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. 2018 Feb 24;8(2):125.
doi: 10.3390/nano8020125.

Effects of Graphene Oxide Nanoparticles on the Immune System Biomarkers Produced by RAW 264.7 and Human Whole Blood Cell Cultures

Kim Lategan  1 , Hend Alghadi  2 , Mohamed Bayati  3 , Maria Fidalgo de Cortalezzi  4 , Edmund Pool  5
Affiliations

Effects of Graphene Oxide Nanoparticles on the Immune System Biomarkers Produced by RAW 264.7 and Human Whole Blood Cell Cultures

Kim Lategan et al. Nanomaterials (Basel). .

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.

Keywords: cytotoxicity; graphene oxide nanoparticles; humoral immune response; macrophage activation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure A1
Figure A1
Fourier-transform infrared spectroscopy (FTIR) analysis of GONPs.
Figure A2
Figure A2
Ultraviolet-visible spectrophotometry (UV-vis) of GONPs, with maximum absorbance at 227 nm.
Figure A3
Figure A3
Visualization of graphene sheets via transmission electron microscopy (TEM).
Figure A4
Figure A4
Zeta potential of GONP under varying pH levels, while maintaining a constant ion concentration of 10 mM NaCl.
Figure 1
Figure 1
Murine macrophage, RAW 264.7 cells after treatment with GONPs. Parameters assessed (a) cell viability; (b) nitric oxide production; (c) Interleukin 6 (IL-6) production; (d) Macrophage inflammatory protein 1 alpha (MIP-1α) levels. Positive control not represented (803.85 ± 353.70 ng/mL MIP-1α); (e) MIP-1β levels. Positive control not represented (1127.19 ± 468.69 ng/mL MIP-1β); and (f) MIP-2 levels. Positive control not represented (307.39 ± 171.86 ng/mL MIP-2). Data represents mean ± SD with n = 9. Bars marked with symbols indicate significant difference to control (p < 0.01). Significance demarcated by: *—significantly different compared to negative control (p < 0.001), #—significantly different compared to positive control (p ≤ 0.005).
Figure 2
Figure 2
The effect of graphene oxide nanoparticles (GONPs) on RAW 264.7 cells. Cells were incubated with (a) media only (negative control); (b) media in the presence of LPS and (c) 15.6 μg/mL GONPs in the absence of a mitogen. Supernatants were probed using the proteome profiler array as described in methods. Cytokines/chemokines that were detected were allocated numbers: 1, 3 and 16 are reference spots; 2-IP-10; 4-G-CSF; 5-TNF-α; 6-GM-CSF; 7-IL-6; 8-JE; 9-sICAM-1; 10-MIP-1α; 11-MIP-1β; 12-IL-1β; 13-MIP-2; 14-IL-1ra; 15-RANTES; 17-IL-27; 18-SDF-1.
Figure 3
Figure 3
Quantification of cytokines secreted by RAW 264.7 cultures not stimulated with LPS after treatment with medium only or medium containing 15.6 μg/mL GONPs. Membranes subjected to chromogenic exposure. Data is represented as mean ± SD. Bars marked with symbols indicate significant differences (p < 0.01). Significance demarcated by: *—significantly different compared to the negative control (p < 0.001), #—significantly different compared to the positive control (p < 0.001).
Figure 4
Figure 4
Cell viability of whole blood cell cultures exposed to GONPs. Data represents mean ± SD with n = 4. Bars marked with symbol indicate significant difference to control (p < 0.01). Significance demarcated by: *—significantly different compared to negative control (p < 0.003).
Figure 5
Figure 5
The inflammatory biomarker expression levels of whole blood cell cultures exposed to GO. NPs in the absence or presence of LPS: (a) IL-6 expression levels and (b) MIP-1β expression levels. Data represents mean ± SD with n = 4. Bars marked with symbols indicate significant differences (p < 0.01). Significance demarcated by: *—significantly different compared to negative control (p < 0.001), #-significantly different compared to positive control (p < 0.001).
Figure 6
Figure 6
The acquired immune system biomarker expression levels of whole blood cell cultures exposed to GONPs in the absence or presence of PHA: (a) IL-10 expression levels and (b) IFNγ expression levels. Data represents mean ± SD with n = 4. Bars marked with symbols indicate significant differences (p < 0.01). Significance demarcated by: *—significantly different compared to negative control (p < 0.001), #—significantly different compared to positive control (p < 0.001).
Figure 7
Figure 7
The effects of GONPs on whole blood cells. Cells were incubated with (a) media only, (b) media and LPS, (c) 5 μg/mL GONPs in the absence of LPS. Cytokines/chemokines that were detected were allocated numbers: 1, 2 and 13 are reference spots; 3-IL-1ra; 4-MIF; 5-MCP-1; 6-Serpin E1; 7-MIP-1α/β; 8-RANTES; 9-ICAM-1, 10-IL-6, 11-IL-8 and 12-IL-1β.
Figure 8
Figure 8
Quantification of cytokines secreted by whole blood cell cultures not stimulated with LPS after treatment with medium only or medium containing 5 μg/mL GONPs. Membranes subjected to chromogenic exposure. Data is represented as mean ± SD. Bars marked with symbols indicate significant differences (p < 0.01). Significance demarcated by: *—significantly different compared to the negative control (p < 0.001), #—significantly different compared to the positive control (p < 0.001).
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