title: Detection of Endotoxin Contamination of Graphene Based Materials Using the TNF-α Expression Test and Guidelines for Endotoxin-Free Graphene Oxide Production.
abstract: Nanomaterials may be contaminated with bacterial endotoxin during production and handling, which may confound toxicological testing of these materials, not least when assessing for immunotoxicity. In the present study, we evaluated the conventional Limulus amebocyte lysate (LAL) assay for endotoxin detection in graphene based material (GBM) samples, including graphene oxide (GO) and few-layered graphene (FLG). Our results showed that some GO samples interfered with various formats of the LAL assay. To overcome this problem, we developed a TNF-α expression test (TET) using primary human monocyte-derived macrophages incubated in the presence or absence of the endotoxin inhibitor, polymyxin B sulfate, and found that this assay, performed with non-cytotoxic doses of the GBM samples, enabled unequivocal detection of endotoxin with a sensitivity that is comparable to the LAL assay. FLG also triggered TNF-α production in the presence of the LPS inhibitor, pointing to an intrinsic pro-inflammatory effect. Finally, we present guidelines for the preparation of endotoxin-free GO, validated by using the TET.
authors.affiliation: Nanosafety & Nanomedicine Laboratory, Division of Molecular Toxicology, Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Sourav P
authors.initials: SP
authors.lastname: Mukherjee
authors.affiliation: Nanomedicine Laboratory, Faculty of Medical & Human Sciences and National Graphene Institute, University of Manchester, Manchester, United Kingdom.
authors.forename: Neus
authors.initials: N
authors.lastname: Lozano
authors.affiliation: Particles-Biology Interactions Laboratory, Swiss Federal Laboratories for Materials Science and Technology, St. Gallen, Switzerland.
authors.forename: Melanie
authors.initials: M
authors.lastname: Kucki
authors.affiliation: Department of Organic Chemistry, University of Castilla-La Mancha, Ciudad Real, Spain.
authors.forename: Antonio E
authors.initials: AE
authors.lastname: Del Rio-Castillo
authors.affiliation: Nanomedicine Laboratory, Faculty of Medical & Human Sciences and National Graphene Institute, University of Manchester, Manchester, United Kingdom.
authors.forename: Leon
authors.initials: L
authors.lastname: Newman
authors.affiliation: Department of Organic Chemistry, University of Castilla-La Mancha, Ciudad Real, Spain.
authors.forename: Ester
authors.initials: E
authors.lastname: Vázquez
authors.affiliation: Nanomedicine Laboratory, Faculty of Medical & Human Sciences and National Graphene Institute, University of Manchester, Manchester, United Kingdom.
authors.forename: Kostas
authors.initials: K
authors.lastname: Kostarelos
authors.affiliation: Particles-Biology Interactions Laboratory, Swiss Federal Laboratories for Materials Science and Technology, St. Gallen, Switzerland.
authors.forename: Peter
authors.initials: P
authors.lastname: Wick
authors.affiliation: Nanosafety & Nanomedicine Laboratory, Division of Molecular Toxicology, Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Bengt
authors.initials: B
authors.lastname: Fadeel
mesh_terms: D001681:Biological Assay / Q000379:methods*; D002478:Cells, Cultured; D004731:Endotoxins / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D000091344:Filaggrin Proteins; D006108:Graphite / Q000737:chemistry*; D017408:Guidelines as Topic; D006801:Humans; D008033:Limulus Test; D008264:Macrophages / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D010087:Oxides / Q000737:chemistry; D011112:Polymyxin B / Q000737:chemistry / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis*
chemical_list: D004731:Endotoxins; C000717249:FLG protein, human; D000091344:Filaggrin Proteins; D010087:Oxides; D014409:Tumor Necrosis Factor-alpha; D006108:Graphite; D011112:Polymyxin B