title: Development of a delivery vehicle for intracellular transport of botulinum neurotoxin antagonists.
abstract: 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.
authors.affiliation: Department of Food Microbiology, Food Research Institute, University of Wisconsin, 1925 Willow Dr., Madison, WI 53706, USA.
authors.forename: Michael C
authors.initials: MC
authors.lastname: Goodnough
authors.forename: George
authors.initials: G
authors.lastname: Oyler
authors.forename: Paul S
authors.initials: PS
authors.lastname: Fishman
authors.forename: Eric A
authors.initials: EA
authors.lastname: Johnson
authors.forename: Elaine A
authors.initials: EA
authors.lastname: Neale
authors.forename: James E
authors.initials: JE
authors.lastname: Keller
authors.forename: William H
authors.initials: WH
authors.lastname: Tepp
authors.forename: Mike
authors.initials: M
authors.lastname: Clark
authors.forename: Stephanie
authors.initials: S
authors.lastname: Hartz
authors.forename: Michael
authors.initials: M
authors.lastname: Adler
mesh_terms: D000818:Animals; D000992:Antitoxins / Q000008:administration & dosage* / Q000494:pharmacology; D001667:Binding, Competitive; D001692:Biological Transport; D019274:Botulinum Toxins, Type A / Q000037:antagonists & inhibitors*; D002478:Cells, Cultured; D003014:Clostridium botulinum / Q000737:chemistry; D004396:Coloring Agents / Q000737:chemistry; D004337:Drug Carriers / Q000737:chemistry / Q000378:metabolism*; D016503:Drug Delivery Systems; D051379:Mice
chemical_list: D000992:Antitoxins; D004396:Coloring Agents; D004337:Drug Carriers; D019274:Botulinum Toxins, Type A