Graphene Oxide Nanoribbons Induce Autophagic Vacuoles in Neuroblastoma Cell Lines

Abstract

Since graphene nanoparticles are attracting increasing interest in relation to medical applications, it is important to understand their potential effects on humans. In the present study, we prepared graphene oxide (GO) nanoribbons by oxidative unzipping of single-wall carbon nanotubes (SWCNTs) and analyzed their toxicity in two human neuroblastoma cell lines. Neuroblastoma is the most common solid neoplasia in children. The hallmark of these tumors is the high number of different clinical variables, ranging from highly metastatic, rapid progression and resistance to therapy to spontaneous regression or change into benign ganglioneuromas. Patients with neuroblastoma are grouped into different risk groups that are characterized by different prognosis and different clinical behavior. Relapse and mortality in high risk patients is very high in spite of new advances in chemotherapy. Cell lines, obtained from neuroblastomas have different genotypic and phenotypic features. The cell lines SK-N-BE(2) and SH-SY5Y have different genetic mutations and tumorigenicity. Cells were exposed to low doses of GO for different times in order to investigate whether GO was a good vehicle for biological molecules delivering individualized therapy. Cytotoxicity in both cell lines was studied by measuring cellular oxidative stress (ROS), mitochondria membrane potential, expression of lysosomial proteins and cell growth. GO uptake and cytoplasmic distribution of particles were studied by Transmission Electron Microscopy (TEM) for up to 72 h. The results show that GO at low concentrations increased ROS production and induced autophagy in both neuroblastoma cell lines within a few hours of exposure, events that, however, are not followed by growth arrest or death. For this reason, we suggest that the GO nanoparticle can be used for therapeutic delivery to the brain tissue with minimal effects on healthy cells.

Quercetin-mediated synthesis of graphene oxide–silver nanoparticle nanocomposites: a suitable alternative nanotherapy for neuroblastoma

“…81 Consistent with our results, previous studies also reported that GO can enter the cells by interacting with the plasma membrane and probably temporarily disrupting the phospholipid bilayer or causing plasma membrane invagination in HepG2 cells and neuroblastoma cell lines, including SK-N-BE(2) and SH-SY5Y. 30,112 Mari et al 30 further demonstrated that GO nanoribbons at low concentrations and within 72 h of exposure induced autophagy in neuroblastoma cell lines. Evidence from the presence of cytoplasmic vacuoles, especially in treated SK-N-BE(2) cells, and the induction of the lysosomal proteins LC3 and beclin, which are markers of autophagy, also confirmed that the autophagy is mainly due to ROS induced by GO.…”

“…65,77 For instance, mitochondrial depolarization is associated with mitochondrial dysfunction and the overproduction of ROS. 68,78 Recently, Mari et al 30 reported that GO nanoribbons induced toxicity in human neuroblastoma cancer cell lines such as SK-N-BE(2) and SH-SY5Y. Altogether, the combination of GO and AgNPs in a single platform such as GO-AgNPs could modulate the regulation of mitochondria and increase the generation of ROS.…”

“…30 In contrast, one study has shown that low concentrations of GO could support embryonic stem cell (ESC) growth, while graphene inhibited the differentiation of ESCs to endoderm, GO promoted their differentiation to endoderm. 62 In addition, GO induced neuronal differentiation of neuroblastoma SH-SY5Y cells.…”

“…63,113 Altogether, the data from previous studies and our studies showed that in neuroblastoma SH-SY5Y cells, GO-AgNPs potentially induce initial oxidative stress and mitochondrial damage to which cells respond by activating the autophagy process in order to remove damaged proteins and mitochondria. 30 Collectively, our studies suggested that GO-AgNPs cause stronger toxicity than that of GO, which is due to different physical and chemical properties of GO-AgNPs and the synergistic effect of GO and AgNPs in a single platform. Our studies clearly suggested that the accumulation of APs and autophagic or enlarged vacuoles may be due to the oxidative stress induced by GO-AgNPs.…”

“…Similarly, our studies have shown the toxicity of GO and biomolecule-functionalized reduced graphene in various types of cancer and non-cancer cells, including mouse embryonic fibroblast cells, 26 human breast cancer cells, 27 and human ovarian cancer cells. 28 GO induced cytotoxicity in human neuroblastoma SH-SY5Y cells at higher concentrations of 80 µg/mL at 96 h in a dose-and timedependent manner, 29 and GO nanoribbons induced toxicity by generation of reactive oxygen species (ROS), impairment of mitochondrial membrane potential (MMP), and formation of autophagosomes (APs), 30 which also depends on the size, charge, surface chemistry, and physical and chemical nature of the synthesized graphene. 27,28,31,32 On the other hand, the biocompatibility was analyzed using a variety of chemically and biologically functionalized graphene in several cell lines.…”