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New Techniques to Assess In Vitro Release of siRNA from Nanoscale Polyplexes

Abstract

Purpose

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.

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Abbreviations

BR50 :

50% binding ratio

cc1 :

Lowest m/m ratio at which complexation exceeds 95%

CLSM:

Confocal laser scanning microscopy

CPP:

Cell penetrating peptide

DAB:

Diaminobutane-dendrimer-(NH2)64

DOTAP:

1,2-dioleoyl-3-trimethylammonium-propane

EMSA:

Electrophoretic mobility shift assay

FACS:

Fluorescence activated cell sorting

FCS:

Fluorescence correlation spectroscopy

FRET:

Förster resonance energy transfer

HPMA:

N-(2-hydroxypropyl) methacrylamide

m/m:

MassParticle/masssiRNA ratio

MnO@SiO2 particle:

Manganese oxide particle covered with silica

MST:

Microscale thermophoresis

PAMAM:

Poly(amido amine)

pDMAEMA:

Poly(2-dimethylamino)ethyl methacrylate

PEI:

Polyethyleneimine

PLL:

Poly-L-lysine

PVA:

Polyvinylalcohol

R/G:

Red/green, ratio of acceptor emission to donor emission

Rh :

Hydrodynamic radius

z Hep/z RNA :

Negative charges of heparin per negative charges of siRNA

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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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Correspondence to Mark Helm.

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Bettina Krieg and Markus Hirsch have an equal contribution to this paper.

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Krieg, B., Hirsch, M., Scholz, E. et al. New Techniques to Assess In Vitro Release of siRNA from Nanoscale Polyplexes. Pharm Res 32, 1957–1974 (2015). https://doi.org/10.1007/s11095-014-1589-7

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  • DOI: https://doi.org/10.1007/s11095-014-1589-7

KEY WORDS

  • FRET
  • nuclease resistance
  • release
  • siRNA
  • thermophoresis