Elsevier

Journal of Neuroscience Methods

Volume 273, 1 November 2016, Pages 160-174
Journal of Neuroscience Methods

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

https://doi.org/10.1016/j.jneumeth.2016.09.003Get rights and content

Highlights

  • Cholera toxin-B (CTB) modified protocells provide a novel delivery method to target motoneurons.

  • CTB-protocells display uptake by presynaptic axon terminals at neuromuscular junctions.

  • CTB-protocells showed greater motoneuron uptake compared to unmodified protocells.

  • CTB-protocells constitute a promising delivery vehicle for therapy in motoneuron diseases.

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 24 h 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.

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

References (83)

  • O. Maier et al.

    Differentiated NSC-34 motoneuron-like cells as experimental model for cholinergic neurodegeneration

    Neurochem. Int.

    (2013)
  • O. Maier et al.

    Differentiated NSC-34 motoneuron-like cells as experimental model for cholinergic neurodegeneration

    Neurochem. Int.

    (2013)
  • C.B. Mantilla et al.

    Neuromuscular adaptations to respiratory muscle inactivity

    Respir. Physiol. Neurobiol.

    (2009)
  • C.B. Mantilla et al.

    Retrograde labeling of phrenic motoneurons by intrapleural injection

    J. Neurosci. Methods

    (2009)
  • A. Matsumoto et al.

    Ganglioside characterization of a cell line displaying motor neuron-like phenotype: GM2 as a possible major ganglioside in motor neurons

    J. Neurol. Sci.

    (1995)
  • C.E. Miller et al.

    Cholera toxin assault on lipid monolayers containing ganglioside GM1

    Biophys. J.

    (2004)
  • L. Nobs et al.

    Current methods for attaching targeting ligands to liposomes and nanoparticles

    J. Pharm. Sci.

    (2004)
  • L. Nobs et al.

    Poly(lactic acid) nanoparticles labeled with biologically active Neutravidin (TM) for active targeting

    Eur. J. Pharm. Biopharm.

    (2004)
  • L. Nobs et al.

    Poly(lactic acid) nanoparticles labeled with biologically active Neutravidin for active targeting

    Eur. J. Pharm. Biopharm.

    (2004)
  • R.W. Oppenheim

    Neurotrophic survival molecules for motoneurons: an embarrassment of riches

    Neuron

    (1996)
  • B. Ruozi et al.

    Neurotrophic factors and neurodegenerative diseases: a delivery issue

    Int. Rev. Neurobiol.

    (2012)
  • D.C. Sieck et al.

    Structure-activity relationships in rodent diaphragm muscle fibers vs. neuromuscular junctions

    Respir. Physiol. Neurobiol.

    (2012)
  • T. Soykan et al.

    Modes and mechanisms of synaptic vesicle recycling

    Curr. Opin. Neurobiol.

    (2016)
  • S.A. Townsend et al.

    Tetanus toxin C fragment-conjugated nanoparticles for targeted drug delivery to neurons

    Biomaterials

    (2007)
  • M. von Zastrow et al.

    Modulating neuromodulation by receptor membrane traffic in the endocytic pathway

    Neuron

    (2012)
  • X.C.S. Wan et al.

    Cholera-toxin and wheat-germ-agglutinin conjugates as neuroanatomical probes—their uptake and clearance, transganglionic and retrograde transport and sensitivity

    Brain Res.

    (1982)
  • N. Weishaupt et al.

    BDNF: the career of a multifaceted neurotrophin in spinal cord injury

    Exp. Neurol.

    (2012)
  • R.G. Zhang et al.

    The 2.4 angstrom crystal-structure of cholera-toxin-B subunit pentamer−choleragenoid

    J. Mol. Biol.

    (1995)
  • S. Alvarez-Argote et al.

    The impact of midcervical contusion injury on diaphragm muscle function

    J. Neurotrauma

    (2016)
  • C.E. Ashley et al.

    The targeted delivery of multicomponent cargos to cancer cells by nanoporous particle-supported lipid bilayers

    Nat. Mater.

    (2011)
  • C.E. Ashley et al.

    Delivery of small interfering RNA by peptide-targeted mesoporous silica nanoparticle-supported lipid bilayers

    ACS Nano

    (2012)
  • M. Boido et al.

    Neuromuscular junctions as key contributors and therapeutic targets in spinal muscular atrophy

    Front. Neuroanat.

    (2016)
  • K.S. Butler et al.

    Protocells modular mesoporous silica nanoparticle-supported lipid bilayers for drug delivery

    Small

    (2016)
  • N.R. Cashman et al.

    Neuroblastoma x spinal cord (NSC) hybrid cell lines resemble developing motor neurons

    Dev. Dyn.

    (1992)
  • L.H. Comley et al.

    Cross-disease comparison of amyotrophic lateral sclerosis and spinal muscular atrophy reveals conservation of selective vulnerability but differential neuromuscular junction pathology

    J. Comp. Neurol.

    (2016)
  • P.J. Dederen et al.

    Retrograde neuronal tracing with cholera toxin B subunit: comparison of three different visualization methods

    Histochem. J.

    (1994)
  • P.N. Durfee et al.

    Mesoporous silica nanoparticle-supported lipid bilayers (protocells) for active targeting and delivery to individual leukemia cells

    ACS Nano

    (2016)
  • O.P. Edupuganti et al.

    Targeted delivery into motor nerve terminals of inhibitors for SNARE-cleaving proteases via liposomes coupled to an atoxic botulinum neurotoxin

    FEBS J.

    (2012)
  • O.P. Edupuganti et al.

    Targeted delivery into motor nerve terminals of inhibitors for SNARE-cleaving proteases via liposomes coupled to an atoxic botulinum neurotoxin

    FEBS J.

    (2012)
  • C.J. Eggett et al.

    Development and characterisation of a glutamate-sensitive motor neurone cell line

    J. Neurochem.

    (2000)
  • M.L. Fanarraga et al.

    Expression of unphosphorylated class III beta-tubulin isotype in neuroepithelial cells demonstrates neuroblast commitment and differentiation

    Eur. J. Neurosci.

    (1999)
  • Cited by (25)

    • A potential delivery system based on cholera toxin: A macromolecule carrier with multiple activities

      2022, Journal of Controlled Release
      Citation Excerpt :

      The modified NPs resisted to proteolysis in fresh plasma and monocyte-macrophage system (MPS) clearance in vivo and showed glioma-targeting ability [63]. Other study also found that CTB conjugated to mesoporous silica nanoparticles (MSNP) obtained specific motoneuron uptake and inhibited lysosomal degradation [64,65]. The toxicity of CTA comes from ADP ribosylation caused by CTA1 and biological activity of the subsequent increase in cAMP.

    • Colloidal bag of marbles: The structure and properties of lipid-coated silica nanoclusters

      2021, Colloids and Surfaces A: Physicochemical and Engineering Aspects
      Citation Excerpt :

      Protocells prepared by the described procedures were tested in-vivo. These experiments showed that protocells have a potential in improving the cellular uptake and prolongation of circulation in the blood stream [17,18] or targeted drug delivery to motoneurons [19,20]. Other studies focused on interaction between silica nanoparticles and lipid membrane [21] or the thermoresponsive release from silica nanoparticles [22,23], where the potential of lipid coated nanoparticles for targeted drug delivery was successfully tested.

    • Nanotechnology in peripheral nerve repair and reconstruction

      2019, Advanced Drug Delivery Reviews
      Citation Excerpt :

      Also, they were well tolerated within the cochlea, with a tissue response that was localized only at the site of implantation in the cochlear base. Gonzales Porras et al., [202] described a novel delivery system to motor neurons using mesoporous SiO2-NPs encapsulated within a lipid bilayer and modified with the non-toxic subunit B of the cholera toxin (CTB). This subunit was described to binds to gangliosides present on neuronal membranes.

    View all citing articles on Scopus
    View full text