Brinker, Jeffrey

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.

Cholera toxin B modified protocells (CTB‐protocells) are a promising, customizable approach for targeting and delivering drugs to motoneurons. Understanding the endocytic mechanism and intracellular fate of CTB‐protocells is essential for safe therapeutic application and cargo delivery efficiency, and also to elucidate characteristics of motoneuron physiology that are not well understood. To assess the energy dependent uptake of CTB‐protocells by motoneurons, intracellular protocell fluorescence was measured at 4°C and 37°C. Confocal analysis demonstrated that after 3 h at 4°C, there is a 40% reduction in CTB‐protocell uptake compared to the uptake observed at 37°C (p<0.001). Pharmacological endocytic pathway inhibitors were used to elucidate the preferential endocytotic mechanism. Results showed that amiloride (macropinocytosis inhibitor) and methyl‐β‐cyclodextrin (inhibitor of cholesterol dependent endocytic processes) significantly reduced the uptake of CTB‐protocells (p<0.001), consistent with endocytosis being dependent on the integrity of lipid rafts. To track intracellular fate, immunofluorescence techniques and live confocal microscopy were used to demonstrate colocalization of CTB‐protocells with lysosomes, endoplasmic reticulum (ER) and Golgi apparatus at 1 h and 3 h. Colocalization of CTB‐protocells with lysosomes was less than 20%. Both, ER and Golgi colocalization increased from ~20 to ~40% in a 2‐h period (from 1 h to 3 h). To evaluate retrograde transport in motoneurons in vivo , CTB‐protocells were intrapleurally injected in rats. Evidence of protocells in phrenic nerve axons measured by protocell fluorescence intensity supported retrograde transport. In conclusion, uptake of CTB‐protocells is energy‐dependent and mediated principally by macropinocytosis. Additionally, CTB‐protocells are able to escape lysosomal degradation, and are trafficked to relevant subcellular compartments after endocytosis.

At present, there are limited delivery systems to target neurons in the central nervous system. Neurological diseases are commonly hindered by treatments lacking drug selectivity to the neurons of interest. In particular, motoneuron diseases will likely require selective targeting of therapeutic interventions, which can be delivered via the peripheral nervous system. Nanoparticles are novel drug delivery systems with exceptional therapeutic potential. Specifically, mesoporous silica nanoparticles coated with a lipid bilayer (protocells) are a suitable treatment vehicle to target the peripheral and central nervous system because they offer advantageous fusion characteristics including 1) increased surface area with larger loading capacity, 2) stability and 3) improved biocompatibility. A number of natural toxins exist that already target the nervous system and could be used as a targeting strategy. Cholera toxin produced by the bacterium Vibrio cholerae has an atoxic subunit (CTB) that binds to gangliosides present on neuronal membranes and is a reliable motoneuron tracer. We hypothesized that CTB‐coated protocells specifically target motoneurons. In culture, CTB uptake is evident in motoneuron‐like NSC‐34 cells but not in L6 muscle cells, consistent with the suitability of CTB as a motoneuron targeting strategy. Fluorescently labeled protocells (130 nm in diameter) were bound to CTB using biotin‐neutravidin. CTB‐protocells showed significantly greater motoneuron uptake compared to non‐CTB coated protocells when quantified by measurements of intracellular protocell fluorescence intensity. Confocal images were used for co‐localization of fluorescently labeled protocells and lysosomes. Protocells effectively escape endo‐lysosomal degradation delivering their cargo intracellularly. Biocompatibility was evidenced by lack of cytotoxicity in cultured motoneurons and unimpaired cellular proliferation. To evaluate the targeting efficacy of CTB‐protocells in tissue, we utilized isolated diaphragm muscle‐phrenic nerve preparations. Evidence of protocell uptake at diaphragm neuromuscular junctions measured by protocell fluorescence intensity supports uptake into phrenic motoneuron axon terminals. In conclusion, CTB‐protocells are a promising, customizable approach for targeting and delivering drugs to motoneurons in vivo.

Diseases of the central nervous system commonly affect motoneurons. In particular, spinal cord injury, aging and neurological disorders can cause motoneuron dysfunction or loss. There are no effective diagnostic tools or treatments that specifically target motoneurons. We hypothesized that nanoparticles can be used to target drug delivery to motoneurons. Specifically, mesoporous silica nanoparticles (MSNPs) may be ideal since they can be independently modified allowing loading of various cargos and surface‐engineered for bio‐functionality. The aim of this study was to characterize the role of nanoparticle size, concentration, time of incubation and surface coating on uptake by cultured motoneurons. Four different fluorescently‐labeled MSNPs were evaluated: PEG‐PEI‐Ace MSNPs of 150 nm and 50 nm, lipid coated MSNPs (protocell ‐ 140 nm) and PEG‐PEI MSNPs (50nm). PEG‐PEI‐Ace showed significant greater motoneuron uptake (p<0.05) after longer periods of incubation (12h for 50 nm & 24h for 150 nm). Protocells and PEG‐PEI achieved maximal uptake by 3h. Differentiated motoneurons displayed significant uptake at cell soma and dendrites. Regardless of cell density and duration of treatment, there was minimal evidence of cytotoxicity by any MSNP. In conclusion, MSNPs can be used to target delivery to motoneurons. Specifically, coating, size and concentration affect motoneuron uptake. Smaller and coated PEG‐PEI‐Ace MSNPs showed substantial uptake by motoneurons. These results may help inform the appropriate characteristics of MSNPs for specific in vivo application.