title: PEGylation of Reduced Graphene Oxide Induces Toxicity in Cells of the Blood-Brain Barrier: An in Vitro and in Vivo Study.
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.
authors.affiliation: Department of Pharmacology, Faculty of Medical Sciences, State University of Campinas , Campinas, SP 13083-881, Brazil.|Department of Biochemistry and Tissue Biology, Institute of Biology, State University of Campinas , Campinas, SP 13083-863, Brazil.
authors.forename: Monique Culturato Padilha
authors.initials: MC
authors.lastname: Mendonça
authors.affiliation: Department of Biochemistry and Tissue Biology, Institute of Biology, State University of Campinas , Campinas, SP 13083-863, Brazil.
authors.forename: Edilene Siqueira
authors.initials: ES
authors.lastname: Soares
authors.affiliation: Department of Biochemistry and Tissue Biology, Institute of Biology, State University of Campinas , Campinas, SP 13083-863, Brazil.
authors.forename: Marcelo Bispo
authors.initials: MB
authors.lastname: de Jesus
authors.affiliation: Department of Semiconductors, Instruments and Photonics, Faculty of Electrical and Computer Engineering, State University of Campinas , Campinas, SP 13083-852, Brazil.
authors.forename: Helder José
authors.initials: HJ
authors.lastname: Ceragioli
authors.affiliation: Department of Food and Nutrition, School of Food Engineerig, State University of Campinas , Campinas, SP 13083-862, Brazil.
authors.forename: Ângela Giovana
authors.initials: ÂG
authors.lastname: Batista
authors.affiliation: Institute of Biophysics, Biological Research Centre, Hungarian Academy of Sciences , Szeged 1051, Hungary.
authors.forename: Ádám
authors.initials: Á
authors.lastname: Nyúl-Tóth
authors.affiliation: Institute of Biophysics, Biological Research Centre, Hungarian Academy of Sciences , Szeged 1051, Hungary.
authors.forename: Judit
authors.initials: J
authors.lastname: Molnár
authors.affiliation: Institute of Biophysics, Biological Research Centre, Hungarian Academy of Sciences , Szeged 1051, Hungary.
authors.forename: Imola
authors.initials: I
authors.lastname: Wilhelm
authors.affiliation: Department of Food and Nutrition, School of Food Engineerig, State University of Campinas , Campinas, SP 13083-862, Brazil.
authors.forename: Mário Roberto
authors.initials: MR
authors.lastname: Maróstica
authors.affiliation: Institute of Biophysics, Biological Research Centre, Hungarian Academy of Sciences , Szeged 1051, Hungary.|Vasile Goldis Western University , Arad, Romania.
authors.forename: István
authors.initials: I
authors.lastname: Krizbai
authors.affiliation: Department of Pharmacology, Faculty of Medical Sciences, State University of Campinas , Campinas, SP 13083-881, Brazil.|Department of Biochemistry and Tissue Biology, Institute of Biology, State University of Campinas , Campinas, SP 13083-863, Brazil.
authors.forename: Maria Alice
authors.initials: MA
authors.lastname: da Cruz-Höfling
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000166:cytology / Q000378:metabolism; D001812:Blood-Brain Barrier / Q000737:chemistry / Q000187:drug effects; D015153:Blotting, Western; D002490:Central Nervous System / Q000187:drug effects / Q000378:metabolism; D006108:Graphite / Q000737:chemistry* / Q000633:toxicity; D007150:Immunohistochemistry; D008297:Male; D049329:Nanostructures / Q000737:chemistry; D018384:Oxidative Stress / Q000502:physiology; D051381:Rats
chemical_list: D006108:Graphite
keywords: PEGylation; central nervous system; graphene-based nanomaterials; nanotoxicity