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Pharmaceutical Research

, Volume 32, Issue 6, pp 1957–1974 | Cite as

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

  • Bettina Krieg
  • Markus Hirsch
  • Erik Scholz
  • Lutz Nuhn
  • Ilja Tabujew
  • Heiko Bauer
  • Sandra Decker
  • Andriy Khobta
  • Manfred Schmidt
  • Wolfgang Tremel
  • Rudolf Zentel
  • Kalina Peneva
  • Kaloian Koynov
  • A. James Mason
  • Mark Helm
Research Paper

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.

KEY WORDS

FRET nuclease resistance release siRNA thermophoresis 

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

zHep/zRNA

Negative charges of heparin per negative charges of siRNA

Notes

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.

Supplementary material

11095_2014_1589_MOESM1_ESM.pdf (848 kb)
ESM 1 (PDF 848 kb)

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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Bettina Krieg
    • 1
  • Markus Hirsch
    • 1
  • Erik Scholz
    • 1
  • Lutz Nuhn
    • 2
  • Ilja Tabujew
    • 3
  • Heiko Bauer
    • 2
  • Sandra Decker
    • 2
  • Andriy Khobta
    • 1
  • Manfred Schmidt
    • 2
  • Wolfgang Tremel
    • 2
  • Rudolf Zentel
    • 2
  • Kalina Peneva
    • 3
  • Kaloian Koynov
    • 3
  • A. James Mason
    • 4
  • Mark Helm
    • 1
  1. 1.Institute of Pharmacy and BiochemistryJohannes Gutenberg-University MainzMainzGermany
  2. 2.Department of ChemistryJohannes Gutenberg-University MainzMainzGermany
  3. 3.Max Planck Institute for Polymer ResearchMainzGermany
  4. 4.Institute of Pharmaceutical ScienceKing’s College LondonLondonUK

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