A targeted
drug carrier (TDC) is described for transferring functional
proteins or
peptides into motor nerve terminals, a pivotal locus for
therapeutics to treat neuromuscular disorders. It exploits the pronounced selectivity of
botulinum neurotoxin type B (BoNT/B) for interacting with acceptors on these
cholinergic nerve endings and becoming internalized. The gene encoding an innocuous BoNT/B
protease-inactive mutant (BoTIM) was fused to that for core
streptavidin, expressed in Escherichia coli and the purified
protein was conjugated to surface-biotinylated
liposomes. Such decorated
liposomes, loaded with
fluorescein as traceable cargo, acquired pronounced specificity for motor nerve terminals in isolated mouse hemidiaphragms and facilitated the intraneuronal transfer of the fluor, as revealed by confocal microscopy. Delivery of the
protease light chain of
botulinum neurotoxin type A (
BoNT/A) via this TDC accelerated the onset of neuromuscular
paralysis, indicative of improved translocation of this
enzyme into the presynaptic cytosol with subsequent proteolytic inactivation of synaptosomal-associated
protein of molecular mass 25 kDa (SNAP-25), an exocytotic soluble N-ethyl-
maleimide-sensitive factor attachment
protein receptor (SNARE) essential for
neurotransmitter release. BoTIM-coupled
liposomes, loaded with
peptide inhibitors of
proteases, yielded considerable attenuation of the neuroparalytic effects of
BoNT/A or BoNT/F as a result of their cytosolic transfer, the first in situ demonstration of the ability of designer
antiproteases to suppress the symptoms of
botulism ex vivo. Delivery of the
BoNT/A inhibitor by
liposomes targeted with the full-length BoTIM proved more effective than that mediated by its C-terminal neuroacceptor-binding domain. This demonstrated versatility of TDC for nonviral cargo transfer into
cholinergic nerve endings has unveiled its potential for direct delivery of functional targets into motor nerve endings.