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. 2016 Nov 1:273:160-174.
doi: 10.1016/j.jneumeth.2016.09.003. Epub 2016 Sep 15.

A novel approach for targeted delivery to motoneurons using cholera toxin-B modified protocells

Maria A Gonzalez Porras  1 , Paul N Durfee  2 , Ashley M Gregory  1 , Gary C Sieck  3 , C Jeffrey Brinker  4 , Carlos B Mantilla  5
Affiliations

A novel approach for targeted delivery to motoneurons using cholera toxin-B modified protocells

Maria A Gonzalez Porras et al. J Neurosci Methods. .

Abstract

Background: Trophic interactions between muscle fibers and motoneurons at the neuromuscular junction (NMJ) play a critical role in determining motor function throughout development, ageing, injury, or disease. Treatment of neuromuscular disorders is hindered by the inability to selectively target motoneurons with pharmacological and genetic interventions.

New method: We describe a novel delivery system to motoneurons using mesoporous silica nanoparticles encapsulated within a lipid bilayer (protocells) and modified with the atoxic subunit B of the cholera toxin (CTB) that binds to gangliosides present on neuronal membranes.

Results: CTB modified protocells showed significantly greater motoneuron uptake compared to unmodified protocells after 24h of treatment (60% vs. 15%, respectively). CTB-protocells showed specific uptake by motoneurons compared to muscle cells and demonstrated cargo release of a surrogate drug. Protocells showed a lack of cytotoxicity and unimpaired cellular proliferation. In isolated diaphragm muscle-phrenic nerve preparations, preferential axon terminal uptake of CTB-modified protocells was observed compared to uptake in surrounding muscle tissue. A larger proportion of axon terminals displayed uptake following treatment with CTB-protocells compared to unmodified protocells (40% vs. 6%, respectively).

Comparison with existing method(s): Current motoneuron targeting strategies lack the functionality to load and deliver multiple cargos. CTB-protocells capitalizes on the advantages of liposomes and mesoporous silica nanoparticles allowing a large loading capacity and cargo release. The ability of CTB-protocells to target motoneurons at the NMJ confers a great advantage over existing methods.

Conclusions: CTB-protocells constitute a viable targeted motoneuron delivery system for drugs and genes facilitating various therapies for neuromuscular diseases.

Keywords: Cholera toxin B; Diaphragm; Drug delivery system; Mesoporous silica nanoparticles; Motoneurons; Nanoparticles; Nanotechnology; Neuromuscular junction; Phrenic nerve.

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Figures

Fig. 1
Fig. 1
(A) Representative transmission electron microscopy (TEM) image of hexagonal anisotropic MSNPs and (B) CryoTEM image of protocells. Scale bar 100 nm.
Fig. 2
Fig. 2
Effect of incubation time, concentration and CTB modification on protocell uptake. Protocell fluorescence per cell was measured after different incubation times and protocell treatment concentrations. Significant CTB-protocell fluorescence was observed with 50 μg/ml after 3 h of incubation. No further increase was observed with longer periods of incubation. (*, p<0.001 compared to control cells). After a 24 h incubation with CTB modified and unmodified protocells (50 μg/ml), increased uptake of CTB-protocells was evident compared to unmodified protocells, which showed minimal evidence of uptake (†, p = 0.008). Mean ± SE values were obtained from two separate experiments carried out in triplicate.
Fig. 3
Fig. 3
Confocal microscopy images of cultured NSC-34 motoneuron-like cells demonstrating intracellular uptake of protocells. Uptake of (A) unmodified protocells and (B) CTB-protocells in differentiated NSC-34 cells stained with beta III tubulin (light blue); bar 10 μm. (C) Quantitative analysis of intracellular volume occupied by protocells (50 μg/ml; 24 h incubation) in cultured NSC-34 cells was significantly greater in CTB-protocells (*, p = 0.008). Mean ± SE values were obtained from 2 different experiments, 5 images per experiment and ~30 cells per image for a total of ~300 NSC-34 cells analyzed per group. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 4
Fig. 4
TEM images of cultured NSC-34 motoneuron-like cells. (A) Internalization of CTB-protocells in NSC-34 motoneuron-like cells after 24h incubation. (B) CTB-protocells were observed in the cell periphery as well as in the cytoplasm. Arrows highlight representative membrane-bound and internalized CTB-protocells. Asterisk highlights an intracellular vacuole (note presence of membrane). (C) Higher magnification image showing internalized particles (arrow) outside vacuoles compartments (asterisk).
Fig. 5
Fig. 5
Selective uptake of CTB in NSC-34 motoneuron-like cells and L6 muscle cells. Single plane confocal images of (A) NSC-34 motoneuron-like cells and (B) L6 muscle cells treated with 15 μg/ml of Alexa 488 cholera toxin subunit B (CTB) show co-localization of CTB (green) to tubulin-labeled (cyan) NSC-34 cells but not in laminin-labeled (orange) L6 cells; bar 20 μm. (C) The percentage of cells displaying CTB uptake was significantly higher in NSC-34 motoneuron-like cells compared to L6 muscle cells (*, p < 0.001). (D) Increasing CTB concentration in NSC-34 motoneuron-like cells did not increase uptake further. Mean ± SE values were obtained from 2 different experiments, ~600 NSC-34 cells and ~1000 L6 cells counted per group. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 6
Fig. 6
Selective uptake of CTB-protocells in NSC-34 motoneuron-like cells and L6 muscle cells. Single plane confocal images of (A) cultured NSC-34 cells and (B) cultured L6 muscle cells incubated with CTB modified and unmodified protocells (both rhodamine labeled); bar 50 μm. Arrows indicate intracellular uptake of CTB-protocells or protocells. Staining of beta III tubulin identifies differentiated motoneurons while laminin staining identifies muscle cells. (C) After 24 h of incubation with protocells, the percentage of NSC-34 cells displaying protocell uptake increased for CTB-protocells compared to unmodified protocells. L6 muscle cells displayed low levels of protocell uptake, and there was no difference with CTB coating (*, p < 0.001 compared to untreated control (CTL) in the same cell type; +, p < 0.001 compared to protocells in NSC-34 cells). Mean ± SE values were obtained from three separate experiments carried out in duplicate.
Fig. 7
Fig. 7
YO-PRO-1 cargo release in NSC-34 motoneuron-like cells. Single plane confocal images of cultured NSC-34 cells treated for 24 h with (A) 50 μg/ml of YO-PRO-1 loaded CTB-protocells and (B) 50 μg/ml of YO-PRO-1 loaded unmodified protocells; bar 10 μm. Note presence of CTB-protocells (red) and YO-PRO-1 (green) in cultured NSC-34 cells (arrow), which are co-stained with DAPI to show nuclei. (C) Intracellular YO-PRO-1 was quantified by the amount of intracellular YO-PRO-1 fluorescence. Mean ±SE values were obtained from three separate experiments (*, p = 0.034 compared to CTL and protocells). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 8
Fig. 8
Lack of CTB-protocell induced toxicity in NSC-34 motoneuron-like cells. (A) Fraction of dead cells at different CTB-protocell concentrations and incubationtimes. Dead cells were identified by dead-cell protease activity using a luminogenic peptide substrate. (B) Total cell luminescence determined using dead cell protease activity luminescence after addition of a lysis reagent (*, p < 0.001 compared to control cells). Note the lack of differences in dead cell fraction with CTB-protocell treatment and unimpaired cell proliferation evidenced by the similar or greater number of total cells following CTB-protocell treatment. Mean values ± SE were obtained from two separate experiments carried out in triplicate.
Fig. 9
Fig. 9
CTB-protocell uptake by axon terminals at diaphragm neuromuscular junctions (NMJs). Representative (A) maximum intensity projection confocal micrographs of diaphragm muscle-phrenic nerve preparations treated with CTB-protocells, labeled with synaptophysin (axon terminal). (B) 3D reconstruction showing varying depth (grayscale, in μm) of pre-synaptic structures, CTB-protocells and presynaptic colocalization. (C) Protocell fluorescence intensity at axon terminals after 24 h incubation with CTB-protocells, unmodified protocells and no treatment (CTL). CTB-protocells had significantly greater uptake at the axon terminals compared to modified protocells (*, p < 0.001 compared to protocells and CTL). (D) Proportion of axon terminals displaying protocell uptake at diaphragm muscle-phrenic nerve preparations (n=3 per group; ~40 NMJs per preparation). CTB-protocell uptake was determined as present at an NMJ after thresholding based on the maximum fluorescence in untreated CTL preparations.
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