Neuronal protection against oxidative insult by polyanhydride nanoparticle-based mitochondria-targeted antioxidant therapy

Abstract

A progressive loss of neuronal structure and function is a signature of many neurodegenerative conditions including chronic traumatic encephalopathy, Parkinson's, Huntington's and Alzheimer's diseases. Mitochondrial dysfunction and oxidative and nitrative stress have been implicated as key pathological mechanisms underlying the neurodegenerative processes. However, current therapeutic approaches targeting oxidative damage are ineffective in preventing the progression of neurodegeneration. Mitochondria-targeted antioxidants were recently shown to alleviate oxidative damage. In this work, we investigated the delivery of biodegradable polyanhydride nanoparticles containing the mitochondria-targeted antioxidant apocynin to neuronal cells and the ability of the nano-formulation to protect cells against oxidative stress. The nano-formulated mitochondria-targeted apocynin provided excellent protection against oxidative stress-induced mitochondrial dysfunction and neuronal damage in a dopaminergic neuronal cell line, mouse primary cortical neurons, and a human mesencephalic cell line. Collectively, our results demonstrate that nano-formulated mitochondria-targeted apocynin may offer improved efficacy of mitochondria-targeted antioxidants to treat neurodegenerative disease.

Keywords: Mito-apocynin; Neurodegeneration; Oxidative stress; Polyanhydride nanoparticles.

Figure 1

Mito-Apo release kinetics from 20:80 CPH:SA nanoparticles. The amount of Mito-Apo released at each time point was normalized by the total amount of Mito-Apo encapsulated within the particles. The experiments were performed in triplicate and are reported as means ± SEM.

Figure 2

Nanoparticle interactions with N27 neuronal cells. Cells were treated with 20:80 CPH:SA particles for 24 hours. a) Cell viability was measured by using the MTS reagent. Data is expressed as percent viability compared to H₂O₂-treated controls. QD-loaded NPs were added separately to N27 cells for 24 hours. Internalization was measured by flow cytometry (b) and confocal microscopy (c). 40% of the cells internalized QD-loaded FA-NP as compared to only 25% of the cells that internalized QD-loaded NP.

Figure 3

Assessment of neuronal viability and nanoparticle internalization. Primary cortical cells were pre-treated for 24 hours with NP, FA-NP, and 2FA-NP. a) MTS assay was used to measure the mitochondrial conversion of the tetrazolium salt to a water-soluble formazan salt. Neurons treated with H₂O₂ for 1.5 hours were used as a control for oxidative stress-induced cell death. b) TEM photomicrographs show presence of QD-loaded NP and FA-NP in the cytosol of cortical cells. Scale bar: 100 nm.

Figure 4

Efficacy of Mito-Apo-encapsulated nano-formulations on primary cortical neurons. Neurons were pre-treated with the various nano-formulations for 24 hours and challenged with H₂O₂ for 1.5 hours. a) Cell viability was measured by MTS reagent. Data is expressed as percent viability compared to untreated controls. b) Neurons were stained for β-III tubulin (green) and cleaved caspase-3 (red). Cell death was quantified by the presence of cleaved caspase-3 and by the reduction in neurite length. Caspase-3-positive cells were quantified as shown in the graph.

Figure 5

Neuroprotective role of M:(FA-NP) against 6-OHDA-induced dopaminergic neuronal degeneration. a) LUHMES cells were treated with 6-OHDA and superoxide levels were measured as fluorescence intensity. b) Neuronal function was assessed by dopamine uptake. Cells were pre-treated with various formulations and exposed to 6-OHDA. Dopamine uptake was measured using a high affinity [³H] assay and expressed as mean values of counts. c) Cell death was quantified by the presence of cleaved caspase-3 in cells exposed to various treatments. Caspase-3-positive cells were quantified as shown in the graph.