Nerve cell-mimicking liposomes as biosensor for botulinum neurotoxin complete physiological activity

- A cell-free in vitro system was used to measure BoNT/B physiological function.
- The system relies on nerve-cell mimicking liposomes as a novel detection system.
- A FRET-based reporter assay is used as final readout of the test system.
- BoNT/B physiological activity was detected at picogram quantities in short time.
- The method opens the possibility to replace animal experimentation in BoNT testing.

Botulinum neurotoxins (BoNT) are the most toxic substances known, and their neurotoxic properties and paralysing effects are exploited for medical treatment of a wide spectrum of disorders. To accurately quantify the potency of a pharmaceutical BoNT preparation, its physiological key activities (binding to membrane receptor, translocation, and proteolytic degradation of SNARE proteins) need to be determined. To date, this was only possible using animal models, or, to a limited extent, cell-based assays. We here report a novel in vitro system for BoNT/B analysis, based on nerve-cell mimicking liposomes presenting motoneuronal membrane receptors required for BoNT binding. Following triggered membrane translocation of the toxin's Light Chain, the endopeptidase activity can be quantitatively monitored employing a FRET-based reporter assay within the functionalized liposomes. We were able to detect BoNT/B physiological activity at picomolar concentrations in short time, opening the possibility for future replacement of animal experimentation in pharmaceutical BoNT testing.