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Tracking siRNA–Nanocarrier Assembly and Disassembly Using FRET

Part of the Methods in Pharmacology and Toxicology book series (MIPT)

Abstract

The success of cellular nucleic acid delivery, such as siRNA, mRNA, or plasmid DNA strongly depends on the selected carrier system. Its attributes must guarantee the incorporation of the nucleic acid into nanosized particles, followed by efficient cellular uptake, resistance to the changing physicochemical environment during endolysosomal trafficking and finally the adequate release of its payload in the cytosol of the cell. While the outcome of the last step can easily be evaluated in case of siRNA delivery via knockdown of a reporter gene, the kinetics experienced by particles on their cellular journey remain mostly unknown.

Fluorescence/Förster resonance energy transfer (FRET) is a versatile tool in nanocarrier-based theranostics and encompasses the transfer of excitation energy from a donor molecule in an excited state to an acceptor molecule in close proximity. To understand the kinetics of cellular nucleic acid delivery, we use the multifunctional PEG-PCL-PEI (PPP) triblock copolymers in combination with a FRET-based nanocarrier reporting system consisting of hydrophobic Quantum Dots 605 (Qdots605) and Alexa Fluor 647 (AF647) labeled siRNA.

FRET based monitoring of both distinct entities allows us on the one hand to monitor the necessary compaction (=assembly of polymer vehicle and cargo) and decompaction (=disassembly and release of cargo) of the nanocarrier and on the other hand enables tracking the cellular trafficking of both molecules individually subsequent to the carrier–siRNA dissociation.

Key words

  • siRNA
  • Polyplexes
  • Gene delivery
  • FRET
  • Polyplex assembly/disassembly

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  • DOI: 10.1007/978-1-0716-1250-7_17
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Abbreviations

PCL:

poly(ɛ-caprolactone)

PEG:

Polyethylene glycol

PEI:

Polyethyleneimine

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Acknowledgments

This work was supported by the ERC Starting Grant ERC-2014-StG—637830 “Novel Asthma Therapy.”

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Correspondence to Olivia M. Merkel .

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Isert, L., Mehta, A., Adams, F., Merkel, O.M. (2021). Tracking siRNA–Nanocarrier Assembly and Disassembly Using FRET. In: Rosania, G.R., Thurber, G.M. (eds) Quantitative Analysis of Cellular Drug Transport, Disposition, and Delivery. Methods in Pharmacology and Toxicology. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1250-7_17

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  • DOI: https://doi.org/10.1007/978-1-0716-1250-7_17

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1249-1

  • Online ISBN: 978-1-0716-1250-7

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