Abstract

Graphene oxide nanoparticles (GONPs) have attracted a lot of attention due to their many applications. These applications include batteries, super capacitors, drug delivery and biosensing. However, few studies have investigated the effects of these nanoparticles on the immune system. In this study, the in vitro effects of GONPs on the immune system was evaluated by exposing murine macrophages, RAW 264.7 cells and human whole blood cell cultures (to GONPs. The effects of GONPs on RAW cells were monitored under basal conditions. The whole blood cell cultures were exposed to GONPs in the presence or absence of the mitogens lipopolysaccharide (LPS) and phytohaemmagglutinin (PHA). A number of parameters were monitored for both RAW and whole blood cell cultures, these included cytotoxicity, inflammatory biomarkers, cytokines of the acquired immune system and a proteome profile analysis. The GONPs were cytotoxic to both RAW and whole blood cell cultures at 500 μg/mL. In the absence of LPS, GONPs elicited an inflammatory response from the murine macrophage, RAW and whole blood cell cultures at 15.6 and 5 μg/mL respectively. This activation was further corroborated by proteome profile analysis of both experimental cultures. GONPs inhibited LPS induced interleukin 6 (IL-6) synthesis and PHA induced interferon gamma (IFNγ) synthesis by whole blood cell cultures in a dose dependent manner. In the absence of mitogens, GONPs stimulated IL-10 synthesis by whole blood cell cultures. The current study shows that GONPs modulate immune system biomarkers and that these may pose a health risk to individuals exposed to this type of nanoparticle.

Highlights

  • Graphene (G) is a two-dimensional (2-D) carbon material, with a hexagonal structure consisting of sp2 hybridized atoms, which has been isolated from its three-dimensional parent material, graphite [1,2,3]

  • A number of immune system biomarkers were monitored such as cytotoxicity, inflammatory biomarkers, cytokines of the acquired immune system and a proteome profile analysis of cytokines and chemokines expressed upon graphene oxide nanoparticles (GONPs) exposure

  • GONPs at concentrations ≤ 31.25 μg/mL did not have an effect on cell viability (Figure 1a)

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Summary

Introduction

Graphene (G) is a two-dimensional (2-D) carbon material, with a hexagonal structure (honeycomb lattice) consisting of sp hybridized atoms, which has been isolated from its three-dimensional parent material, graphite [1,2,3]. These oxidized graphene nanoparticles possess unique characteristics such as electronic and thermal conductivity, superior mechanical strength and optical properties [6] Due to these exceptional physiochemical properties of graphene oxide nanoparticles (GONPs), it has a broad range of applications in a number of fields. These fields include electrochemistry, biomedicine, biosensing, drug delivery, high energy capacity batteries and super capacitors to name a few. IFNγ is produced mainly by activated CD4+ or CD8+ T cells and natural killer (NK) cells and is recognized as the principal mediator of innate and adaptive immunity [14,15] Validating their selection as GONPs could potentially result in immunotoxicity. A number of immune system biomarkers were monitored such as cytotoxicity, inflammatory biomarkers, cytokines of the acquired immune system and a proteome profile analysis of cytokines and chemokines expressed upon GONPs exposure

Results
Materials and Methods
Preparation of GONPs
Cytotoxicity Assay
NO Determination
Mouse Proteome Profiling Assay
Quantification of Pixel Density for Cytokine and Chemokine Membranes
Blood Collection
Cell Culture
LDH Assay
Cytokine Analysis Using DAS ELISAs
Human MIP-1β DAS ELISAs
Human Proteome Profiling
Statistical Analysis

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