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Toxins (Basel). 2014 May 22;6(5):1644-66. doi: 10.3390/toxins6051644.

Reporter assay for endo/lysosomal escape of toxin-based therapeutics.

Author information

1
Institute of Laboratory Medicine, Clinical Chemistry and Pathobiochemistry, Charité-Universitätsmedizin Berlin, Campus Virchow-Klinikum, Augustenburger Platz 1, Berlin D-13353, Germany. roger.gilabert-oriol@charite.de.
2
Institute of Laboratory Medicine, Clinical Chemistry and Pathobiochemistry, Charité-Universitätsmedizin Berlin, Campus Virchow-Klinikum, Augustenburger Platz 1, Berlin D-13353, Germany. mayank.thakur@charite.de.
3
Institute of Laboratory Medicine, Clinical Chemistry and Pathobiochemistry, Charité-Universitätsmedizin Berlin, Campus Virchow-Klinikum, Augustenburger Platz 1, Berlin D-13353, Germany. benedicta.von-mallinckrodt@charite.de.
4
Institute of Laboratory Medicine, Clinical Chemistry and Pathobiochemistry, Charité-Universitätsmedizin Berlin, Campus Virchow-Klinikum, Augustenburger Platz 1, Berlin D-13353, Germany. cheenu.bhargava@charite.de.
5
Leibnizinstitut für Molekulare Pharmakologie (FMP), Berlin D-13125, Germany. wiesner@fmp-berlin.de.
6
Leibnizinstitut für Molekulare Pharmakologie (FMP), Berlin D-13125, Germany. eichhorst@fmp-berlin.de.
7
Institute of Pharmacy, Freie Universität Berlin, Königin-Luise-Straße 2 + 4, Berlin D-14195, Germany. melzig@zedat.fu-berlin.de.
8
Institute of Laboratory Medicine, Clinical Chemistry and Pathobiochemistry, Charité-Universitätsmedizin Berlin, Campus Virchow-Klinikum, Augustenburger Platz 1, Berlin D-13353, Germany. hendrik.fuchs@charite.de.
9
Institute of Laboratory Medicine, Clinical Chemistry and Pathobiochemistry, Charité-Universitätsmedizin Berlin, Campus Virchow-Klinikum, Augustenburger Platz 1, Berlin D-13353, Germany. a.weng@ucl.ac.uk.

Abstract

Protein-based therapeutics with cytosolic targets are capable of exhibiting their therapeutic effect once they have escaped from the endosomes or lysosomes. In this study, the reporters-horseradish peroxidase (HRP), Alexa Fluor 488 (Alexa) and ricin A-chain (RTA)-were investigated for their capacity to monitor the endo/lysosomal escape of the ribosome-inactivating protein, saporin. The conjugates-saporin-HRP, (Alexa)saporin and saporin-KQ-RTA-were constructed, and the endo/lysosomal escape of these conjugates alone (lack of endo/lysosomal release) or in combination with certain structurally-specific triterpenoidal saponins (efficient endo/lysosomal escape) was characterized. HRP failed in reporting the endo/lysosomal escape of saporin. Contrastingly, Alexa Fluor 488 successfully allowed the report of the process at a toxin concentration of 1000 nM. In addition, single endo/lysosome analysis facilitated the determination of the amount of (Alexa)saporin released from each vesicle. RTA was also successful in reporting the endo/lysosomal escape of the enzymatically inactive mutant, saporin-KQ, but in this case, the sensitivity of the method reached a toxin concentration of 10 nM. In conclusion, the simultaneous usage of Alexa Fluor 488 and RTA as reporters may provide the possibility of monitoring the endo/lysosomal escape of protein-based therapeutics in the concentration range of 10-1000 nM.

PMID:
24859158
PMCID:
PMC4052257
DOI:
10.3390/toxins6051644
[Indexed for MEDLINE]
Free PMC Article

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