Durfee, Paul N.

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.

BackgroundThe ability to isolate extracellular vesicles (EVs) such as exosomes or microparticles is an important method that is currently not standardized. While commercially available kits offer purification of EVs from biofluids, such purified EV samples will also contain non‐EV entities such as soluble protein and nucleic acids that could confound subsequent experimentation. Ideally, only EVs would be isolated and no soluble protein would be present in the final EV preparation.MethodsWe compared commercially available EV isolation kits with immunoaffinity purification techniques and evaluated our final EV preparations using atomic force microscopy (AFM) and nanoscale flow cytometry (NFC). AFM is the only modality capable of detecting distinguishing soluble protein from EVs which is important for downstream proteomics approaches. NFC is the only technique capable of quantitating the proportion of target EVs to non‐target EVs in the final EV preparation.ResultsTo determine enrichment of prostate derived EVs relative to non‐target MPs, anti‐PSMA (Prostate Specific Membrane Antigen) antibodies were used in NFC. Antibody‐based immunoaffinity purification generated the highest quality of prostate derived EV preparations due to the lack of protein and RNA present in the samples. All kits produced poor purity EV preparations that failed to deplete the sample of plasma protein.ConclusionsWhile attractive due to their ease of use, EV purification kits do not provide substantial improvements in isolation of EVs from biofluids such as plasma. Immunoaffinity approaches are more efficient and economical and will also eliminate a significant portion of plasma proteins which is necessary for downstream approaches.