Polyethylene glycol copolymer nanocarriers: Biocompatibility, uptake and intracellular trafficking in neurons
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Spinal cord injury (SCI) causes neuronal death and leads to persistent loss of motor and sensory functions. Treatment of SCI is challenging as axon regeneration from damaged neurons is largely inhibited in the central nervous system (CNS). Moreover, targeting therapeutics to damaged CNS neurons is difficult due to barriers, including the blood brain barrier and injury-induced inhibitors of regeneration. This prompts the exploration of new treatment strategies for SCI. Due to their unique properties, nanomaterial-based drug delivery systems (nanocarriers) are promising excipients for targeted drug delivery to neurons. We have developed four types of nanocarriers and performed experiments to further optimize their potential use in SCI treatment. One of them is a polymer (PEG copolymer) encapsulated magnetic nanocarrier (PE-MNC) and was tested for its biocompatibility in a neuron model (PC12 cells), as well as in chick dorsal root ganglion (DRG) sensory neurons, cells that are damaged during SCI. We have performed time- and dose-dependent studies showing that treatment of up to 150 µg/mL PE-MNC for 72 to 96 hours does not affect the morphology and neurite outgrowth in DRG sensory neurons and neuronal cell line, as assessed using immunocytochemistry and confocal microscopy. The other three nanomaterial systems are surface functionalized nanocarriers (SFNCs) made of fluorescently-labeled PEG copolymers without the magnetic core. One system is derivatized by covalently-attached amino groups, producing SFNCs of 150 nm diameter (N150). The other two systems are functionalized with covalently-attached carboxyl groups, producing SFNCs with diameters of 150 (C150) and 750 (C750) nm. We have used these SFNCs to study the effect of nanocarrier size and charge in their clathrin-mediated endocytosis (CME), intracellular trafficking and uptake efficiency in neurons and glia, assessed using immunocytochemistry, confocal microscopy and live cell imaging. We have observed that, irrespective of size and charge, a portion of all SFNCs are internalized by CME in neurons, where they follow endo-lysosomal trafficking in B35 cells, but not in PC12 cells. Moreover, the efficiency by which SFNCs are taken up into cortical neurons is higher than that of glia, significantly for the uptake of C750 system. We conclude that modifying PE-MNC with C750 and/or N150 properties provides a potential nanocarrier for drug delivery to SCI-damaged neurons in vivo.