1. Introduction
1.3 Contrast Agent
1.3.3 Superparamagnetic Agents
1.3.3.3 Recent Advancement in Superparamagnetic Agents
Magnetic nanoparticle probes are emerging as a next generation contrast medical imaging. Super paramagnetic iron oxide (SPIO) particles were suggested as potential liver specific MR contrast agents as early as the mid 1980s [56]. Recent research shows SPIO particles have potentially to generate higher contrast enhancement in MRI than conventional paramagnetic Gd-based contrast agents. Numerous chemical methods have been reported for synthesize of SPIO nanoparticles for medical imaging applications:
microemulsions [57], sol-gel syntheses [58], sonochemical reactions [59], hydrothermal reactions [60], hydrolysis and thermolysis of precursors [61], flow injection syntheses [62], and electrospray syntheses [63]. Aqueous co-precipitation process in the presence of the coating material has been frequently employed for the synthesis of SPIO and USPIO.
The main advantage of this process is that a large amount of nanoparticles can be synthesized. However, the control of particle size distribution is limited, because only kinetic factors are controlling the growth of the crystal [64-66]. The saturation magnetizations (Ms) Values of nanoparticles obtained by these methods are in the range of 30–50 emu/g, which is lower than the 90 emu/g reported for their bulk form. The low Ms value is due to incorporation of impurities hampering the crystal structure and surface effect [67]. Monodisperse magnetite nanoparticles has been synthesized at high-temperature by decomposition reaction of iron(III) acetylacetonate with 1,2-hexadecanediol in the presence of oleic acid and oleylamine to. The particle diameter can be tuned from 4 to 20 nm by varying the reaction temperature [68]. However, use of hydrophobic oleic acid and oleylamine surfactants in the process results in a hydrophobic coating on the particle surface, amphiphilic polymer or surface surfactant exchange have been utilized to overcome this problem [69]. In order to meet the demand of excellent
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magnetic property for the applications such as, molecular imaging, metal doped iron oxide nanoparticles have drawn much attention due to their enhanced magnetic properties. Spinel metal ferrites with a composition of MFe2O, where M is +2 cation of Mn, Fe, Co or Ni, have been fabricated by various methods to tune specific magnetic properties. Comprehensive study conducted by Lee et al. on 12 nm ferrite nanoparticles (MnFe2O4, Fe3O4, CoFe2O4, NiFe2O4) for MRI application shows that 12 nm MnFe2O4
NPs have the highest mass magnetization value of 110 (emu per mass of magnetic atoms) among MnFe2O4, Fe3O4, CoFe2O4, NiFe2O4 as shown in Figure 5. Furthermore their results also suggest that MnFe2O nanoparticles are nontoxic in vitro and possess higher magnetic susceptibility than magnetite nanoparticles, suggesting that they may be used as an ultrasensitive MR imaging probe [70].
Figure 5 Magnetism-engineered iron oxide (MEIO) nanoparticles. TEM images of MnFe2O4 (MnMEIO), Fe3O4 (MEIO), CoFe2O4 (CoMEIO) and NiFe2O4 (NiMEIO).
Scale bar, 50 nm [70]
Recently, Seo et al. reported synthesis of bimetallic FeCo core of 7 nm and 4 nm with a single-graphitic shell through chemical vapor deposition (CVD), a systematic graphical
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design shown in Figure 6. The Ms of the 7 nm and 4 nm nanocrystals were 215 emu/g and 162 emu/g, respectively with excellent r1 and r2 relaxivities [24].
Figure 6 Systematic diagram of a FeCo/GC nanocrystal [24]
The direct use of SPION as in vivo MRI contrast agents results in biofouling of the particles in blood plasma and formation of aggregates due to high surface energy that triggered by the “opsonization” process resulting in fast clearance of SPIO nanoparticles [71]. Therefore, it is essential to engineer the surface of the SPION to minimize biofouling and aggregation of the particles in physiological conditions for long periods.
Poly (ethylene glycol) (PEG) is widely used polymer for nanoparticle coating. Kohler et al. developed bifunctional PEG silanes capable of forming self-assembled monolayers (SAMs), furthermore terminal functional group (amine or carboxyl) extending out from the nanoparticle surface provide sites for conjugation of functional ligand.
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1.3.4 Carbon Based Materials for Future MRI Contrast Agent
In last two decades a wide range of all-carbon nanostructures have been discovered for instance, fullerenes (1985), carbon nanotubes (1991), graphene (2004). Since the discovery of carbon nanotube (CNT) in 1991 [72], it has been used extensively used for various technological applications [73-74]. Chemically modifiable outer surface have been used as diagnostic and therapeutic agents in medicine [75-76]. Single-walled carbon nanotubes (SWNTs), fabricated from single sheets of graphene have been extensively investigated forms of carbon nanotubes for biological and medical and other technological application owing to its intriguing physical properties such as quantum electronic transport [77], a tunable band gap [78], extremely high mobility [79], high elasticity [80] and electromechanical modulation [81].
Although there has been doubt as to whether the ferromagnetic features of some of the graphitic materials was due to magnetic impurities such as iron. Recent study shows occurrence of intrinsic magnetism in carbon-based materials possessing the sp2 network [82]. Carbon-based magnetic materials would bring a new prospective to technologies relying on magnetism for instance MR imaging. Surprisingly, the first organic ferromagnet, the γ-phase p-nitrophenyl nitronyl nitroxide (p-NPNN) was discovered only in 1991 [83]. Such materials may have low density, be transparent or environment-friendly.
Graphene, a two-dimensional honeycomb lattice of sp-bonded carbon atoms, has shown a wealth of exceptional properties. Since the discovery of the first isolated graphene in 2004, it has attracted major interest, because of its high charge mobility and crystal quality [84]. Xie et al. and Yu et al. have reported synthesis of carbon nanoscrolls
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[85] and carbon nanotube [86] from monolayer graphene respectively. Schematic representation of CNS formation is shown in Figure 7.
Figure 7 Schematic representation of the formation of CNS. Step1: surface strain is induced in graphene after it is immersed in IPA solution. Step2: the edge of graphene is lifted up with the help of the surface strain and the intercalation of IPA solution. Step3:
the initial bending of the graphene is energetically unfavorable and might be caused by perturbations. Once the graphene gets selfstacked, the scrolling process will be easier.
Step4: the graphene continued to roll up until a CNS is formed. [85]
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Richerd et al. have reported noncovalent functionalization of carbon nanotube with amphiphilic Gd(III) chelates, a systematic scheme shown in Figure 8. The study shows T2 frequencies at any GdL concentration are remarkably lower than that of pure water, and they are practically independent of both the frequency and the Gd(III) concentration (8.5-13.8 ms for MWNT/GdL versus 2500 ms for water). Interestingly, the transverse relaxation times, T2 values of suspensions containing MWNT and the amphiphilic ligand L at various concentrations without any Gd(III) are in the same range as those measured in the solutions containing Gd(III) [87].
Figure 8 Carbon nanotubes noncovalently functionalized by amphiphilic Gd3+
chelates[87]
Recently, Ananta et al. have report that raw HiPco SWNTs (r-SWNTs), purified SWNTs (p-SWNTs), and US-tubes show inherently high performance T2-weighted MRI contrast agents by virtue of their superparamagnetic character, with the US-tubes being the most efficacious of the materials by far. The high-efficacy contrast performance is due to contributions from both the iron catalyst nanoparticles (originating from the synthesis of SWNT materials) and the carbon SWNT material itself. Table 3 shows relaxation time of Aqueous SWNT solution along with clinically-used SPIO (Ferumoxtran) T2 agent measured at 1.41 T and 37 °C [88].
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Table 3 Relaxation Times of Aqueous SWNT Solutions (dispersed using pluronic surfactant)a
aData were collected with a Bruker Minispec Mq-60 spectrometer at 1.41 T and 37 °C.
For comparison, a clinically-used SPIO T2 agent (Ferumoxtran) is also included . b Based on the Fe concentration. c Based on the SWNT concentration [88]
Wang et al. has reported synthesis of soluble carbon nanotubes by the sonication of graphene oxide nanosheets. Systematic diagram of synthesis procedure is depicted in Figure 9.
Figure 9 systematic illustration of the mechanism for transforming GO nanosheets into carbon nanoparticles and nanotubes following their ultrasonication in acid. (I) Oxidative cutting of graphene oxide produces PAH molecules in concentrated HNO3. In the dehydrating acidic medium, the polyaromatic fragments fuse and nucleate into (II) carbon nanoparticles or (III) nanotubes via acid-catalyzed intramolecular or intermolecular dehydration reactions. [89]
sample [Fe] mM T1(ms) T2(ms) r1 (mM-1 s-1)b r2 (mM-1 s-1)b r2/r1 T2(ms ·mg-1)c
r-SWNT 0.449 34.6 63.6 4.6
p-SWNT 0.167 242.2 46.5 22.8 126.8 5.6 5.1
US-tube 0.075 1000 67.7 8.9 192.5 21.6 31.7
Ferumoxtran-10 9.9 65 6.6
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The saturation magnetization of CNT synthesized by Wang et al. increased from 25 µemu cm-2 to a maximum of 100 µemu cm-2 upon annealing to 400 °C and gradually dropped to 41 µemu cm-2 on annealing at higher temperatures [89].
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Chapter 2
Experimental Section
2.1 Instruments and Regents 2.1.1 Instruments
1. Particles diameter analyzer (Dynamic Light Scattering): English Malvern Instruments Company, Model: ZetaSizer 3000 HAS
2. NMR relaxometer (NMS-120 Minispec, Bruker) 3. CO2 incubator (Japan SANYO, Model: MCO-20AIC)
4. MR scanner (3.0 T) (Sigma; GE Medical Systems, Milwaukee, WI) 5. MPCVD (ASTeX type microwave plasma CVD)
6. XRD
7. Field-emission scanning electron microscopy (FESEM, JEOL JSM-6700F) 8. Transmission electron microscope (Philips TEM)
9. Raman microscopy (LABRAMHR).
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2.1.2 Reagents
1. Zymed Laboratories:
Mouse anti- MUC4, clone 1G8 2. HyClone
RPMI-1640 medium 3. Malliockrodt Methanol 99.7 % 4. TEDIA
Dimethyl sulfoxideDMSO99.9 %
5. GIBCO:
Dulbecco's modified Eagle's medium
2.1.3 Cell Culture Medium
Culture Medium for BxPC-3
RPMI 1640 medium with 2 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 90%; fetal bovine serum, 10%
Culture Medium for PANC-1
90% Dulbecco's modified Eagle's medium with 4 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate and 4.5 g/L glucose + 10% fetal bovine serum
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2.2 Experimental Methods
2.2.1 Immobilization of 8G7 mAb on SPIO-mPEG-NH
2One hundred microliters of the SPIO-mPEG-NH2 at a concentration of 4 mg Fe/mL was added to 400 lL of 8G7 mAb (ZYMED Laboratories, UK), using 1-hydroxybenzotriazole and (benzotriazol-1-yloxy) tripyrrolidinophosphonium hexafluorophosphate as catalysts, and the mixture was stirred for 24 h at room temperature. The solution was separated from unbound 8G7 mAb by dialysis.
2.2.2 Measurement of Particle Size
The hydrodynamic diameter of the SPIO-mPEG-NH2 particles was measured using a Zetasizer Nano-z (Malvern Instruments, Malvern, UK) through dynamic light scattering (DLS).
2.2.3 Relaxation Time Measurement
The relaxation times (T1 and T2) of aqueous solutions of SPIO-mPEG-8G7 nanoparticle complexes were measured to determine relaxivity, r1 and r2. All measurements were made using a NMR relaxometer operating at 20 MHz and 37.0 ± 0.1 °C (NMS-120 Minispec, Bruker). Before each measurement the relaxometer was tuned and calibrated.
The values of r1 and r2 were determined from eight data points generated by inversion recovery and a Carr–Purcell–Meiboom–Gill pulse sequence, respectively.
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2.2.4 In vitro MRI
BxPC3 and PANC were cultured at 37 °C in humidified 5% CO2 atmosphere. MRI was performed with a clinical 3.0-T magnetic resonance scanner (Sigma; GE Medical Systems, Milwaukee, WI, USA) and a knee coil. All cell lines contained 2 × 106 cells and were incubated with SPIO-mPEG-8G7 nanoparticle (diluted in 1 mL medium, 0.3 mM Fe) for 30 min in an ice bath and then washed three times with PBS. All samples were scanned by a fast gradient echo pulse sequence (TR/TE/flip angel 3,000/90/10◦
.
)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.
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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
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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.
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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.
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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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