PEGylation of Reduced Graphene Oxide Induces Toxicity in Cells of the Blood–Brain Barrier: Anin Vitroandin VivoStudy

Abstract

Polyethylene glycol (PEG) coating has been frequently used to improve the pharmacokinetic behavior of nanoparticles. Studies that contribute to better unravel the effects of PEGylation on the toxicity of nanoparticle formulation are therefore highly relevant. In the present study, reduced graphene oxide (rGO) was functionalized with PEG, and its effects on key components of the blood-brain barrier, such as astrocytes and endothelial cells, were analyzed in culture and in an in vivo rat model. The in vitro studies demonstrated concentration-dependent toxicity. The highest concentration (100 μg/mL) of non-PEGylated rGO had a lower toxic influence on cell viability in primary cultures of astrocytes and rat brain endothelial cells, while PEGylated rGO induced deleterious effects and cell death. We assessed hippocampal BBB integrity in vivo by evaluating astrocyte activation and the expression of the endothelial tight and adherens junctions proteins. From 1 h to 7 days post-rGO-PEG systemic injection, a notable and progressive down-regulation of protein markers of astrocytes (GFAP, connexin-43), the endothelial tight (occludin), and adherens (β-catenin) junctions and basal lamina (laminin) were observed. The formation of intracellular reactive oxygen species demonstrated by increases in the enzymatic antioxidant system in the PEGylated rGO samples was indicative of oxidative stress-mediated damage. Under the experimental conditions and design of the present study the PEGylation of rGO did not improve interaction with components of the blood-brain barrier. In contrast, the attachment of PEG to rGO induced deleterious effects in comparison with the effects caused by non-PEGylated rGO.

Graphene oxide and reduced graphene oxide induced neural pheochromocytoma-derived PC12 cell lines apoptosis and cell cycle alterations via the ERK signaling pathways

“…12 These nanomaterials may exert differing degrees of toxicity in animals or cell models depending on the administration route or penetration through physiological barriers such as the BBB. 11,13,14 However, toxicological information on GO materials remains limited. In this study, we examined the neurotoxic effects of graphene family nanomaterials using the PC12 cell model.…”

Cytotoxicity profile of pristine graphene on brain microvascular endothelial cells

“…In addition, reduced GO conjugated with PEG but not unconjugated GO induced a significant decrease in viability at 100 μg/mL in primary isolated rBMVECs. This decrease in viability was related to a decrease in the expression of claudin‐5 and OC (Mendonca et al, ). In the present study, we also observed an increase in BBB permeability and decrease in viability at 50 and 100 μg/mL as well as a decrease in the expression of OC at the highest concentration tested, suggesting that even when the type of graphene used in all three studies was different, all of them still induced deleterious effects to the BBB.…”

“…Administration of reduced GO conjugated with polyethylene glycol (PEG) also induced a decrease in the expression in OC in vivo. In addition, reduced GO conjugated with PEG, but not reduced GO alone decreased the viability of rat (r)BMVECs in culture, and decreased the expression of claudin-5 and OC (Mendonca et al, 2016), suggesting that reduced GO induced a disruption of the BBB.…”

“…The strong interaction of these proteins confers the BBB its low permeability, and alterations of these proteins may lead to an increased permeability and subsequently, brain damage (Ebnet, 2008;Forster, 2008;Hawkins & Davis, 2005). Only a couple of studies have analyzed the effects of graphene on the function of the BBB (Mendonca et al, 2015;Mendonca et al, 2016). Intravenous administration of reduced GO at 7 mg/kg to Wistar rats induced an increase in BBB permeability and a decrease in the expression of the TJ protein OC (Mendonca et al, 2015).…”

Clinical Applications of Carbon Nanomaterials in Diagnostics and Therapy

“…Few‐layer graphene‐induced DNA damage in primary human umbilical vein endothelial cells . Reduced graphene oxide have been shown to have significant degree of toxicity on rat brain endothelial cells although the toxicity could be dampened with PEGylation . Graphite, multiwalled carbon nanotubes, and fullerenes inhibit vascular endothelial growth factor—and fibroblast growth factor–induced angiogenesis may further aggravate the potent effects of exposing the endothelium to these nanomaterials .…”