Targeted delivery and endosomal cellular uptake of DARPin-siRNA bioconjugates: Influence of linker stability on gene silencing.
- Lorenzer C Department of Pharmaceutical Chemistry, University of Vienna, Althanstraße 14, 1090 Vienna, Austria.
- Streußnig S Department of Pharmaceutical Chemistry, University of Vienna, Althanstraße 14, 1090 Vienna, Austria.
- Tot E Department of Pharmaceutical Chemistry, University of Vienna, Althanstraße 14, 1090 Vienna, Austria.
- Winkler AM Department of Pharmaceutical Chemistry, University of Vienna, Althanstraße 14, 1090 Vienna, Austria.
- Merten H Department of Biochemistry, University of Zurich, Winterthurerstraße 190, 8057 Zurich, Switzerland.
- Brandl F Department of Biochemistry, University of Zurich, Winterthurerstraße 190, 8057 Zurich, Switzerland.
- Sayers EJ Cardiff School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Cardiff, Wales CF10 3NB, United Kingdom.
- Watson P School of Biosciences, Cardiff University, Cardiff, Wales CF10 3AX, United Kingdom.
- Jones AT Cardiff School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Cardiff, Wales CF10 3NB, United Kingdom.
- Zangemeister-Wittke U Department of Biochemistry, University of Zurich, Winterthurerstraße 190, 8057 Zurich, Switzerland; Institute of Pharmacology, University of Bern, Inselspital INO-F, 3010 Bern, Switzerland.
- Plückthun A Department of Biochemistry, University of Zurich, Winterthurerstraße 190, 8057 Zurich, Switzerland.
- Winkler J Department of Pharmaceutical Chemistry, University of Vienna, Althanstraße 14, 1090 Vienna, Austria; Department of Cardiology, Medical University of Vienna, Währinger Gürtel 18-20, 1090 Vienna, Austria. Electronic address: johannes.winkler@univie.ac.at.
- 2019-05-20
Published in:
- European journal of pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V. - 2019
Bioconjugation
DARPins
Endocytosis
Targeting
siRNA
Alanine
Cell Line, Tumor
Click Chemistry
Endosomes
Epithelial Cell Adhesion Molecule
Gene Silencing
HeLa Cells
Humans
MCF-7 Cells
Maleimides
Muscle Proteins
Nuclear Proteins
RNA, Small Interfering
Sulfhydryl Compounds
Triazoles
English
Specific cell targeting and efficient intracellular delivery are major hurdles for the widespread therapeutic use of nucleic acid technologies, particularly siRNA mediated gene silencing. To enable receptor-mediated cell-specific targeting, we designed a synthesis scheme that can be generically used to engineer Designed Ankyrin Repeat Protein (DARPin)-siRNA bioconjugates. Different linkers, including labile disulfide-, and more stable thiol-maleimide- and triazole- (click chemistry) tethers were employed. Crosslinkers were first attached to a 3'-terminal aminohexyl chain on the siRNA sense strands. On the protein side thiols of a C-terminal cysteine were used as anchoring sites for disulfide- and thiol-maleimide conjugate formations, while strain-promoted azido-alkyne cycloadditions were carried out at a metabolically introduced N-terminal azidohomoalanine. After establishing efficient purification methods, highly pure products were obtained. Bioconjugates of EpCAM-targeted DARPins with siRNA directed at the luciferase gene were evaluated for cell-specific binding, uptake and gene silencing. As shown by flow cytometry and fluorescence microscopy, all constructs retained the highly specific and high-affinity antigen recognition properties of the native DARPin. As expected, internalization was observed only in EpCAM-positive cell lines, and predominantly endolysosomal localization was detected. Disulfide linked conjugates showed lower serum stability against cleavage at the linker and thus lower internalization into endosomes compared to thiol-maleimide- and triazole-linked conjugates, yet induced more pronounced gene silencing. This indicates that the siRNA payload needs to be liberated from the protein in the endosome. Our data confirm the promise of DARPin-siRNA bioconjugates for tumor targeting, but also identified endosomal retention and limited cytosolic escape of the siRNA as the rate-limiting step for more efficient gene silencing.
- Language
-
- English
- Open access status
- green
- Identifiers
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- DOI 10.1016/j.ejpb.2019.05.015
- PMID 31103742
- Persistent URL
- https://sonar.ch/global/documents/278879
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