Premium
Analysis of the intracellular barriers encountered by nonviral gene carriers in a model of spatially controlled delivery to neurons
Author(s) -
Bergen Jamie M.,
Pun Suzie H.
Publication year - 2008
Publication title -
the journal of gene medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.689
H-Index - 91
eISSN - 1521-2254
pISSN - 1099-498X
DOI - 10.1002/jgm.1137
Subject(s) - gene delivery , transfection , internalization , neurite , intracellular , reporter gene , soma , microbiology and biotechnology , axoplasmic transport , neuron , biology , green fluorescent protein , axon , gene expression , neuroscience , cell culture , cell , gene , biochemistry , genetics , in vitro
Background Neuron‐specific, nonviral gene delivery vehicles are useful tools for the potential treatment of neurological disease and spinal cord injury. For minimally invasive, peripheral administration, gene carriers must efficiently mediate uptake at axon terminals, retrograde axonal transport, vesicular escape, and nuclear entry. The design of improved vehicles will benefit from an understanding of the barriers that limit nonviral delivery to neurons. Here, we demonstrate a detailed analysis of intracellular trafficking of both a lipid‐based and a polymer‐based delivery vehicle following site‐specific exposure to neuron‐like cells. Methods Site‐specific exposure of gene carriers to soma or neurites of neuron‐like PC‐12 cells was accomplished using a microfluidic, compartmented culture chamber. Binding and internalization of vehicles at neurites and soma were quantified using an environmentally sensitive fluorescent marker. The intracellular transport of gene carriers was analyzed by time‐lapse particle tracking in live cells, and transfection efficiencies were measured using green fluorescent protein (GFP) as a reporter gene. Results While the lipid‐based carrier mediated measurable transfection when delivered to neuronal soma, neuritic delivery of this formulation failed to produce reporter gene expression due to limited internalization and transport. In contrast, the polymeric nanoparticles displayed active retrograde transport toward neuronal soma, but failed to produce measurable reporter gene expression. Conclusions These results highlight distinct intracellular barriers preventing efficient neuronal transfection by the nonviral carriers examined, and provide a basis for the rational improvement of existing nonviral systems. Copyright © 2007 John Wiley & Sons, Ltd.