to intracellular persistence.
从纳米级多聚体中释放小干扰RNA（siRNA）是一个关键但却鲜少被研究的过程，在治疗研究的各个阶段都很重要。在此，我们开发了新方法，以便在新型材料研发的早期阶段对多聚体稳定性进行表征。
我们使用双荧光标记的siRNA来比较一系列化学性质高度多样的纳米级多聚体的结合和稳定性，这些多聚体包括肽、脂质、纳米水凝胶、聚-L-赖氨酸刷、聚甲基丙烯酸羟乙酯嵌段共聚物和氧化锰颗粒。将传统的电泳迁移率变动分析（EMSA）和肝素竞争法与新开发的微量热泳动（MST）分析进行对比，MST分析是一种近平衡方法，可自由选择缓冲条件。在核酸酶存在的情况下、在细胞培养基中以及在活细胞内监测荧光共振能量转移（FRET）标记的siRNA的完整性。该方法可表征从多聚体稳定性、摄取到体外敲低能力的所有相关步骤。
不同的多聚体结合特性揭示了传统EMSA和肝素竞争分析的缺点，而MST和基于FRET的siRNA完整性测量能更好地区分不同的结合强度。由于细胞培养基使所有多聚体中的siRNA基本保持完整，因此可以确定相关的降解事件发生在细胞内。各种多聚体的不同结合强度仅部分与细胞内降解相关。在RNA干扰中最成功的化合物在我们的分析中显示出中等结合强度。
我们引入了新方法，用于高效且有信息价值地表征siRNA多聚体，特别关注稳定性。比较不同多聚体中FRET标记的siRNA发现，从制剂到细胞内持久性的各个步骤中，成功的敲低与中等的siRNA稳定性相关。
