Development of a delivery vehicle for intracellular transport of botulinum neurotoxin antagonists 1

FEBS Letters

Tepp, William H¹  Neale, Elaine A⁴  Adler, Michael³  Keller, James E⁴  Fishman, Paul S²  Hartz, Stephanie³  Johnson, Eric A¹  Clark, Mike³  Oyler, George²  Goodnough, Michael C¹ 

[1] Department of Food Microbiology, Food Research Institute, University of Wisconsin, 1925 Willow Dr., Madison, WI 53706, USA;Department of Neurology, University of Maryland School of Medicine, Veteran's Affairs Medical Center, Baltimore, MD 21201, USA;Neurotoxicology Branch, US Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD 21010, USA;Laboratory of Developmental Neurobiology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA

关键词: Botulinum neurotoxin;    Targeted drug delivery;    Laser confocal microscopy;    Fluorescent probe;    DV;    delivery vehicle;    BoNT;    botulinum neurotoxin;    HC;    heavy chain;    LC;    light chain;    PBS;    phosphate-buffered saline;    PDPH;    3-(2-pyridylthio)-propionyl hydrazide;    SEC;    size-exclusion chromatography;    EDTA;    ethylenediaminetetraacetic acid;    SDS–PAGE;    sodium dodecyl sulfate–polyacrylamide gel electrophoresis;   

DOI  :  10.1016/S0014-5793(02)02268-8

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown