2. Experimental Section
2.2 Experimental Methods
2.2.5 Synthesis of Graphene Nanosheet
GNSs have been successively synthesized on GaN/Sapphire template, which was carried out in an ASTeXtype microwave plasma CVD system. In order to optimize the microwave discharge and the extension of the bias discharge over the entire substrate, we used a dome-shaped Mo anode which was placed above the substrate as counter-electrode. The ~3 µm thickness of GaN has been formed on sapphire substrate by metal-organic CVD. Prior to the deposition GNSs over it, the template was ultrasonically cleaned with acetone and alcohol for 12 min each. For carburization of template, we used 4 % CH4 with 20 torr and microwave power was 550W. For the deposition of graphene over it, we used 2.9 % CH4 with 40 torr and microwave power was 800W and bias voltage of - 100 V for 30 min and followed by further deposition for ~2 hr without bias the template. The GNSs was analyzed by a standard x-ray diffractometer (XRD) with a Cu K
source. The microstructure of theGNSs /GaN sample was evaluated with Raman microscopy (LABRAMHR)
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Chapter 3
Results and Discussion
3.1 Physical Characteristics of SPIO-mPEG-8G7
The physical parameters of SPIO nanoparticle used for the study are shown in Table 4.
Table 4 Physical properties of MnFe2O4a
Parameter MnFe
2O
4TEM (nm) 12.4 ± 0.9
b
DLS(nm) 30.3 ± 5.7
b
Relaxivity(r
2/r
1) 238.4/36.9
Magnetization (emu/g) 84
aData obtained from Mr. John thesis. bThe DLS and relaxivity data are based on SPIO-mPEG.
The –NH2 terminal of SPIO-mPEG-NH2 has been used to conjugate 8G7 mAb on the surface of SPIO nanoparticle (Scheme 1). The Bicinchoninic Acid (BCA) protein assay was performed to confirm the presence of 8G7 mAb on the surface modification of SPIO-mPEG-NH2. Scheme 2 shows BCA-protein reaction mechanism. BCA serves the purpose of the Folin reagent in the Lowry assay, namely to react with complexes between copper ions and peptide bonds to produce a purple end product [90,91]. The change in color of BCA protein assay solution on adding SPIO-mPEG-8G7 from light blue to purple confirms the presence of 8G7 mAb on surface modification of SPIOThe hydrodynamic size distribution of SPIO-mPEG-NH2 and SPIO-mPEG-anti-MUC4 was investigated by dynamic laser scattering (DLS) analysis system shown in Figure 10. The average diameter
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for SPIO-mPEG-NH2 and SPIO-mPEG-8G7 nanoparticle is 33.1 ± 2.3 nm and 45.4 ± 4.4 nm respectively.
Scheme 1 Conjugation of 8G7 mAb on the surface of SPIO-mPEG-NH2, PyBop (benzotriazol-yloxy) tripyrrolidinophosphonium hexafluorophosphate, HoBt 1-hydroxybenzotriazole
Scheme 2 BCA-Protein Reaction Mechanism
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Figure 10 Hydrodynamic diameters distribution (a) SPIO-mPEG-NH2 (b) SPIO-mPEG-8G7
3.2 Relaxivity of SPIO-mPEG-8G7
In aqueous solution, the relaxivity values, r1 and r2, of the SPIO-mPEG-8G7 at 37.0 ± 0.1
◦C and 20 MHz are 24.97 and 206.01 mM-1 s-1, respectively. The r2 value of SPIO-mPEG-8G7 is higher than that of clinically used Resovist (r2 = 164 mM-1 s-1) [92]. The r1 value of SPIO-mPEG-8G7 and Resovist are similar (r1 = 26 mM-1 s-1) [92].
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Figure 11 T1 relaxation time of SPIO-mPEG-8G7
Figure 12 T2 relaxation time of SPIO-mPEG-8G7
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3.3 In vitro MRI
The targeting ability of SPIO-mPEG-8G7 nanoparticles was confirmed by in vitro MRI, as shown in Figure 13. BxPC3 which has a relatively high MUC4 expression level showed noticeable magnetic resonance contrast. However no contrast observed was observed in case of negatively express MUC4 mucine cell line, Panc1.
Figure 13 T2-weighted images of positive and negative cells for MUC4 expression after the treatment with or without 0.3 mM SPIO-mPEG-8G7 nanoparticles. The upper rows show cells without contrast agent treatment. The lower rows show cells treated with contrast agent. B color-map MRI
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3.4 Structural Characterization of GNSs
The phase structure of the as-prepared final product was characterized by XRD. Figure14 shows a typical XRD pattern of the as-prepared 3-D GNSs/spherical carbon/GaN. A sharp and intense XRD diffraction peak at about 2θ = 26.6○ can be indexed as the (002) diffraction reveals the high-quality graphitic nature of nanosheets. The weak and very sharp peaks at about 2θ = 34.8○ and 2θ = 45○ could be due to the GaN substrate and these two diffraction peaks are corresponding to (002) and (101) planes, respectively.
Figure 14 XRD patterns of the GNSs/spherical carbon/GaN sample.
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The thickness of the graphitic nanosheets along the (001) direction (i.e. the average crystallite size along the (001) direction) is about 56.6 nm estimated from the half-peak width of the (002) reflection peak using the Scherrer equation. This indicates that graphitization is complete and the degree of long-range order of these nanostructures is similar to that of bulk graphite [93]. The interlayer spacing is calculated to be ~0.34 nm from the position of (002) reflection peak are similar to those observed for bulk hexagonal graphite (~0.335 nm) [93]. Later in, high-resolution transmission electron microscope (HRTEM) analysis was performed to confirm the interlayer spacing.
The morphologies of the 3-D GNSs/spherical carbon/GaN sample obtained under typical synthesis conditions were examined by using field-emission scanning electron microscopy (FESEM, JEOL JSM-6700F), transmission electron microscope (Philips TEM), selected-area electron diffraction (SAED), and HRTEM. Figure 15 (A-D) shows the typical FESEM images of the product prepared by microwave plasma CVD in presence of methane/hydrogen gas mixture.
36
Figure 15 (A) Side-view FESEM image of the GNSs/spherical carbons/GaN; (B-C) plane-view FESEM images of the GNSs/spherical carbons/GaN sample at different magnifications; (D) CNS.
Figure 15(A) and 14(B) show the low-resolution side view and plane view FESEM images of the 3-D GNS/spherical carbon/GaN sample. As shown in the FESEM image in Figure 15(A), the as-obtained 3-D GNS consists of spheres with diameters ranging from 9 to 10 µm. The magnified FESEM images (Figure 15(B) shows that the surfaces of spheres are not smooth. And the microspheres look like completely covered by the 3-D GNSs. Figure 15(C) shows the high-resolution SEM image of the 3-D GNS/spherical carbon/GaN. According to Figure 15(C), transparent individual graphite clearly overlaps
37
on the other graphite structure. The higher magnification FESEM image (Figure 15C) clearly reveals that the GNSs have a thickness range 1 to 5 nm.
We have tried to nanoscrolls monolayer GNSs using isopropyl alcohol solution shown in Figure 15(D). Although preliminary result show formation of nanoscrolls from monolayer graphene however to confirm the result further investigation is in process. We believed that this can help us in drug loading.
Figure 16 TEM image of the individual GNS and the corresponding SAED pattern is shown in the inset, and (B) HRTEM of the individual GNS.
38
Figure 16(A) and 16(B) show the TEM image of an individual GNS and its corresponding SAED pattern with the electron beam directed along the individual GNS.
The SAD pattern from the individual GNS shows few bright spots. The clearly visible bright spots confirm that the GNSs are single crystals. The HRTEM image taken at the top edge of the individual GNS shows that the interlayer distance about ~0.34 nm, as shown in Figure 16(B). The lattice spacing of ~0.34 nm corresponds to the (002) plane.
This result is consistent with XRD data.
All the forms of carbon materials such as amorphous carbon, fullerenes, carbon nanotubes, polycrystalline carbon etc. have been characterized by Raman spectroscopy.
The positions, half widths, and relative areas of spectral bands are governed by the nature of the chemical bonds of carbon. Therefore, the Raman spectrum may provide additional information about the as-prepared 3-D GNSs/spherical carbon/GaN structure. Raman spectra taken on GNSs, as shown in Figure 17, are similar to those observed for graphitic carbon [94]. Second order modes in the range of 2000–3000 cm-1 are also present in Figure 17 shows that it has two strong peaks at 1363, and 1582 cm-1. The peak at around 1363 cm-1 is the D-band associated with vibrations of carbon atoms with dangling bonds in plane terminations of the disordered graphite. The peak at 1576 cm-1 (G band) is attributed to the vibration of sp2-bonded carbon atoms in a two-dimensional hexagonal lattice [95,96]. Figure 17 also shows that the strong peak at about 2716 cm-1, is attributed to the disorder mode 2D band.
39
Figure 17 Raman spectrums of the GNSs/spherical carbons/GaN sample.
From, Figure 17 we can see that, the G-band peak is stronger than the D-band peak and their intensity ratio is about 1.4 unambiguously suggests that the 3-D GNSs have high degree of graphitization. In addition, the area ratio between the two bands (AD/AG) allows the degree of ordering or graphitization of the carbon structure to be characterized [97,98]. In the spectra of highly crystalline graphite, D-band is absent, which indicates the 100 %-degree of graphitization. It should be noted that the AD/AG value of GNSs (1.02) was smaller than that of Vulcan XC-72 and AP-carbon [99].
Furthermore, a similar value of AD/AG between GNSs (1.02) and MWCNT (1.03) [99]
confirms that the 3-D GNSs retained similar graphitic characteristics to the MWCNT.
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Conclusion
The preliminary data of this study suggest SPIO-mPEG-8G7 nanoparticles are highly specific to MUC4 expression and it can be successfully used for early diagnosis of pancreatic cancer. This finding will be taken into account in highest priority for the development of carbon based T2 MRI contrast agent for early diagnosis of pancreatic cancer. As for now we have successfully synthesized GNSs in high yield.
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