Search

A targeted delivery vehicle (DV) was developed for intracellular transport of emerging botulinum neurotoxin (BoNT) antagonists. The DV consisted of the isolated heavy chain (HC) of BoNT/A coupled to a 10-kDa amino dextran via the heterobifunctional linker 3-(2-pyridylthio)-propionyl hydrazide. The HC served to target BoNT-sensitive cells and promote internalization of the complex, while the dextran served as a platform to deliver model therapeutic molecules to the targeted cells. To determine the ability of this chimeric glycoprotein to enter neurons, dextran and HC were labeled independently with the fluorescent dyes Oregon green 488 and Cy3, respectively. Internalization of DV was monitored in primary cortical cells using laser confocal microscopy. Incubation of cells for 24 h with DV resulted in discrete punctate labeling of both soma and processes. The Cy3 and Oregon green 488 signals were generally co-localized, suggesting that the complex remained in the same intracellular compartment during the initial 24 h. The DV-associated fluorescence was reduced progressively by co-application of increasing concentrations of unlabeled BoNT/A holotoxin. The results suggest that the BoNT/A HC is able to mediate internalization of a coupled dextran, even though the latter bears no resemblance to the BoNT/A light chain (LC). The HC of BoNT/A thus offers promise as a selective carrier to deliver BoNT antagonists to the nerve terminal cytoplasm for inhibiting the proteolytic activity of internalized BoNT/A LC.

Primary dissociated fetal mouse spinal cord cultures were used to study the mechanisms underlying the differences in persistence of botulinum neurotoxin A (BoNT/A) and botulinum neurotoxin/E (BoNT/E) activities. Spinal cord cultures were exposed to BoNT/A (0.4 pM) for 2-3 days, which converted approximately half of the SNAP-25 to an altered form lacking the final nine C-terminal residues. The distribution of toxin-damaged to control SNAP-25 remained relatively unchanged for up to 80 days thereafter. Application of a high concentration of BoNT/E (250 pM) either 25 or 60 days following initial intoxication with BoNT/A converted both normal and BoNT/A-truncated SNAP-25 into a single population lacking the final 26 C-terminal residues. Excess BoNT/E was removed by washout, and recovery of intact SNAP-25 was monitored by Western blot analysis. The BoNT/E-truncated species gradually diminished during the ensuing 18 days, accompanied by the reappearance of both normal and BoNT/A-truncated SNAP-25. Return of BoNT/A-truncated SNAP-25 was observed in spite of the absence of BoNT/A in the culture medium during all but the first 3 days of exposure. These results indicate that proteolytic activity associated with the BoNT/A light chain persists inside cells for > 11 weeks, while recovery from BoNT/E is complete in < 3 weeks. This longer duration of enzymatic activity appears to account for the persistence of serotype A action.