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.

Graphene Oxide as 2D Platform for Complexation and Intracellular Delivery of siRNA

“…Bare GO complexes siRNA. Endotoxin-free GO sheets were synthesized in house following a modified Hummers method [13][14] (see Experimental). A summary of the physicochemical characterization of the GO dispersion in water, reported elsewhere 15 , is provided in Table S1.…”

“…Graphene oxide (GO). GO was produced in house following the modified Hummer's method as previously described [13][14] . Full characterization of the material has been provided in a previous study 15 where it was termed small GO (s-GO) in order to differentiate it from larger counterparts that have not been used in the present study, and is summarized in Table S1.…”

“…In this study, we aimed to investigate whether bare GO could interact with a short, double-stranded nucleic acid of biological relevance, siRNA, and act as its efficient intracellular delivery vector without further functionalization. To address this, we used endotoxin-free, small GO flakes (mean lateral size < 1µm) obtained synthetically by a modified Hummer's method [13][14] and assessed complexation at different GO:siRNA mass ratios, both experimentally and via molecular dynamics (MD) simulation. We interrogated the intracellular fate of both components, carrier and cargo, in a primary mouse cell culture system, as well as the capacity of the oligonucleotide to desorb from the GO lattice and induce gene silencing.…”

Endotoxin‐Free Preparation of Graphene Oxide and Graphene‐Based Materials for Biological Applications

“…The main contamination sources include air, water, glassware and plastic used in the synthesis, and contact with skin (Mukherjee et al 2016). Precursors, intermediates and products may be exposed to these sources of contamination at various steps of the synthesis.…”

Exposure to graphene oxide sheets alters the expression of reference genes used for real-time RT-qPCR normalization

“…This range was selected based on previous studies from our laboratory that confirmed lack of toxicity in a number of cell lines (data not shown). Full characterization of the material, produced in house by a modified Hummer's method as previously described (27,28), has been reported in a previous publication (29) and the most relevant parameters are summarized in Table S1. In brief, lateral dimensions did not surpass the 2 μ m threshold and thickness corresponded to 1-2 single GO layers.…”