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, Alexasaporin 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 Alexasaporin 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. View Full-Tex