Graphene Oxide and Reduced Derivatives, as Powder or Film Scaffolds, Differentially Promote Dopaminergic Neuron Differentiation and Survival.

Noela Rodriguez-Losada,M Carmen Ocaña,Jose A Narvaez,Miguel A Arraez,Rune Wendelbob,Roberto Guzman De Villoría,Ernest Arenas,Pedro Gonzalez-Alegre,Miguel A Medina,Amelia Diaz Casares,Jose A Aguirre Gomez

doi:10.3389/fnins.2020.570409

Abstract

Emerging scaffold structures made of carbon nanomaterials, such as graphene oxide (GO) have shown efficient bioconjugation with common biomolecules. Previous studies described that GO promotes the differentiation of neural stem cells and may be useful for neural regeneration. In this study, we examined the capacity of GO, full reduced (FRGO), and partially reduced (PRGO) powder and film to support survival, proliferation, differentiation, maturation, and bioenergetic function of a dopaminergic (DA) cell line derived from the mouse substantia nigra (SN4741). Our results show that the morphology of the film and the species of graphene (GO, PRGO, or FRGO) influences the behavior and function of these neurons. In general, we found better biocompatibility of the film species than that of the powder. Analysis of cell viability and cytotoxicity showed good cell survival, a lack of cell death in all GO forms and its derivatives, a decreased proliferation, and increased differentiation over time. Neuronal maturation of SN4741 in all GO forms, and its derivatives were assessed by increased protein levels of tyrosine hydroxylase (TH), dopamine transporter (DAT), the glutamate inward rectifying potassium channel 2 (GIRK2), and of synaptic proteins, such as synaptobrevin and synaptophysin. Notably, PRGO-film increased the levels of Tuj1 and the expression of transcription factors specific for midbrain DA neurons, such as Pitx3, Lmx1a, and Lmx1b. Bioenergetics and mitochondrial dysfunction were evaluated by measuring oxygen consumption modified by distinct GO species and were different between powder and film for the same GO species. Our results indicate that PRGO-film was the best GO species at maintaining mitochondrial function compared to control. Finally, different GO forms, and particularly PRGO-film was also found to prevent the loss of DA cells and the decrease of the α-synuclein (α-syn) in a molecular environment where oxidative stress has been induced to model Parkinson's disease. In conclusion, PRGO-film is the most efficient graphene species at promoting DA differentiation and preventing DA cell loss, thus becoming a suitable scaffold to test new drugs or develop constructs for Parkinson's disease cell replacement therapy.

Highlights

Over the last decade, it has become increasingly apparent that it is essential to provide cells with appropriate physical substrates to allow regeneration
The temperature of the fully reduced GO (FRGO)-film is significantly higher than that of the partially reduced GO (PRGO)-film (1,100 and 300◦C, respectively), carbon particles or other impurities might have been formed during the reduction process
We found that while control ells SN4741 cells retain a fibroblast-like morphology (Figure 5A), GO derivatives (GOd) treatments induced the formation of neurite-like processes (Figure 5B); and the cytoarchitecture of cells treated with graphene oxide (GO) powder changed dramatically (Figure 5C)

Summary

Introduction

It has become increasingly apparent that it is essential to provide cells with appropriate physical substrates to allow regeneration. Scaffolds aid in supporting cellular architecture and may regulate processes, such as cell polarization and differentiation, contributing to regeneration. Graphene has recently emerged as a reliable material to create scaffolds for the neural tissue (Fabbro et al, 2016) because of its biocompatibility and electroconductive and physicochemical properties. It is known that graphene’s electric conductivity improves the neuronal differentiation of neural stem cells (Park et al, 2011). In foam form, graphene has inherent mechanical properties and capability for adsorption of proteins and substances with low molecular weight (Yavari et al, 2011), which facilitates cellular interactions as well as cell differentiation and proliferation (Lim et al, 2011). Graphene has been shown to activate apoptosis in a glioblastoma cell line (U118), suggesting that it does not support tumor growth and that its use is safe (Jaworski et al, 2013)

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