New Techniques to Assess In Vitro Release of siRNA from Nanoscale Polyplexes
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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.
KEY WORDSFRET nuclease resistance release siRNA thermophoresis
50% binding ratio
Lowest m/m ratio at which complexation exceeds 95%
Confocal laser scanning microscopy
Cell penetrating peptide
Electrophoretic mobility shift assay
Fluorescence activated cell sorting
Fluorescence correlation spectroscopy
Förster resonance energy transfer
- MnO@SiO2 particle
Manganese oxide particle covered with silica
Red/green, ratio of acceptor emission to donor emission
Negative charges of heparin per negative charges of siRNA
ACKNOWLEDGMENTS AND DISCLOSURES
This work was supported by the DFG in the frame of the collaborative research center, featuring project grant A7 to M.H., A6 to M.S., B2 to K.K., A3 to W.T., A4 to R.Z. The cylindrical brush sample was synthesized by Dr. Mike Sahl, Institute for Physical Chemistry, University Mainz, which is gratefully acknowledged. Flow cytometry was kindly supported by the Cytometry Core Facility of the Institute of Molecular Biology (IMB), Mainz.
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