title: New Techniques to Assess In Vitro Release of siRNA from Nanoscale Polyplexes.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Institute of Pharmacy and Biochemistry, Johannes Gutenberg-University Mainz, Staudinger Weg 5, 55128, Mainz, Germany.
authors.forename: Bettina
authors.initials: B
authors.lastname: Krieg
authors.forename: Markus
authors.initials: M
authors.lastname: Hirsch
authors.forename: Erik
authors.initials: E
authors.lastname: Scholz
authors.forename: Lutz
authors.initials: L
authors.lastname: Nuhn
authors.forename: Ilja
authors.initials: I
authors.lastname: Tabujew
authors.forename: Heiko
authors.initials: H
authors.lastname: Bauer
authors.forename: Sandra
authors.initials: S
authors.lastname: Decker
authors.forename: Andriy
authors.initials: A
authors.lastname: Khobta
authors.forename: Manfred
authors.initials: M
authors.lastname: Schmidt
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Tremel
authors.forename: Rudolf
authors.initials: R
authors.lastname: Zentel
authors.forename: Kalina
authors.initials: K
authors.lastname: Peneva
authors.forename: Kaloian
authors.initials: K
authors.lastname: Koynov
authors.forename: A James
authors.initials: AJ
authors.lastname: Mason
authors.forename: Mark
authors.initials: M
authors.lastname: Helm
mesh_terms: D001692:Biological Transport; D024202:Electrophoretic Mobility Shift Assay; D031541:Fluorescence Resonance Energy Transfer; D005786:Gene Expression Regulation; D055785:Gene Knockdown Techniques; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D053758:Nanoparticles; D036103:Nanotechnology / Q000379:methods*; D034622:RNA Interference; D020871:RNA Stability; D034741:RNA, Small Interfering / Q000737:chemistry / Q000378:metabolism*; D014162:Transfection / Q000379:methods*
chemical_list: D034741:RNA, Small Interfering; D049452:Green Fluorescent Proteins