New Techniques to Assess In Vitro Release of siRNA from Nanoscale Polyplexes
- Publikationstyp:
- Zeitschriftenaufsatz
- Metadaten:
-
- Autoren
- 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
- Autoren-URL
- https://www.webofscience.com/api/gateway?GWVersion=2&SrcApp=fis-test-1&SrcAuth=WosAPI&KeyUT=WOS:000354098600009&DestLinkType=FullRecord&DestApp=WOS_CPL
- DOI
- 10.1007/s11095-014-1589-7
- eISSN
- 1573-904X
- Externe Identifier
- Clarivate Analytics Document Solution ID: CH5TQ
- PubMed Identifier: 25488263
- ISSN
- 0724-8741
- Ausgabe der Veröffentlichung
- 6
- Zeitschrift
- PHARMACEUTICAL RESEARCH
- Schlüsselwörter
- FRET
- nuclease resistance
- release
- siRNA
- thermophoresis
- Paginierung
- 1957 - 1974
- Datum der Veröffentlichung
- 2015
- Status
- Published
- Titel
- New Techniques to Assess In Vitro Release of siRNA from Nanoscale Polyplexes
- Sub types
- Article
- Ausgabe der Zeitschrift
- 32
Datenquelle: Web of Science (Lite)
- Andere Metadatenquellen:
-
- Autoren
- 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
- DOI
- 10.1007/s11095-014-1589-7
- eISSN
- 1573-904X
- ISSN
- 0724-8741
- Ausgabe der Veröffentlichung
- 6
- Zeitschrift
- Pharmaceutical Research
- Sprache
- en
- Online publication date
- 2014
- Paginierung
- 1957 - 1974
- Datum der Veröffentlichung
- 2015
- Status
- Published
- Herausgeber
- Springer Science and Business Media LLC
- Herausgeber URL
- http://dx.doi.org/10.1007/s11095-014-1589-7
- Datum der Datenerfassung
- 2020
- Titel
- New Techniques to Assess In Vitro Release of siRNA from Nanoscale Polyplexes
- Ausgabe der Zeitschrift
- 32
Datenquelle: Crossref
- Abstract
- <h4>Purpose</h4>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.<h4>Methods</h4>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.<h4>Results</h4>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.<h4>Conclusions</h4>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.
- Addresses
- Institute of Pharmacy and Biochemistry, Johannes Gutenberg-University Mainz, Staudinger Weg 5, 55128, Mainz, Germany.
- Autoren
- 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
- DOI
- 10.1007/s11095-014-1589-7
- eISSN
- 1573-904X
- Externe Identifier
- PubMed Identifier: 25488263
- Open access
- false
- ISSN
- 0724-8741
- Ausgabe der Veröffentlichung
- 6
- Zeitschrift
- Pharmaceutical research
- Schlüsselwörter
- Hela Cells
- Humans
- Green Fluorescent Proteins
- RNA, Small Interfering
- Electrophoretic Mobility Shift Assay
- Fluorescence Resonance Energy Transfer
- Transfection
- Gene Expression Regulation
- RNA Interference
- RNA Stability
- Biological Transport
- Kinetics
- Genes, Reporter
- Nanotechnology
- Nanoparticles
- Gene Knockdown Techniques
- Sprache
- eng
- Medium
- Print-Electronic
- Online publication date
- 2014
- Paginierung
- 1957 - 1974
- Datum der Veröffentlichung
- 2015
- Status
- Published
- Datum der Datenerfassung
- 2014
- Titel
- New Techniques to Assess In Vitro Release of siRNA from Nanoscale Polyplexes.
- Sub types
- Research Support, Non-U.S. Gov't
- Journal Article
- Ausgabe der Zeitschrift
- 32
Datenquelle: Europe PubMed Central
- 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.
- Date of acceptance
- 2014
- Autoren
- 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
- Autoren-URL
- https://www.ncbi.nlm.nih.gov/pubmed/25488263
- DOI
- 10.1007/s11095-014-1589-7
- eISSN
- 1573-904X
- Ausgabe der Veröffentlichung
- 6
- Zeitschrift
- Pharm Res
- Schlüsselwörter
- Biological Transport
- Electrophoretic Mobility Shift Assay
- Fluorescence Resonance Energy Transfer
- Gene Expression Regulation
- Gene Knockdown Techniques
- Genes, Reporter
- Green Fluorescent Proteins
- HeLa Cells
- Humans
- Kinetics
- Nanoparticles
- Nanotechnology
- RNA Interference
- RNA Stability
- RNA, Small Interfering
- Transfection
- Sprache
- eng
- Country
- United States
- Paginierung
- 1957 - 1974
- Datum der Veröffentlichung
- 2015
- Status
- Published
- Datum, an dem der Datensatz öffentlich gemacht wurde
- 2016
- Titel
- New Techniques to Assess In Vitro Release of siRNA from Nanoscale Polyplexes.
- Sub types
- Journal Article
- Research Support, Non-U.S. Gov't
- Ausgabe der Zeitschrift
- 32
Datenquelle: PubMed
- Beziehungen:
- Eigentum von