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Detection of Toxin Translocation into the Host Cytosol by Surface Plasmon Resonance
Published on: January 3, 2012
Reporter assay for endo/lysosomal escape of toxin-based therapeutics
Roger Gilabert-Oriol1, Mayank Thakur2, Benedicta von Mallinckrodt3
1Institute 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.
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.
Insights
Researchers evaluated reporter molecules like Alexa Fluor 488 and ricin A-chain (RTA) to track the endosomal escape of protein therapeutics. Alexa Fluor 488 and RTA effectively monitored saporin release, enabling therapeutic monitoring within a 10-1000 nM range.
Area of Science:
- Biochemistry
- Molecular Biology
- Drug Delivery
Background:
- Protein therapeutics targeting the cytosol require endosomal escape for efficacy.
- Monitoring this escape is crucial for developing effective drug delivery systems.
- Saporin, a ribosome-inactivating protein, serves as a model therapeutic.
Purpose of the Study:
- To investigate the utility of horseradish peroxidase (HRP), Alexa Fluor 488, and ricin A-chain (RTA) as reporters for endo/lysosomal escape.
- To characterize the endo/lysosomal escape of saporin conjugates using these reporters.
- To determine the sensitivity and applicability of each reporter system.
Main Methods:
- Construction of saporin conjugates: saporin-HRP, (Alexa)saporin, and saporin-KQ-RTA.
- Characterization of endo/lysosomal escape with and without saponin-mediated enhancement.
- Utilizing single endo/lysosome analysis for quantitative measurements.
Main Results:
- Horseradish peroxidase (HRP) was ineffective in reporting saporin's endo/lysosomal escape.
- Alexa Fluor 488 successfully reported escape at 1000 nM, enabling vesicle-specific release quantification.
- Ricin A-chain (RTA) reported escape for saporin-KQ at 10 nM, demonstrating high sensitivity.
Conclusions:
- Alexa Fluor 488 and RTA are effective reporters for monitoring endo/lysosomal escape of protein therapeutics.
- The combined use of Alexa Fluor 488 and RTA allows monitoring across a broad concentration range (10-1000 nM).
- This dual-reporter system enhances the study of drug release mechanisms for cytosolic delivery.
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