A novel approach for targeted delivery to motoneurons using cholera toxin-B modified protocells
Introduction
Targeted delivery systems to motoneurons are critical in developing effective and safe treatments for motoneuron diseases (e.g. amyotrophic lateral sclerosis), as well as in understanding causes of muscle denervation (e.g. spinal cord injury, spinal muscular atrophy or ageing muscle) (Boido and Vercelli, 2016, Comley et al., 2016, Dupuis and Loeffler, 2009, Hepple and Rice, 2015, Mantilla and Sieck, 2009). Despite efforts in the field, treatments remain hindered by lack of drug selectivity to neurons in the central nervous system (CNS), difficulty in targeted cellular delivery, poor penetration through biological membranes/barriers and insufficient stability (Misra et al., 2003). There is an advantage in targeting motoneurons over other CNS neurons in that they have peripherally located nerve terminals at neuromuscular junctions (NMJs). This characteristic makes motoneurons accessible to treatments that exploit retrograde neuronal transport. However, the transfer of promising molecules (e.g., trophic factors) into the desired sites of action with high efficiency and uncompromised activity, while minimizing adverse reactions caused by their off-target effects, remains challenging (Weishaupt et al., 2012).
Nanoparticles are novel drug delivery systems with exceptional therapeutic potential (Simonato et al., 2013) that can encapsulate a variety of compounds and deliver them to target cells or tissues often with favorable safety profiles. In particular, mesoporous silica nanoparticles (MSNPs) have unique properties that make them a suitable treatment vehicle to target motoneurons, including: (1) the ability to independently modify pore size and the surface chemistry to enhance cargo loading when compared to other common drug delivery systems (e.g., liposomes); and, (2) the possibility to engineer bio-functionality and bio-compatibility by modifying the MSNPs surface (Ashley et al., 2011, Tarn et al., 2013). MSNPs encapsulated within a supported lipid bilayer (so-called protocells) exhibit the combined beneficial features of MSNPs and liposomes with versatile cargo loading, controlled release and the possibility to introduce strategic targeting ligands in the supported lipid bilayer to enable cell specific delivery of molecular components (Ashley et al., 2011).
A number of natural toxins exist that target the nervous system and could be employed in a targeting strategy (Edupuganti et al., 2012b). Cholera toxin produced by the bacterium Vibrio cholerae has an atoxic subunit (CTB) formed by five identical B-subunit monomers each composed of 103 amino acids (Miller et al., 2004). CTB binds a cell-surface receptor, ganglioside GM1, present on neuronal membranes (Sheikh et al., 1999, Zhang et al., 1995), and is effectively transported retrogradely in neurons. Indeed, CTB has been extensively used as a reliable neuronal tracer (Dederen et al., 1994, Mantilla et al., 2009, Wan et al., 1982). We hypothesized that cargo loaded, CTB modified protocells (CTB-protocells) will target motoneurons and show axon terminal uptake at NMJs. In the present study, we demonstrate that CTB conjugated protocells using biotin-NeutrAvidin, predominantly target motoneurons in vitro compared to muscle cell controls. We also show that there is CTB-protocell uptake into nerve terminals at diaphragm muscle NMJs. In addition, we validate intracellular cargo delivery using a membrane impermeable molecule, demonstrating the efficacy of CTB-protocells as a vehicle for targeting and delivering cargo to motoneurons.
Section snippets
MSNP synthesis
Fluorescently labeled MSNPs with hexagonal prismatic shape composed of close packed 2.8 nm diameter cylindrical pores were synthesized via a solution-based surfactant-directed self-assembly method, as reported by Lin et al. (2005). Briefly, MSNPs were fluorescently modified by dissolving 1 mg of rhodamine B isothiocyanate (Sigma-Aldrich, St. Louis, MO) in 1 mL of N,N-dimethyl formamide (DMF; Sigma-Aldrich) followed by addition of 1 μL 3-aminopropyltriethoxysilane (APTES; Sigma-Aldrich). Next, 290 mg
Physicochemical characterization of protocells modified with CTB
Synthesized MSNP cores and protocells were characterized by dynamic light scattering, TEM, cryo-TEM and zeta potential. Hexagonal MSNPs cores were obtained of uniform size (∼110 nm) evident by a low polydispersity index (Table 1) and TEM imaging (Fig. 1A). Fusion of a lipid bilayer to silica cores was achieved by adding the highly lipophilic MSNP framework to liposomes in aqueous buffer (PBS), permitting spontaneous fusion largely driven by electrostatic interactions and van der Waals attractive
Discussion
The current study presents a novel drug delivery system to specifically target motoneurons, viz CTB-modified mesoporous silica-supported lipid bilayer nanoparticles (protocells). CTB-protocells demonstrate primarily uptake at cultured motoneurons (compared to muscle cells), effective intracellular delivery of a small molecule cargo, as well as uptake by presynaptic axon terminals at diaphragm NMJs in tissue preparations. The availability of effective vehicles to target motoneurons fills an
Conclusions
In summary, CTB-protocells provide a promising delivery vehicle for therapy in motoneuron diseases and neuromuscular disorders. The ability of CTB-protocells to enter motoneurons at the NMJ confers a great advantage over existing formulations. The demonstrated biocompatibility of CTB-protocells with motoneurons suggests that CTB-protocells constitute a viable targeted cell delivery system suitable for drugs and genes. Protocell based delivery systems permit interventions, e.g., in the treatment
Acknowledgments
This project was supported by internal funding from the Mayo Foundation. C.J.B. acknowledges the U.S. Department of Energy (DOE), Office of Basic Energy Sciences (BES), and the Division of Materials Sciences and Engineering for support of fundamental structure-property relationship studies. C.J.B also acknowledges the Air Force Office of Scientific Research grant FA 9550-1-14-066, the National Science Foundation Grant#1344298, and the University of California's Center for Environmental
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