Purpose: Traumatic spinal cord injury (SCI) leads to neuronal damage and results in varying levels of functional impairment. Chondroitin sulfate proteoglycan (CSPG), an inhibitory molecule is upregulated following SCI and may contribute to unsuccessful axon regeneration. Previous studies have shown that CSPGs activate Rho GTPase and that inhibition of Rho kinase (ROCK) activity with Y-27632 reverses or reduces CSPG inhibition. Targeting therapeutics specifically to corticospinal motor neurons (CSMNs) that are damaged during SCI is challenging. Nanomaterials have the potential to function as drug delivery systems (nanocarriers) to deliver therapeutics specifically to the injured neurons. We have constructed a thermoresponsive, polymer encapsulated magnetic nanocarrier (PE-MNC) system that can deliver therapeutics specifically to damaged neurons. However, biocompatibility of PE-MNCs and their drug delivery efficacy needs to be determined. We hypothesized that our PE-MNCs are biocompatible with motor neurons and deliver drugs to CSMNs at physiological temperatures.
Methods: To test this hypothesis, we measured several aspects of neurite outgrowth (initiation, elongation and arborization) from digital images, obtained using a Nikon A1 confocal microscope, of cultured CSMNs from P0 rat and B35 neurblastoma cells exposed to different concentrations of PE-MNCs for up to 96 hours. Then, we treated B35 cells and P0 rat CSMNs plated on patterned substrata of CSPG stripes by delivering Y-27632 using PE-MNCs at physiological temperature (37 0C). Following treatment, neurite outgrowth of B35 cells and CSMNs on inhibitory substrate is quantified and compared with that of cells treated with Y-27632 alone.
Results: Thermo-responsive PE-MNCs have a diameter of 230 nm at room temperature and 180 nm at physiological temperature. Up to 150 µg/mL of PE-MNC treatment did not negatively affect the morphology and neurite outgrowth in B35 cells or CSMNs (after 96 hours of exposure). In accordance with this, labeling for cytoskeletal components (microtubules or actin filaments) did not indicate any significant differences between B35 cells or CSMNs not exposed to PE-MNCs and those exposed to any concentration of PE-MNCs for any length of time tested. Also, we could find significant neurite outgrowth of CSMNs over inhibitory substrata following treatment using Y-27632 delivered by PE-MNCs.
Conclusion: Together, these results provide evidence that of PE-MNCs are feasible for targeting drugs to encourage axon regeneration in CSMNs, cells likely to be damaged in SCI.