DETOX: An innovative dendritic nanosystem for the delivery of BoNT/A LC domain to ischemic neurons

Ischemic stroke is a leading cause of mortality and morbidity worldwide. Nevertheless, current treatments are very ineffective as they only restore the blood flow, lacking neuroprotective properties. Excitotoxicity, primarily mediated by the overaccumulation of glutamate in the synaptic space, plays a key role in infarct expansion. Therefore, we propose a novel neuroprotective strategy to limit excessive glutamate release using the light-chain (LC) of Botulinum neurotoxin type A (BoNT/A), known to block neurotransmitters exocytosis, transported and delivered with proprietary fully biodegradable PEG-dendritic block copolymers (fbB). Here, the synthesis of 'taggable' dendrimers at their PEG end was further explored, where a Cy5.5 molecule was tagged through this point to allow for the tracking of nanoparticles in complex biological systems. A highly pure and catalytically active LC protein was produced and successfully complexed with the fbB dendrimers, forming DETOX nanoparticles. These systems, with nanosizes and mildly positive net charges, achieved up to 84 % LC-loading efficiency. DETOX significantly enhanced the cleavage of SNAP-25 - the LC's target - in neurons compared to BoNT/A and demonstrated a faster onset of action than the full-length neurotoxin, without inducing cytotoxicity. Importantly, DETOX capacity to reduce glutamate exocytosis was demonstrated in an in vitro model of ischemia. Moreover, DETOX nanoparticles showed their remarkable ability to permeate a 3D full-human blood-brain barrier model, indicating their promising application in humans. These findings position DETOX nanosystems as a groundbreaking neuroprotective strategy, offering a novel, clinically relevant approach for mitigating excitotoxicity in ischemic stroke, paving the way for advanced CNS therapeutics.