Endo‐lysosomal Escape of Cholera Toxin B‐Modified Protocells in Motoneurons

Mesoporous silica nanoparticles coated with a lipid bilayer (protocells) are a stable, versatile and biocompatible delivery platform that allows surface engineering to target specific cells and intracellular cargo delivery. The endo‐lysosomal escape of this type of drug carriers is important to enhance the therapeutic efficacy of their cargo. Motoneurons can be specifically targeted by surface‐coating protocells with the subunit B of cholera toxin (CTB‐protocells). The central hypothesis was that CTB‐protocells escape endo‐lysosomal compartments after internalization by motoneurons. In a first study, fixed NSC‐34 motoneuron‐like cells were treated with fluorescently labeled CTB‐protocells (for 1, 3, 6 and 24 hours), immunostained for lysosomes (with anti‐LAMP1) or early endosomes (with anti‐EEA1) and imaged using multicolor confocal microscopy. In a second study, NSC‐34 cells treated with CTB‐protocells and labeled with the acidic vesicle tracker LysoTracker were visualized using time lapse confocal imaging. In these two studies, colocalization of CTB‐protocells with lysosomes or endosomes was analyzed using ImageJ and found to remain less than 20% up to 24 hours. The fraction of CTB‐protocells in endo‐lysosomes increased only slightly between 1 and 24 hours. In a third study, transmission electron microscopy was used to confirm the intracellular localization of CTB‐protocells in NSC‐34 motoneuron‐like cells. CTB‐protocells were visualized in the cytosol and were not detected within larger vacuole compartments after 24‐hour incubation. In conclusion, CTB‐protocells escape lysosomal degradation in motoneurons making them a suitable vehicle to target drug delivery to motoneurons.