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. 2014 Jun 14;2(22):3519-3530.
doi: 10.1039/C4TB00326H.

Synthesis, Characterization, In Vitro Phantom Imaging, and Cytotoxicity of A Novel Graphene-Based Multimodal Magnetic Resonance Imaging - X-Ray Computed Tomography Contrast Agent

Gaurav Lalwani  1 , Joe Livingston Sundararaj  2 , Kenneth Schaefer  1 , Terry Button  3 , Balaji Sitharaman  1
Affiliations

Synthesis, Characterization, In Vitro Phantom Imaging, and Cytotoxicity of A Novel Graphene-Based Multimodal Magnetic Resonance Imaging - X-Ray Computed Tomography Contrast Agent

Gaurav Lalwani et al. J Mater Chem B. .

Abstract

Graphene nanoplatelets (GNPs), synthesized using potassium permanganate-based oxidation and exfoliation followed by reduction with hydroiodic acid (rGNP-HI), have intercalated manganese ions within the graphene sheets, and upon functionalization with iodine, show excellent potential as biomodal contrast agents for magnetic resonance imaging (MRI) and computed tomography (CT). Structural characterization of rGNP-HI nanoparticles with low- and high-resolution transmission electron microscope (TEM) showed disc-shaped nanoparticles (average diameter, 200 nm, average thickness, 3 nm). Energy dispersive X-ray spectroscopy (EDX) analysis confirmed the presence of intercalated manganese. Raman spectroscopy and X-ray diffraction (XRD) analysis of rGNP-HI confirmed the reduction of oxidized GNPs (O-GNPs), absence of molecular and physically adsorbed iodine, and the functionalization of graphene with iodine as polyiodide complexes (I3- and I5-). Manganese and iodine content were quantified as 5.1 ± 0.5 and 10.54 ± 0.87 wt% by inductively-coupled plasma optical emission spectroscopy and ion-selective electrode measurements, respectively. In vitro cytotoxicity analysis, using absorbance (LDH assay) and fluorescence (calcein AM) based assays, performed on NIH3T3 mouse fibroblasts and A498 human kidney epithelial cells, showed CD50 values of rGNP-HI between 179-301 µg/ml, depending on the cell line and the cytotoxicity assay. CT and MRI phantom imaging of rGNP-HI showed high CT (approximately 3200% greater than HI at equimolar iodine concentration) and MRI (approximately 59% greater than equimolar Mn2+ solution) contrast. These results open avenues for further in vivo safety and efficacy studies towards the development of carbon nanostructure-based multimodal MRI-CT contrast agents.

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Figures

Figure 1
Figure 1
(A, B) High-resolution TEM images of rGNP-HI depicting multiple layers of graphene nanoplatelets. (C) Low-resolution TEM image depicting a single rGNP-HI. (D) AFM topographical scan of rGNP-HI. (E) AFM height profile showing the thickness of rGNP-HI (F) HRTEM-EDX showing the presence of iodine and manganese. (G, H, I) SEM images of O-GNPs, rGNP-N2H2 and r-GNP-HI with the corresponding EDX spectra of the region marked with a red box (inset). (J) SEM-EDX of rGNP-HI reduced using 31.1, 16.05 and 8.02 mM HI.
Figure 1
Figure 1
(A, B) High-resolution TEM images of rGNP-HI depicting multiple layers of graphene nanoplatelets. (C) Low-resolution TEM image depicting a single rGNP-HI. (D) AFM topographical scan of rGNP-HI. (E) AFM height profile showing the thickness of rGNP-HI (F) HRTEM-EDX showing the presence of iodine and manganese. (G, H, I) SEM images of O-GNPs, rGNP-N2H2 and r-GNP-HI with the corresponding EDX spectra of the region marked with a red box (inset). (J) SEM-EDX of rGNP-HI reduced using 31.1, 16.05 and 8.02 mM HI.
Figure 1
Figure 1
(A, B) High-resolution TEM images of rGNP-HI depicting multiple layers of graphene nanoplatelets. (C) Low-resolution TEM image depicting a single rGNP-HI. (D) AFM topographical scan of rGNP-HI. (E) AFM height profile showing the thickness of rGNP-HI (F) HRTEM-EDX showing the presence of iodine and manganese. (G, H, I) SEM images of O-GNPs, rGNP-N2H2 and r-GNP-HI with the corresponding EDX spectra of the region marked with a red box (inset). (J) SEM-EDX of rGNP-HI reduced using 31.1, 16.05 and 8.02 mM HI.
Figure 1
Figure 1
(A, B) High-resolution TEM images of rGNP-HI depicting multiple layers of graphene nanoplatelets. (C) Low-resolution TEM image depicting a single rGNP-HI. (D) AFM topographical scan of rGNP-HI. (E) AFM height profile showing the thickness of rGNP-HI (F) HRTEM-EDX showing the presence of iodine and manganese. (G, H, I) SEM images of O-GNPs, rGNP-N2H2 and r-GNP-HI with the corresponding EDX spectra of the region marked with a red box (inset). (J) SEM-EDX of rGNP-HI reduced using 31.1, 16.05 and 8.02 mM HI.
Figure 1
Figure 1
(A, B) High-resolution TEM images of rGNP-HI depicting multiple layers of graphene nanoplatelets. (C) Low-resolution TEM image depicting a single rGNP-HI. (D) AFM topographical scan of rGNP-HI. (E) AFM height profile showing the thickness of rGNP-HI (F) HRTEM-EDX showing the presence of iodine and manganese. (G, H, I) SEM images of O-GNPs, rGNP-N2H2 and r-GNP-HI with the corresponding EDX spectra of the region marked with a red box (inset). (J) SEM-EDX of rGNP-HI reduced using 31.1, 16.05 and 8.02 mM HI.
Figure 1
Figure 1
(A, B) High-resolution TEM images of rGNP-HI depicting multiple layers of graphene nanoplatelets. (C) Low-resolution TEM image depicting a single rGNP-HI. (D) AFM topographical scan of rGNP-HI. (E) AFM height profile showing the thickness of rGNP-HI (F) HRTEM-EDX showing the presence of iodine and manganese. (G, H, I) SEM images of O-GNPs, rGNP-N2H2 and r-GNP-HI with the corresponding EDX spectra of the region marked with a red box (inset). (J) SEM-EDX of rGNP-HI reduced using 31.1, 16.05 and 8.02 mM HI.
Figure 1
Figure 1
(A, B) High-resolution TEM images of rGNP-HI depicting multiple layers of graphene nanoplatelets. (C) Low-resolution TEM image depicting a single rGNP-HI. (D) AFM topographical scan of rGNP-HI. (E) AFM height profile showing the thickness of rGNP-HI (F) HRTEM-EDX showing the presence of iodine and manganese. (G, H, I) SEM images of O-GNPs, rGNP-N2H2 and r-GNP-HI with the corresponding EDX spectra of the region marked with a red box (inset). (J) SEM-EDX of rGNP-HI reduced using 31.1, 16.05 and 8.02 mM HI.
Figure 1
Figure 1
(A, B) High-resolution TEM images of rGNP-HI depicting multiple layers of graphene nanoplatelets. (C) Low-resolution TEM image depicting a single rGNP-HI. (D) AFM topographical scan of rGNP-HI. (E) AFM height profile showing the thickness of rGNP-HI (F) HRTEM-EDX showing the presence of iodine and manganese. (G, H, I) SEM images of O-GNPs, rGNP-N2H2 and r-GNP-HI with the corresponding EDX spectra of the region marked with a red box (inset). (J) SEM-EDX of rGNP-HI reduced using 31.1, 16.05 and 8.02 mM HI.
Figure 2
Figure 2
Raman spectroscopic analysis of (a) pristine graphite, (b) O-GNPs, (c) rGNP-N2H2, and (d) rGNP-HI.
Figure 3
Figure 3
Powder x-ray diffraction patterns of (a) pristine graphite, (b) O-GNPs, and (c) rGNP-HI.
Figure 4
Figure 4
Cytotoxicity analysis of rGNP-HI at concentrations ranging from 10–500 µg/ml against NIH3T3 cells and A498 cells after 24 and 48 hours of exposure. (A, B) Percent LDH release (normalized to positive controls, i.e. lysed cells). (C, D) Percentage of cell viability (normalized to positive controls, i.e. cells cultured on TCPS in the absence of rGNP-HI treatment) as measured by quantification of calcein fluorescence. Data are presented as mean ± SD for n = 6 groups. Groups with a significant difference (p < 0.05) compared to the TCPS controls at each time point are indicated with ‘*’, and those with a statistical difference between the 24 and 48 hour time points are indicated with ‘#’. (E) Representative fluorescence images of NIH3T3 cells stained with calcein-AM (green) and EthD-1 (red) after 48 hours of exposure to rGNP-HI at 1 µg/ml [(c) & (d)], 10 µg/ml [(e) & (f)], 25 µg/ml [(g) & (h)] and 50 µg/ml [(i) & (j)]. Images (a) & (b) are control cells (no exposure). The size of the scale bars are 100 µm.
Figure 4
Figure 4
Cytotoxicity analysis of rGNP-HI at concentrations ranging from 10–500 µg/ml against NIH3T3 cells and A498 cells after 24 and 48 hours of exposure. (A, B) Percent LDH release (normalized to positive controls, i.e. lysed cells). (C, D) Percentage of cell viability (normalized to positive controls, i.e. cells cultured on TCPS in the absence of rGNP-HI treatment) as measured by quantification of calcein fluorescence. Data are presented as mean ± SD for n = 6 groups. Groups with a significant difference (p < 0.05) compared to the TCPS controls at each time point are indicated with ‘*’, and those with a statistical difference between the 24 and 48 hour time points are indicated with ‘#’. (E) Representative fluorescence images of NIH3T3 cells stained with calcein-AM (green) and EthD-1 (red) after 48 hours of exposure to rGNP-HI at 1 µg/ml [(c) & (d)], 10 µg/ml [(e) & (f)], 25 µg/ml [(g) & (h)] and 50 µg/ml [(i) & (j)]. Images (a) & (b) are control cells (no exposure). The size of the scale bars are 100 µm.
Figure 4
Figure 4
Cytotoxicity analysis of rGNP-HI at concentrations ranging from 10–500 µg/ml against NIH3T3 cells and A498 cells after 24 and 48 hours of exposure. (A, B) Percent LDH release (normalized to positive controls, i.e. lysed cells). (C, D) Percentage of cell viability (normalized to positive controls, i.e. cells cultured on TCPS in the absence of rGNP-HI treatment) as measured by quantification of calcein fluorescence. Data are presented as mean ± SD for n = 6 groups. Groups with a significant difference (p < 0.05) compared to the TCPS controls at each time point are indicated with ‘*’, and those with a statistical difference between the 24 and 48 hour time points are indicated with ‘#’. (E) Representative fluorescence images of NIH3T3 cells stained with calcein-AM (green) and EthD-1 (red) after 48 hours of exposure to rGNP-HI at 1 µg/ml [(c) & (d)], 10 µg/ml [(e) & (f)], 25 µg/ml [(g) & (h)] and 50 µg/ml [(i) & (j)]. Images (a) & (b) are control cells (no exposure). The size of the scale bars are 100 µm.
Figure 4
Figure 4
Cytotoxicity analysis of rGNP-HI at concentrations ranging from 10–500 µg/ml against NIH3T3 cells and A498 cells after 24 and 48 hours of exposure. (A, B) Percent LDH release (normalized to positive controls, i.e. lysed cells). (C, D) Percentage of cell viability (normalized to positive controls, i.e. cells cultured on TCPS in the absence of rGNP-HI treatment) as measured by quantification of calcein fluorescence. Data are presented as mean ± SD for n = 6 groups. Groups with a significant difference (p < 0.05) compared to the TCPS controls at each time point are indicated with ‘*’, and those with a statistical difference between the 24 and 48 hour time points are indicated with ‘#’. (E) Representative fluorescence images of NIH3T3 cells stained with calcein-AM (green) and EthD-1 (red) after 48 hours of exposure to rGNP-HI at 1 µg/ml [(c) & (d)], 10 µg/ml [(e) & (f)], 25 µg/ml [(g) & (h)] and 50 µg/ml [(i) & (j)]. Images (a) & (b) are control cells (no exposure). The size of the scale bars are 100 µm.
Figure 4
Figure 4
Cytotoxicity analysis of rGNP-HI at concentrations ranging from 10–500 µg/ml against NIH3T3 cells and A498 cells after 24 and 48 hours of exposure. (A, B) Percent LDH release (normalized to positive controls, i.e. lysed cells). (C, D) Percentage of cell viability (normalized to positive controls, i.e. cells cultured on TCPS in the absence of rGNP-HI treatment) as measured by quantification of calcein fluorescence. Data are presented as mean ± SD for n = 6 groups. Groups with a significant difference (p < 0.05) compared to the TCPS controls at each time point are indicated with ‘*’, and those with a statistical difference between the 24 and 48 hour time points are indicated with ‘#’. (E) Representative fluorescence images of NIH3T3 cells stained with calcein-AM (green) and EthD-1 (red) after 48 hours of exposure to rGNP-HI at 1 µg/ml [(c) & (d)], 10 µg/ml [(e) & (f)], 25 µg/ml [(g) & (h)] and 50 µg/ml [(i) & (j)]. Images (a) & (b) are control cells (no exposure). The size of the scale bars are 100 µm.
Figure 5
Figure 5
(A) CT phantom imaging of DI water, hydroiodic acid, MnCl2, and rGNP-HI using a GE 64 slice Lightspeed VCT CT scanner. (B) MRI phantom imaging of hydroiodic acid, MnCl2, and rGNP-HI using a GE HTXT 1.5T clinical MRI scanner.
Figure 5
Figure 5
(A) CT phantom imaging of DI water, hydroiodic acid, MnCl2, and rGNP-HI using a GE 64 slice Lightspeed VCT CT scanner. (B) MRI phantom imaging of hydroiodic acid, MnCl2, and rGNP-HI using a GE HTXT 1.5T clinical MRI scanner.
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