Lipid and cationic polymer based transduction of botulinum holotoxin, or toxin protease alone, extends the target cell range and improves the efficiency of intoxication

Abstract

Botulinum neurotoxin (BoNT) heavy chain (Hc) facilitates receptor-mediated endocytosis into neuronal cells and transport of the light chain (Lc) protease to the cytosol where neurotransmission is inhibited as a result of SNARE protein cleavage. Here we show that the role of BoNT Hc in cell intoxication can be replaced by commercial lipid-based and polycationic polymer DNA transfection reagents. BoNT "transduction" by these reagents permits efficient intoxication of neuronal cells as well as some non-neuronal cell lines normally refractory to BoNT. Surprisingly, the reagents facilitate delivery of recombinant BoNT Lc protease to the cytosol of both neuronal and non-neuronal cells in the absence of BoNT Hc, and with sensitivities approaching that of BoNT holotoxin. Transduction of BoNT, as with natural intoxication, is inhibited by bafilomycin A1, methylamine and ammonium chloride indicating that both pathways require endosome acidification. DNA transfection reagents facilitate intoxication by holotoxins, or isolated Lc proteases, of all three BoNT serotypes tested (A, B, E). These results suggest that lipid and cationic polymer transfection reagents facilitate cytosolic delivery of BoNT holotoxins and isolated Lc proteases by an endosomal uptake pathway.

A novel method for culturing stellate astrocytes reveals spatially distinct Ca2+ signaling and vesicle recycling in astrocytic processes

“…11668027; Invitrogen) was prepared in 50 µl medium in one tube, and 0.7 µg plasmid DNA was prepared in 50 µl medium in a second tube and incubated at room temperature. For tetanus toxin (TeNT) experiments, 800 nM purified TeNT light chain peptides were also introduced to cells by transfection, which had previously been successful (Kuo et al, 2010); TeNT peptides were a gift from Y. Park and R. Jahn (Max Planck Institute for Biophysical Chemistry, Göttingen, Germany).…”

Synergistic Antitumor Activity from Two-Stage Delivery of Targeted Toxins and Endosome-Disrupting Nanoparticles

“…9−12 A few molecules of such toxins in the cytosol are sufficient to kill a cell, 5,10,11 and this high lethality has made these molecules candidate anticancer therapeutics. However, by themselves, type I toxins such as gelonin lacking any cell-binding or cytoplasmic delivery domains are limited by their inability to cross the plasma membrane at therapeutically useful levels.…”

“…12,13,16−19 Many chaperone molecules that efficiently aid transport of macromolecules into the cytosol are formulated with drug cargos by physical complexation or chemical conjugation of the chaperone and drug. In the case of gelonin, a variety of cytosolic delivery strategies have been tested including conjugation to folate, antibodies, peptides, proteins, or polymers, as well as entrapment in liposomes or polymers designed to deliver the toxin to the cytosol of cancer cells.…”

Basic Tetrapeptides as Potent Intracellular Inhibitors of Type A Botulinum Neurotoxin Protease Activity

“…A comparison of the results at the high and low inhibitor concentrations does not indicate significant differences. In contrast, phosphorylated MAPK p38 increased after 60 min of incubation with phorbol myristate acetate (26), indicating that the assay detected activation of p38 (Fig. 6, ϩ control).…”

“…Primary Rat and Mouse Cerebellar Neurons-Neuronal cerebellar granule cells were isolated from 7-8-day-old rat or mice (CD-1) as previously described (26). Cells were suspended in DMEM/F-12 medium (Invitrogen) supplemented with 50 units/ml penicillin, 50 g/ml of streptomycin, 10% FBS, N2 supplement, and 25 mM KCl.…”