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.
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Abbreviations
- PCL:
-
poly(ɛ-caprolactone)
- PEG:
-
Polyethylene glycol
- PEI:
-
Polyethyleneimine
References
Fang J, Nakamura H, Maeda H (2011) The EPR effect: unique features of tumor blood vessels for drug delivery, factors involved, and limitations and augmentation of the effect. Adv Drug Deliv Rev 63(3):136–151
Shrestha D et al (2015) Understanding FRET as a research tool for cellular studies. Int J Mol Sci 16(4):6718–6756
Endres TK et al (2011) Self-assembled biodegradable amphiphilic PEG–PCL–lPEI triblock copolymers at the borderline between micelles and nanoparticles designed for drug and gene delivery. Biomaterials 32(30):7721–7731
Endres T et al (2014) Amphiphilic biodegradable PEG-PCL-PEI triblock copolymers for FRET-capable in vitro and in vivo delivery of siRNA and quantum dots. Mol Pharm 11(4):1273–1281
Greco CT et al (2017) Anionic polymer and quantum dot excipients to facilitate siRNA release and self-reporting of disassembly in stimuli-responsive Nanocarrier formulations. Biomacromolecules 18(6):1814–1824
Liu Y et al (2009) A new synthesis method and degradation of hyper-branched polyethylenimine grafted polycaprolactone block mono-methoxyl poly (ethylene glycol) copolymers (hy-PEI-g-PCL-b-mPEG) as potential DNA delivery vectors. Polymer 50(16):3895–3904
Beck-Broichsitter M et al (2010) Preparation of nanoparticles by solvent displacement for drug delivery: a shift in the "ouzo region" upon drug loading. Eur J Pharm Sci 41(2):244–253
Behzadi S et al (2017) Cellular uptake of nanoparticles: journey inside the cell. Chem Soc Rev 46(14):4218–4244
Debus H et al (2010) Delivery of messenger RNA using poly(ethylene imine)–poly(ethylene glycol)-copolymer blends for polyplex formation: biophysical characterization and in vitro transfection properties. J Control Release 148(3):334–343
Mittal R, Bruchez MP (2009) Calibration of flow cytometry for quantitative quantum dot measurements. Curr Protoc Cytom 49(1):6.26.1–6.26.7
Acknowledgments
This work was supported by the ERC Starting Grant ERC-2014-StG—637830 “Novel Asthma Therapy.”
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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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