Exfoliation in Endotoxin‐Free Albumin Generates Pristine Graphene with Reduced Inflammatory Properties

Abstract

mechanical strength. Within the GBMs, pristine graphene (pG) presents key advantages; its virtually defect-free surface enhances its mechanical properties and the absence of oxygen groups increases its electrical and optical conductivity compared to functionalized graphene, [7] presenting opportunities to develop new biomedical devices. Moreover, since no additional chemical modifications are required, the use of pG would have a positive environmental and economic impact over functionalized graphenes, such as graphene oxide (GO). Nevertheless, pG has been less studied in a biological context, due to its intrinsic hydrophobicity, restricting dispersibility and stability in aqueous solution, unless potentially toxic stabilizers are added. Thus, the use of biocompatible dispersants is crucial.We currently lack a complete understanding of the toxic and inflammatory effects of GBMs and conflicting results have been reported. While the toxicity of other graphene-based materials (e.g., GO, rGO), has been extensively studied, [1,[8][9][10] limited work has been done on pristine graphene. [11][12][13] Studies highlight key roles for factors such as lateral size (small vs large), [10] surface functionalization (e.g., GO vs pG), [12,14] purity [15] and hydrophobicity [16] on GBM-cell interaction, thus it is vital to address the intrinsic toxicological effects of pG and elucidate its immunomodulatory properties in order to assess its specific potential as a material for biomedical applications. According Graphene-based materials are under consideration as a new generation of biomaterials for medical applications, owning to their distinctive physicochemical properties. A major obstacle to clinical translation of novel biomaterials is the presence of residual endotoxin contaminants that can trigger deleterious inflammatory responses and unrelated immunomodulatory effects. To overcome this challenge, a liquid exfoliation methodology to prepare endotoxin-free bovine serum albumin (BSA)-exfoliated pristine graphene (EF-Gr) was developed and compared with conventional BSA-exfoliated graphene (C-Gr) and sodium cholate-exfoliated graphene (SC-Gr). In contrast to SC-Gr, protein-exfoliated graphene exhibits low toxicity. Moreover, BSAexfoliated EF-Gr does not trigger cytokine secretion or increase costimulatory molecule expression in dendritic cells (DCs) or macrophages; however it retains the ability to activate the NLRP3 inflammasome in DCs primed withToll-like receptor (TLR) agonists. The results suggest that some reported immunostimulatory effects of graphene preparations may have resulted from contaminants in the formulations. When injected into mice, the inflammatory response induced is comparable in magnitude to that induced by the safe and effective vaccine adjuvant alum. Overall, the study demonstrates the feasibility of producing endotoxin-free pristine graphene that facilitates assessment of the potential of pristine graphene for novel biomedical applications without the confounding effects of toxic and inflammatory conta...

Few Layer Graphene Does Not Affect Cellular Homeostasis of Mouse Macrophages

“…We found that FLG was internalized within the cells through passive diffusion or phagocytosis. As inflammasome activation is induced by a wide range of stimuli, such as microorganisms, metabolic dysregulation as well as by nanomaterials [34], phagocytosed FLG was predicted to evoke inflammasome activity, however, in this study we did not observe any significant increase in inflammasome-dependant IL-1β production. This was accompanied by no significant secretion of other pro-inflammatory cytokines such as IL-6 and TNF-α.…”

Macrophage innate training induced by IL-4 and IL-13 activation enhances OXPHOS driven anti-mycobacterial responses

“…Bone-marrow-derived macrophages (BMDMs) were generated as described previously by our group ( Lebre et al, 2018 ). Briefly, bone marrow cells were extracted from the leg bones and were cultured in high glucose DMEM, supplemented with 8% v/v fetal bovine serum (FBS), 2 mM L-glutamine, 50 U ml –1 penicillin, 50 μg ml –1 streptomycin (hereafter referred to as complete DMEM [cDMEM]).…”

Macrophage Innate Training Induced by IL-4 and IL-13 Activation Enhances OXPHOS Driven Anti-Mycobacterial Responses

“…Bone marrow-derived macrophages (BMDMs) were generated as described previously by our group (Lebre, Hanlon, Boland, Coleman, & Lavelle, 2018). Briefly, bone marrow cells were extracted from the leg bones and were cultured in high glucose DMEM, supplemented with 8% v/v fetal bovine serum (FBS), 2 mM L-glutamine, 50 U ml -1 penicillin, 50 μg ml -1 streptomycin (hereafter referred to as complete DMEM [cDMEM]).…”

“…Bone marrow-derived macrophages (BMDMs) were generated as described previously by our group. (Lebre, Hanlon, Boland, Coleman, & Lavelle, 2018) Briefly, bone marrow cells were extracted from the leg bones and were cultured in high glucose DMEM, supplemented with 8% v/v fetal bovine serum (FBS), 2 mM L-glutamine, 50 U ml -1 penicillin, 50 μg ml -1 streptomycin (hereafter referred to as complete DMEM [cDMEM]). Cells were plated on non-tissue cultured treated petri dishes (Corning) and supplemented with 25% v/v of L929 cell line conditioned medium containing macrophage colonystimulating factor (M-CSF) on day -8.…”